Note: Descriptions are shown in the official language in which they were submitted.
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10
NOVEL PROTEASES
The present invention claims priority to provisional application serial no.
60/201,879, filed May 4, 2000, which is hereby incorporated by reference in
its
entirety.
FIELD OF THE INVENTION
The present invention relates to protease polypeptides, nucleotide sequences
encoding the protease polypeptides, as well as various products and methods
useful
for the diagnosis and treatment of various protease-related diseases and
conditions.
BACKGROUND OF THE INVENTION
Proteases and Human Disease
"Protease," "proteinase," and "peptidase" are synonymous terms applying to
all enzymes that hydrolyse peptide bonds, i.e. proteolytic enzymes. Proteases
are an
exceptionally important group of enzymes in medical research and
biotechnology.
They are necessary for the survival of all living creatures, and are encoded
by 1-2%
of all mammalian genes. Rawlings and Barrett (MEROPS: the peptidase database.
Nucleic Acids Res., 1999, 27:325-331) (http://www.babraham.co.uk/Merops/
Merops.htm (Which is incorporated herein by reference in its entirety
including any
figures, tables, or drawings.)) have classified peptidases into 157 families
based on
structural similarity at the catalytic core sequence. These families are
further classed
into 26 clans, based on indications of common evolutionary relationship.
Peptidases
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play key roles in both the normal physiology and disease-related pathways in
mammalian cells. Examples include the modulation of apoptosis (caspases),
control
of blood pressure (renin, angiotensin-converting enzymes), tissue remodeling
and
tumor invasion (collagenase), the development of Alzheimer's Disease (~i-
secretase), protein turnover and cell-cycle regulation (proteosome), and
inflammation (TNF-a, convertase). (Barrett et al., Handbook of Proteolytic
Enzymes, 1995, Academic Press, San Diego which is incorporated herein by
reference in its entirety including any figures, tables, or drawings.)
Peptidases axe classed as either exopeptidases or endopeptidases. The
exopeptidases act only near the ends of polypeptide chains: aminopeptidases
act at
the free N-terminus and carboxypeptidases at the free C-terminus. The
endopeptidases are divided, on the basis of their mechanism of action, into
six sub-
subclasses: aspartyl endopeptidases (3.4.23), cysteine endopeptidases
(3.4.22),
metalloendopeptidases (3.4.24), serine endopeptidases (3.4.21), threonine
endopeptidases (3.4.25), and a final group that could not be assigned to any
of the
above classes (3.4.99). (Enzyme nomenclature and numbering are based on
"Recommendations of the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-ICTBMB) 1992,
(http://www.chem.qmw.ac.uk/iubmb/enzyme/EC34/intro.html).)
In serine-, threonine- and cysteine-type peptidases, the catalytic nucleophile
is the reactive group of an amino acid side chain, either a hydroxyl group
(serine-
and threonine-type peptidases) or a sulthydryl group (cysteine-type
peptidases). In
aspartic-type and metallopeptidases, the nucleophile is commonly an activated
water
molecule. In aspartic-type peptidases, the water molecule is directly bound by
the
side chains of aspartate residues. In metallopeptidases, one or two metal ions
hold
the water molecule in place, and charged amino acid side chains are ligands
for the
metal ions. The metal may be zinc, cobalt or manganese. One metal ion is
usually
attached to three amino acid ligands. Families of peptidases are referred to
by use of
the numbering system of Rawlings & Barrett (Rawlings, N. D. & Barrett, A. J.
MEROPS: the peptidase database. Nucleic Acids Reseal ch 27 (1999) 325-331,
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which is incorporated herein by reference in its entirety including any
figures, tables,
or drawings).
Protease Families
1. As~ariyl proteases (Prosite number PS00141)
Aspartyl proteases, also known as acid proteases, are a widely distributed
family of proteolytic enzymes in vertebrates, fungi, plants, retroviruses and
some
plant viruses. Aspartate proteases of eukaryotes are monomeric enzymes which
consist of two domains. Each domain contains an active site centered on a
catalytic
aspartyl residue. The two domains most probably evolved from the duplication
of
an ancestral gene encoding a primordial domain. Enzymes in this class include
cathepsin E, renin, presenilin (PS1), and the APP secretases.
2. C st~proteases (Prosite PDOC00126)
Eukaryotic cysteine proteases are a family of proteolytic enzymes which
contain an active site cysteine. Catalysis proceeds through a thioester
intermediate
and is facilitated by a nearby histidine side chain; an asparagine completes
the
essential catalytic triad. Peptidases in this family with important roles in
disease
include the caspases, calpain, hedgehog, ubiquitin hydrolases, and papain.
3. Metallo roteases Prosite PDOC00129)
The metalloproteases are a class which includes matrix metalloproteases
(MMPs), collagenasa, stromelysin, gelatinase, neprylisin, carboxypeptidase,
dipeptidase, and membrane-associated metalloproteases, such as those of the
ADAM
family. They require a metal co-factor for activity; frequently the required
metal ion
is zinc but some metalloproteases utilize cobalt and manganese.
Proteins of the extracellular matrix interact directly with cell surface
receptors thereby initiating signal transduction pathways and modulating those
triggered by growth factors, some of which may require binding to the
extracellular
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matrix for optimal activity. Therefore the extracellular matrix has a profound
effect
on the cells encased by it and adjacent to it. Remodeling of the extracellular
matrix
requires protease of several families, including metalloproteases (NIIVIPs).
S 4. Serine proteases (S1)~Prosite PS00134 trypsin-his; PS0013S try sin-ser)
The catalytic activity of the serine proteases from the trypsin family is
provided by a charge relay system involving an aspartic acid residue hydrogen-
bonded to a histidine, which itself is hydrogen-bonded to a serine. The
sequences in
the vicinity of the active site serine and histidine residues are well
conserved in this
family of proteases. A partial list of proteases known to belong to this large
and
important family include: blood coagulation factors VII, IX, X, XI and XII;
thrombin; plasminogen; complement components Clr, Cls, C2; complement factors
B, D and I; complement-activating component of RA-reactive factor; elastases
1, 2,
3A, 3B (protease E); hepatocyte growth factor activator; glandular (tissue)
1S kallikreins including EGF-binding protein types A, B, and C; NGF-y hain, y-
renin,
and prostate specific antigen (PSA); plasma kallikrein; mast cell proteases;
myeloblastin (proteinase 3) (Wegener's autoantigen); plasminogen activators
(urokinase-type, and tissue-type); and the trypsins I, II, III, and IV. These
peptidases play key roles in coagulation, tumorigenesis, control of blood
pressure,
release of growth factors, and other roles.
S. Threonine peptidases Tll~Prosite PDOC00326/PDOC00668)
Threonine proteases are characterized by their use of a hydroxyl group of a
threonine residue in the catalytic site of these enyzmes. Only a few of these
2S enzymes have been characterized thus far, such as the 20S proteasome from
the
archaebacterium Thermoplasma acidophilum (Seemuller et al., 1995, Science,
268:579-82, and chapter 167 of Barrett et al., Handbook of Proteol is Enzymes,
1998, Academic Press, San Diego).
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SUMMARY OF THE INVENTION
This invention concerns the isolation and characterization of novel sequences
of human proteases. These sequences are obtained via bioinformatics searching
strategies on the predicted amino acid translations of new human genetic
sequences.
These sequences, now identified as proteases, are translated into polypeptides
which
are fuxther characterized. Additionaly, the nucleic acid sequences of these
proteases
are used to obtain full-length cDNA clones of the proteases. The partial or
complete
sequences of these proteases are presented here, together with their
classification,
predicted or deduced protein structure.
Modulation of the activities of these proteases will prove useful
therapeutically. Additionally, the presence or absence of these proteases or
the
DNA sequence encoding them will prove useful in diagnosis or prognosis of a
variety of diseases. In this regard, Example 8 describes the chromosomal
localization of proteases of the present invention, and describes diseases
mapping to
the chromosomal locations of the proteases of the invention.
A first aspect of the invention features an identified, isolated, enriched, or
purified nucleic acid molecule encoding a protease polypeptide having an amino
acid sequence selected from the group consisting of those set forth in SEQ ID
N0:36, SEQ ID N0:37, SEQ ID NO:38, SEQ ID N0:39, SEQ ID NO:40, SEQ ID
N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID NO:45, SEQ ID
N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID
NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID
N0:56, SEQ ID N0:57, SEQ,ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID
N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID
N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70The
term "identified" in reference to a nucleic acid is meant that a sequence was
selected
from a genomic, EST, or cDNA sequence database based on being predicted to
encode a portion of a previously unknown or novel protease.
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By "isolated" in reference to nucleic acid is meant a polymer of 10
(preferably 21, more preferably 39, most preferably 75) or more nucleotides
conjugated to each other, including DNA and RNA that is isolated from a
natural
source or that is synthesized as the sense or complementary antisense strand.
In
certain embodiments of the invention, longer nucleic acids are preferred, for
example those of 300, 600, 900, 1200, 1500, or more nucleotides and/or those
having at least 50%, 60%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identity to a sequence selected from the group consisting of
those
set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID
NO:S, SEQ ID NO:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID NO:15,
SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20,
SEQ ID N0:21, SEQ ID N0:22, SEQ ID NO:23, SEQ ID N0:24, SEQ ID N0:25,
SEQ ID N0:26, SEQ ID NO:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30,
SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, and SEQ ID
NO:35.
It is understood that by nucleic acid it is meant, without limitation, DNA,
RNA or cDNA, and where the nucleic acid is RNA, the thymine (T) will be uracil
The isolated nucleic acid of the present invention is unique in the sense that
it is not found in a pure or separated state in nature. Use of the term
"isolated"
indicates that a naturally occurring sequence has been removed from its normal
cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-
free
solution or placed in a different cellular environment. The term does not
imply that
the sequence is the only nucleotide chain present, but that it is essentially
free
(preferably about 90% pure, more preferably at least about 95% pure) of non-
nucleotide material naturally associated with it, and thus is distinguished
from
isolated chromosomes.
By the use of the term "enriched" in reference to nucleic acid is meant that
the specific DNA or RNA sequence constitutes a significantly higher fraction
(2- to
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5-fold) of the total DNA or RNA present in the cells or solution of interest
than in
normal or diseased cells or in the cells from which the sequence was taken.
This
could be caused by a person by preferential reduction in the amount of other
DNA or
RNA present, or by a preferential increase in the amount of the specific DNA
or
RNA sequence, or by a combination of the two. However, it should be noted that
enriched does not imply that there are no other DNA or RNA sequences present,
just
that the relative amount of the sequence of interest has been significantly
increased.
The term "significant" is used to indicate that the level of increase is
useful to the
person making such an increase, and generally means an increase relative to
other
nucleic acids of about at least 2-fold, more preferably at least 5-fold, more
preferably at least 10-fold or even more. The term also does not imply that
there is
no DNA or RNA from other sources. The DNA from other sources may, for
example, comprise DNA from a yeast or bacterial genome, or a cloning vector
such
as pUCl9. This term distinguishes from naturally occurring events, such as
viral
infection, or tumor-type growths, in which the level of one mRNA may be
naturally
increased relative to other species of mRNA. That is, the term is meant to
cover
only those situations in which a person has intervened to elevate the
proportion of
the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in
purified form. The term "purified" in reference to nucleic acid does not
require
absolute purity (such as a homogeneous preparation). Instead, it represents an
indication that the sequence is relatively more pure than in the natural
environment
(compared to the natural level this level should be at least 2- to 5-fold
greater, e.g.,
in terms of mg/mL). Individual clones isolated from a cDNA library may be
purified to electrophoretic homogeneity. The claimed DNA molecules obtained
from these clones could be obtained directly from total DNA or from total RNA.
The cDNA clones are not naturally occurring, but rather are preferably
obtained via
manipulation of a partially purified naturally occurnng substance (messenger
RNA).
The construction of a cDNA library from mRNA involves the creation of a
synthetic
substance (cDNA) and pure individual cDNA clones can be isolated from the
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synthetic library by clonal selection of the cells carrying the cDNA library.
Thus,
the process which includes the construction of a cDNA library from mRNA and
isolation of distinct cDNA clones yields an approximately 10~-fold
purification of
the native message. Thus, purification of at least one order of magnitude,
preferably
two or three orders, and more preferably four or five orders of magnitude is
expressly contemplated.
By a " protease polypeptide" is meant 32 (preferably 40, more preferably 45,
most preferably 55) or more contiguous amino acids in a polypeptide having an
amino acid sequence selected from the group consisting of those set forth in
SEQ ID
N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID
N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID
N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID
NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID
N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID NO:59, SEQ ID N0:60, SEQ ID
N0:61, SEQ ID N0:62, SEQ ID NO:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID
NO:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70. In
certain aspects, polypeptides of 100, 200, 300, 400, 450, 500, 550, 600, 700,
800,
900 or more amino acids are preferred. The protease polypeptide can be encoded
by
a full-length nucleic acid sequence or any portion of the full-length nucleic
acid
sequence, so long as a functional activity of the polypeptide is retained. It
is well
known in the art that due to the degeneracy of the genetic code numerous
different
nucleic acid sequences can code for the same amino acid sequence. Equally, it
is
also well known in the art that conservative changes in amino acid can be made
to
arrive at a protein or polypeptide which retains the functionality of the
original.
Such substitutions may include the replacement of an amino acid by a residue
having similar physicochemical properties, such as substituting one aliphatic
residue
(Ile, Val, Leu or Ala) for another, or substitution between basic residues Lys
and
Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl
residues
Ser and Tyr, or aromatic residues Phe and Tyr. Further information regarding
making amino acid exchanges which have only slight, if any, effects on the
overall
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protein can be found in Bowie et al., Science, 1990, 247:1306-1310, which is
incorporated herein by reference in its entirety including any figures,
tables, or
drawings. In all cases, all permutations are intended to be covered by this
disclosure.
The amino acid sequence of the protease peptide of the invention will be
substantially similar to a sequence having an amino acid sequence selected
from the
group consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID
N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID
N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID
NO:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID
N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID
N0:68, SEQ ID N0:69, and SEQ ID N0:70, or the corresponding full-length amino
acid sequence, or fragments thereof.
A sequence that is substantially similar to a sequence selected from the group
consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43,
SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48,
SEQ ID NO:49, SEQ ID N0:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID N0:53,
SEQ ID N0:54, SEQ ID N0:55, SEQ ID NO:56, SEQ ID N0:57, SEQ ID N0:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID NO:69, and SEQ ID N0:70 will preferably have at least 50%, 60%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a
sequence selected from the group consisting of SEQ ID N0:36, SEQ ID N0:37,
SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID NO:41, SEQ m N0:42,
SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47,
SEQ ID N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ ID N0:51, SEQ ID N0:52,
SEQ ID N0:53, SEQ DJ N0:54, SEQ ID N0:55, SEQ ID N0:56, SEQ m N0:57,
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SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID NO:62,
SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67,
SEQ ID NO:68, SEQ ID N0:69, and SEQ ID N0:70. Preferably the protease
polypeptide will have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to one of the aforementioned sequences.
By "identity" is meant a property of sequences that measures their similarity
or relationship. Identity is measured by dividing the number of identical
residues by
the total number of residues and gaps and multiplying the product by 100.
"Gaps"
are spaces in an alignment that are the result of additions or deletions of
amino
acids. Thus, two copies of exactly the same sequence have 100% identity, but
sequences that are less highly conserved, and have deletions, additions, or
replacements, may have a lower degree of identity. Those skilled in the axt
will
recognize that several computer programs are available for determining
sequence
identity using standard parameters, for example Gapped BLAST or PSI-BLAST
(Altschul, et al. (1997) Nucleic Acids Res. 25:3389-3402), BLAST (Altschul, et
al.
(1990) J. Mol. Biol. 215:403-410), and Smith-Waterman (Smith, et al. (1981) J.
Mol. Biol. 147:195-197). Preferably, the default settings of these programs
will be
employed, but those skilled in the art recognize whether these settings need
to be
changed and know how to make the changes.
"Similarity" is measured by dividing the number of identical residues plus
the number of conservatively substituted residues (see Bowie, et al. Science,
1999),
247:1306-1310, which is incorporated herein by reference in its entirety,
including
any drawings, figures, or tables) by the total number of residues and gaps and
multiplying the product by 100.
In preferred embodiments, the invention features isolated, enriched, or
purified nucleic acid molecules encoding a protease polypeptide comprising a
nucleotide sequence that: (a) encodes a polypeptide having an amino acid
sequence
selected from the group consisting of those set forth in SEQ ID N0:36, SEQ ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID
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N0:47, SEQ ID N0:48, SEQ ID SEQ ID NO:50, SEQ ID NO:51,
N0:49, SEQ ID
N0:52, SEQ m N0:53, SEQ ID SEQ ID NO:55, SEQ ID N0:56,
N0:54, SEQ ID
N0:57, SEQ ID N0:58, SEQ ID SEQ ID N0:60, SEQ ID N0:61,
N0:59, SEQ ID
N0:62, SEQ ID N0:63, SEQ ID SEQ ID N0:65, SEQ ID N0:66,
N0:64, SEQ ID
N0:67,SEQ ID N0:68, SEQ ID and SEQ ID N0:70; (b) is the
N0:69, complement
of the
nucleotide
sequence
of (a);
(c)
hybridizes
under
highly
stringent
conditions
to
the nucleotide molecule of (a) and encodes a naturally occurnng protease
polypeptide.
In preferred embodiments, the invention features isolated, enriched or
purified
nucleic acid molecules comprising a nucleotide sequence substantially
identical to a
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:2, SEQ
ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID
N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID
N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID
N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID
N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID NO:27, SEQ ID
N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID
N0:33, SEQ ID N0:34, and SEQ ID N0:35. Preferably the sequence has at least
50%, 60%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to the above listed sequences.
The term "complement" refers to two nucleotides that can form multiple
favorable interactions with one another. For example, adenine is complementary
to
thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine
are
complementary since they can form three hydrogen bonds. A nucleotide sequence
is
the complement of another nucleotide sequence if all of the nucleotides of the
first
sequence are complementary to all of the nucleotides of the second sequence.
Various low or high stringency hybridization conditions may be used
depending upon the specificity and selectivity desired. These conditions are
well
known to those skilled in the art. Under stringent hybridization conditions
only
highly complementary nucleic acid sequences hybridize. Preferably, such
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conditions prevent hybridization of nucleic acids having more than 1 or 2
mismatches out of 20 contiguous nucleotides, more preferably, such conditions
prevent hybridization of nucleic acids having more than 1 or 2 mismatches out
of 50
contiguous nucleotides, most preferably, such conditions prevent hybridization
of
nucleic acids having more than 1 or 2 mismatches out of 100 contiguous
nucleotides. In some instances, the conditions may prevent hybridization of
nucleic
acids having more than 5 mismatches in the full-length sequence.
By stringent hybridization assay conditions is meant hybridization assay
conditions at least as stringent as the following: hybridization in 50%
formamide,
SX SSC, 50 mM NaH2P04, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm
DNA, and SX Denhardt's solution at 42 °C overnight; washing with 2X
SSC, 0.1%
SDS at 45 °C; and washing with 0.2X SSC, 0.1% SDS at 45 °C.
Under some of the
most stringent hybridization assay conditions, the second wash can be done
with
O.1X SSC at a temperature up to 70 °C (Berger et al. (1987) Guide to
Molecular
Cloning_Technielues pg 421, hereby incorporated by reference herein in its
entirety
including any figures, tables, or drawings.). However, other applications may
require the use of conditions falling between these sets of conditions.
Methods of
determining the conditions required to achieve desired hybridizations are well
known to those with ordinary skill in the art, and are based on several
factors,
including but not limited to, the sequences to be hybridized and the samples
to be
tested. Washing conditions of lower stringency frequently utilize a lower
temperature during the washing steps, such as 65 °C, 60 °C, 55
°C, 50 °C, or 42 °C.
The term "activity" means that the polypeptide hydrolyzes peptide bonds.
The term "catalytic activity", as used herein, defines the rate at which a
protease catalytic domain cleaves a substrate. Catalytic activity can be
measured,
for example, by determining the amount of a substrate cleaved as a function of
time.
Catalytic activity can be measured by methods of the invention by holding time
constant and determining the concentration of a cleaved substrate after a
fixed
period of time. Cleavage of a substrate occurs at the active site of the
protease. The
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active site is normally a cavity in which the substrate binds to the protease
and is
cleaved.
The term "substrate" as used herein refers to a polypeptide or protein which
is cleaved by a protease of the invention. The term "cleaved" refers to the
severing
of a covalent bond between amino acid residues of the backbone of the
polypeptide
or protein.
The term "insert" as used herein refers to a portion of a protease that is
absent from a close homolog. Inserts may or may not be the product alternative
splicing of exons. Inserts can be identified by using a Smith-Waterman
sequence
alignment of the protein sequence against the non-redundant protein database,
or by
means of a multiple sequence alignment of homologous sequences using the
DNAStar program Megalign (Preferably, the default settings of this program
will be
used, but those skilled in the art will recognize whether these settings need
to be
changed and know how to make the changes.). Inserts may play a functional role
by
presenting a new interface for protein-protein interactions, or by interfering
with
such interactions.
In other preferred embodiments, the invention features isolated, enriched, or
purified nucleic acid molecules encoding protease polypeptides, further
comprising
a vector or promoter effective to initiate transcription in a host cell. The
invention
also features recombinant nucleic acid, preferably in a cell or an organism.
The
recombinant nucleic acid may contain a sequence selected from the group
consisting
of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4,
SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ
ID NO:10, SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ
ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ
ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ
DJ N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ
ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, and
SEQ ID N0:35, or a functional derivative thereof and a vector or a promoter
effective to initiate transcription in a host cell. The recombinant nucleic
acid can
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alternatively contain a transcriptional initiation region functional in a
cell, a
sequence complementary to an RNA sequence encoding a protease polypeptide and
a transcriptional termination region functional in a cell. Specific vectors
and host
cell combinations are discussed herein.
The term "vector" relates to a single or double-stranded circular nucleic acid
molecule that can be transfected into cells and replicated within or
independently of
a cell genome. A circular double-stranded nucleic acid molecule can be cut and
thereby linearized upon treatment with restriction enzymes. An assortment of
nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide
sequences cut by restriction enzymes are readily available to those skilled in
the art.
A nucleic acid molecule encoding a protease can be inserted into a vector by
cutting
the vector with restriction enzymes and ligating the two pieces together.
The term "transfecting" defines a number of methods to insert a nucleic acid
vector or other nucleic acid molecules into a cellular organism. These methods
involve a variety of techniques, such as treating the cells with high
concentrations of
salt, an electric field, detergent, or DMSO to render the outer membrane or
wall of
the cells permeable to nucleic acid molecules of interest or use of various
viral
transduction strategies.
The term "promoter" as used herein, refers to nucleic acid sequence needed
for gene sequence expression. Promoter regions vary from organism to organism,
but are well known to persons skilled in the art for different organisms. For
example, in prokaryotes, the promoter region contains both the promoter (which
directs the initiation of RNA transcription) as well as the DNA sequences
which,
when transcribed into RNA, will signal synthesis initiation. Such regions will
normally include those 5'-non-coding sequences involved with initiation of
transcription and translation, such as the TATA box, capping sequence, CART
sequence, and the like.
In preferred embodiments, the isolated nucleic acid comprises, consists
essentially of, or consists of a nucleic acid sequence selected from the group
consisting of those set forth in SEQ m NO:1, SEQ m N0:2, SEQ B7 N0:3, SEQ
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ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID N0:1 l, SEQ ID N0:12, SEQ ID N0:13, SEQ ID
N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID
N0:19, SEQ ID NO:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID
N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID
N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:34, and SEQ ID N0:35 which encodes an amino acid sequence selected from
the group consisting of those set forth in SEQ ID N0:36, SEQ ID NO:37, SEQ ID
N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID
N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID
NO:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID
N0:58, SEQ ID N0:59, SEQ ID NO:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID
N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID
N0:68, SEQ ID N0:69, and SEQ ID N0:70, a functional derivative thereof, or at
least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids selected
from
the group consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ TD
N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID
N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID
N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID
N0:63, SEQ ID N0:64, SEQ ID NO:65, SEQ ID N0:66, SEQ ID N0:67, SEQ TD
N0:68, SEQ ID N0:69, and SEQ ID N0:70. The nucleic acid may be isolated from
a natural source by cDNA cloning or by subtractive hybridization. The natural
source may be mammalian, preferably human, blood, semen, or tissue, and the
nucleic acid may be synthesized by the triester method or by using an
automated
DNA synthesizer.
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The term "mammal" refers preferably to such organisms as mice, rats,
rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs,
monkeys, and
apes, and most preferably to humans.
In yet other preferred embodiments, the nucleic acid is a conserved or unique
region, for example those useful for: the design of hybridization probes to
facilitate
identification and cloning of additional polypeptides, the design of PCR
probes to
facilitate cloning of additional polypeptides, obtaining antibodies to
polypeptide
regions, and designing antisense oligonucleotides.
By "conserved nucleic acid regions", are meant regions present on two or
more nucleic acids encoding a protease polypeptide, to which a particular
nucleic
acid sequence can hybridize wider lower stringency conditions. Examples of
lower
stringency conditions suitable for screening for nucleic acid encoding
protease
polypeptides are provided in Wahl et al. Meth. Enzym. 152:399-407 (1987) and
in
Wahl et al. Meth. E~ezym. 152:415-423 (1987), which are hereby incorporated by
reference herein in its entirety, including any drawings, figures, or tables.
Preferably, conserved regions differ by no more than 5 out of 20 nucleotides,
even
more preferably 2 out of 20 nucleotides or most preferably 1 out of 20
nucleotides.
By "unique nucleic acid region" is meant a sequence present in a nucleic acid
coding for a protease polypeptide that is not present in a sequence coding for
any
other naturally occurnng polypeptide. Such regions preferably encode 32
(preferably 40, more preferably 45, most preferably 55) or more contiguous
amino
acids set forth in a full-length amino acid sequence selected from the group
consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43,
SEQ ID NO:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48,
SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53,
SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID N0:69, and SEQ ID N0:70 in a sample. The nucleic acid probe contains a
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nucleotide base sequence that will hybridize to the sequence selected from the
group
consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ
ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID
N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID
N0:19, SEQ 117 N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID
N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID
N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:34, and SEQ ID N0:35, or a functional derivative thereof.
In preferred embodiments, the nucleic acid probe hybridizes to nucleic acid
encoding at least 12, 32, 75, 90, 105, 120, 150, 200, 250, 300 or 350
contiguous
amino acids of a full-length sequence selected from the group consisting of
those set
forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID NO:38, SEQ ID N0:39, SEQ ID
N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID
NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID
NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID
N0:60, SEQ 117 NO:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID
N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ
ID N0:70, or a functional derivative thereof.
Methods for using the probes include detecting the presence or amount of
protease RNA in a sample by contacting the sample with a nucleic acid probe
under
conditions such that hybridization occurs and detecting the presence or amount
of
the probe bound to protease RNA. The nucleic acid duplex formed between the
probe and a nucleic acid sequence coding for a protease polypeptide may be
used in
the identification of the sequence of the nucleic acid detected (Nelson et
al., in
Nonisotobic DNA Probe Technigues, Academic Press, San Diego, Kricka, ed., p.
275, 1992, hereby incorporated by reference herein in its entirety, including
any
drawings, figures, or tables). Kits for performing such methods may be
constructed
to include a container means having disposed therein a nucleic acid probe.
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Methods for using the probes also include using these probes to find the full-
length clone of each of the predicted proteases by techniques known to one
skilled in
the art. These clones will be useful for screening for small molecule
compounds that
inhibit the catalytic activity of the encoded protease with potential utility
in treating
cancers, immune-related diseases and disorders, cardiovascular disease, brain
or
neuronal-associated diseases, and metabolic disorders. More specifically
disorders
including cancers of tissues, blood, or hematopoietic origin, particularly
those
involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or
kidney;
central or peripheral nervous system diseases and conditions including
migraine,
pain, sexual dysfunction, mood disorders, attention disorders, cognition
disorders,
hypotension, and hypertension; psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia, severe mental
retardation and dyskinesias, such as Huntington's disease or Tourette's
Syndrome;
neurodegenerative diseases including Alzheimer's, Parkinson's, multiple
sclerosis,
and amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-
1,
HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms;
metabolic
disorders including Diabetes and obesity and their related syndromes, among
others;
cardiovascular disorders including reperfusion restenosis, coronary
thrombosis,
clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular
disease
including glaucoma, retinopathy, and macular degeneration; inflammatory
disorders
including rheumatoid arthritis, chronic inflammatory bowel disease, chronic
inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis,
psoriasis,
atherosclerosis, rhinitis, autoimmunity, and organ transplant rejection.
In another aspect, the invention describes a recombinant cell or tissue
comprising a nucleic acid molecule encoding a protease polypeptide having an
amino acid sequence selected from the group consisting of those set forth in
SEQ m
N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID
N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ m N0:44, SEQ ID N0:45, SEQ ID
N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ m N0:49, SEQ m NO:50, SEQ ID
NO:51, SEQ m N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID
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N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID
N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID
N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70. In
such calls, the nucleic acid may be under the control of the genomic
regulatory
elements, or may be under the control of exogenous regulatory elements
including
an exogenous promoter. By "exogenous" it is meant a promoter that is not
normally
coupled ih vivo transcriptionally to the coding sequence for the protease
polypeptides.
The polypeptide is preferably a fragment of the protein encoded by a full-
length amino acid sequence selected from the group consisting of those set
forth in
SEQ ID N0:36; SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40,
SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45,
SEQ ID N0:46, SEQ ID NO:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID NO:54, SEQ ID NO:55,
SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60,
SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65,
SEQ ID NO:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID
N0:70. By "fragment," is meant an amino acid sequence present in a protease
polypeptide. Preferably, such a sequence comprises at least 32, 45, 50, 60,
100, 200,
or 300 contiguous amino acids of a full-length sequence selected from the
group
consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43,
SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48,
SEQ ID N0:49, SEQ ID NO:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID N0:53,
SEQ ID N0:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID N0:57, SEQ ID N0:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID N0:69, and SEQ ID N0:70.
In another aspect, the invention features an isolated, enriched, or purified
protease polypeptide having the amino acid sequence selected from the group
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consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43,
SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48,
SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53,
SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID N0:69, and SEQ ID N0:70.
By "isolated" in reference to a polypeptide is meant a polymer of 6
(preferably 12, more preferably 18, most preferably 25, 32, 40, or 50) or more
amino
acids conjugated to each other, including polypeptides that are isolated from
a
natural source or that are synthesized. In certain aspects longer polypeptides
are
preferred, such as those with 100, 200, 300, 400, 450, 500, 550, 600, 700,
800, 900
or more contiguous amino acids of a full-length sequence selected from the
group
consisting of those set forth in SEQ ID N0:36, SEQ ID NO:37, SEQ ID N0:38,
SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43,
SEQ ID NO:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48,
SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53,
SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID NO:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID NO:62, SEQ ID NO:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID NO:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID N0:69, and SEQ ID N0:70, and/or those polypeptides having at least 50%,
60%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to a sequence selected from the group consisting of SEQ ID N0:36, SEQ
ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID NO:45, SEQ ID N0:46, SEQ ID
N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID
N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID
N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
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N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID
NO:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID NO:70.
The isolated polypeptides of the present invention are unique in the sense
that they are not found in a pure or separated state in nature. Use of the
term
"isolated" indicates that a naturally occurring sequence has been removed from
its
normal cellular environment. Thus, the sequence may be in a cell-free solution
or
placed in a different cellular environment. The term does not imply that the
sequence is the only amino acid chain present, but that it is essentially free
(at least
about 90% pure, more preferably at least about 95% pure or more) of non-amino
acid-based material naturally associated with it.
By the use of the term "enriched" in reference to a polypeptide is meant that
the specific amino acid sequence constitutes a significantly higher fraction
(2- to 5-
fold) of the total amino acid sequences present in the cells or solution of
interest than
in normal or diseased cells or in the cells from which the sequence was taken.
This
could be caused by a person by preferential reduction in the amount of other
amino
acid sequences present, or by a preferential increase in the amount of the
specific
amino acid sequence of interest, or by a combination of the two. However, it
should
be noted that enriched does not imply that there are no other amino acid
sequences
present, just that the relative amount of the sequence of interest has been
significantly increased. The term significant here is used to indicate that
the level of
increase is useful to the person making such an increase, and generally means
an
increase relative to other amino acid sequences of about at least 2-fold, more
preferably at least 5- to 10-fold or even more. The term also does not imply
that
there is no amino acid sequence from other sources. The other source of amino
acid
sequences may, for example, comprise amino acid sequence encoded by a yeast or
bacterial genome, or a cloning vector such as pUCl9. The term is meant to
cover
only those situations in which man has intervened to increase the proportion
of the
desired amino acid sequence.
It is also advantageous for some purposes that an amino acid sequence be in
purified form. The term "purified" in reference to a polypeptide does' not
require
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absolute purity (such as a homogeneous preparation); instead, it represents an
indication that the sequence is relatively purer than in the natural
environment.
Compared to the natural level this level should be at least 2-to 5-fold
greater (e.g., in
terms of mg/mL). Purification of at least one order of magnitude, preferably
two or
three orders, and more preferably four or five orders of magnitude is
expressly
contemplated. The substance is preferably free of contamination at a
functionally
significant level, for example 90%, 95%, or 99% pure.
In preferred embodiments, the protease polypeptide is a fragment of the
protein encoded by a full-length amino acid sequence selected from the group
consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0:39~ SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43,
SEQ ID N0:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID N0:47, SEQ ID N0:48,
SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53,
SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID N0:69, and SEQ ID NO:70. Preferably, the protease polypeptide contains
at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of a full-
length
sequence selected from the group consisting of those set forth in SEQ ID
NO:36,
SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID NO:41,
SEQ ID N0:42, SEQ ID N0:43, SEQ 117 N0:44, SEQ ID N0:45, SEQ ID N0:46,
SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56,
SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61,
SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66,
SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70, or a
functional derivative thereof.
In preferred embodiments, the protease polypeptide comprises an amino acid
sequence having an amino acid sequence selected from the group consisting of
those
set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ
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ID N0:40, SEQ m N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ
ID N0:45, SEQ ID N0:46, SEQ m N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ
ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ
ID NO:55, SEQ ID N0:56, SEQ 117 N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ
ID NO:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ
ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and
SEQ ID N0:70.
The polypeptide can be isolated from a natural source by methods well-
known in the art. The natural source may be mammalian, preferably human,
blood,
semen, or tissue, and the polypeptide may be synthesized using an automated
polypeptide synthesizer.
In some embodiments the invention includes a recombinant protease
polypeptide having (a) an amino acid sequence selected from the group
consisting of
those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39,
SEQ ID N0:40, SEQ ID N0:41, SEQ ID NO:42, SEQ ID N0:43, SEQ ID N0:44,
SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54,
SEQ ID NO:55, SEQ ID N0:56, SEQ ID NO:57, SEQ ID N0:58, SEQ ID N0:59,
SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64,
SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69,
and SEQ ID N0:70. By "recombinant protease polypeptide" is meant a polypeptide
produced by recombinant DNA techniques such that it is distinct from a
naturally
occurring polypeptide either in its location (e.g., present in a different
cell or tissue
than found in nature), purity or structure. Generally, such a recombinant
polypeptide will be present in a cell in an amount different from that
normally
observed in nature.
The polypeptides to be expressed in host cells may also be fusion proteins
which include regions from heterologous proteins. Such regions may be included
to
allow, e.g., secretion, improved stability, or facilitated purification of the
polypeptide. For example, a sequence encoding an appropriate signal peptide
can be
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incorporated into expression vectors. A DNA sequence for a signal peptide
(secretory leader) may be fused in-frame to the polynucleotide sequence so
that the
polypeptide is translated as a fusion protein comprising the signal peptide. A
signal
peptide that is functional in the intended host cell promotes extracellular
secretion of
the polypeptide. Preferably, the signal sequence will be cleaved from the
polypeptide upon secretion of the polypeptide from the cell. Thus, preferred
fusion
proteins can be produced in which the N-terminus of a protease polypeptide is
fused
to a carrier peptide.
In one embodiment, the polypeptide comprises a fusion protein which
includes a heterologous region used to facilitate purification of the
polypeptide.
Many of the available peptides used for such a function allow selective
binding of
the fusion protein to a binding partner. A preferred binding partner includes
one or
more of the IgG binding domains of protein A are easily purified to
homogeneity by
affnuty chromatography on, for example, IgG-coupled Sepharose. Alternatively,
many vectors have the advantage of carrying a stretch of histidine residues
that can
be expressed at the N-terminal or C-terminal end of the target protein, and
thus the
protein of interest can be recovered by metal chelation chromatography. A
nucleotide sequence encoding a recognition site for a proteolytic enzyme such
as
enterokinase, factor X procollagenase or thrombine may immediately precede the
sequence for a protease polypeptide to permit cleavage of the fusion protein
to
obtain the mature protease polypeptide. Additional examples of fusion-protein
binding partners include, but are not limited to, the yeast I-factor, the
honeybee
melatin leader in sf3 insect cells, 6-His tag, thioredoxin tag, hemaglutinin
tag, GST
tag, and OrnpA signal sequence tag. As will be understood by one of skill in
the art,
the binding partner which recognizes and binds to the peptide may be any ion,
molecule or compound including metal ions (e.g., metal affinity columns),
antibodies, or fragments thereof, and any protein or peptide which binds the
peptide,
such as the FLAG tag.
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Antibodies
In another aspect, the invention features an antibody (e.g., a monoclonal or
polyclonal antibody) having specific binding affinity to a protease
polypeptide or a
protease polypeptide domain or fragment where the polypeptide is selected from
the
group having a sequence at least about 90% identical to an amino acid sequence
selected from the group consisting of those set forth in SEQ ID N0:36, SEQ ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID NO:41, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID NO:46, SEQ ID
N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID
N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID
N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID NO:65, SEQ ID N0:66, SEQ ID
NO:67, SEQ ID NO:68, SEQ ID N0:69, and SEQ ID N0:70. By "specific binding
affinity" is meant that the antibody binds to the target protease polypeptide
with
greater affinity than it binds to other polypeptides under specified
conditions.
Antibodies or antibody fragments are polypeptides that contain regions that
can bind
other polypeptides. The term "specific binding affinity" describes an antibody
that
binds to a protease polypeptide with greater affinity than it binds to other
polypeptides under specified conditions. Antibodies can be used to identify an
endogenous source of protease polypeptides, to monitor cell cycle regulation,
and
for immuno-localization of protease polypeptides within the cell.
The term "polyclonal" refers to antibodies that are heterogenous populations
of antibody molecules derived from the sera of animals immunized with an
antigen
or an antigenic functional derivative thereof. For the production of
polyclonal
antibodies, various host animals may be immunized by inj ection with the
antigen.
Various adjuvants may be used to increase the immunological response,
depending
on the host species.
"Monoclonal antibodies" are substantially homogenous populations of
antibodies to a particular antigen. They may be obtained by any technique
which
provides for the production of antibody molecules by continuous cell lines in
CA 02408105 2002-11-O1
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culture. Monoclonal antibodies may be obtained by methods known to those
skilled
in the art (Kohler et al., NatuYe, 1975, 256:495-497, and U.S. Patent No.
4,376,110,
both of which are hereby incorporated by reference herein in their entirety
including
any figures, tables, or drawings).
An antibody of the present invention includes "humanized" monoclonal and
polyclonal antibodies. Humanized antibodies are recombinant proteins in which
non-human (typically marine) complementarity determining regions of an
antibody
have been transferred from heavy and light variable chains of the non-human
(e.g.
marine) immunoglobulin into a human variable domain, followed by the
replacement of some human residues in the framework regions of their marine
counterparts. Humanized antibodies in accordance with this invention are
suitable
for use in therapeutic methods. General techniques for cloning marine
immunoglobulin variable domains are described, for example, by the publication
of
Orlandi et al., PYOG. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniques for
producing humanized monoclonal antibodies are described, for example, by Jones
et
al., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988), Verhoeyen
et
al., Science 239:1534 (1988), Carter et al., Proc. Nat'Z Acad. Sci. USA
89:4285
(1992), Sandhu, C~it. Rev. Biotech. 12:437 (1992), and Singer et al., J.
Immuh.
150:2844 (1993).
~ The term "antibody fragment" refers to a portion of an antibody, often the
hypervariable region and portions of the surrounding heavy and light chains,
that
displays specific binding affinity for a particular molecule. A hypervariable
region
is a portion of an antibody that physically binds to the polypeptide target.
An antibody fragment of the present invention includes a "single-chain
antibody," a phrase used in this description to denote a linear polypeptide
that binds
antigen with specificity and that comprises variable or hypervariable regions
from
the heavy and light chain chains of an antibody. Such single chain antibodies
can be
produced by conventional methodology. The Vh and Vl regions of the Fv fragment
can be covalently joined and stabilized by the insertion of a disulfide bond.
See
Glockshuber, et al., Biochemistry 1362 (1990). Alternatively, the Vh and Vl
regions
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can be joined by the insertion of a peptide linker. A gene encoding the Vh, Vl
and
peptide linker sequences can be constructed and expressed using a recombinant
expression vector. See Colcher, et al., J. Nat'l CahceY Inst. 82: 1191 (1990).
Amino acid sequences comprising hypervariable regions from the Vh and Vl
antibody chains can also be constructed using disulfide bonds or peptide
linkers.
Antibodies or antibody fragments having specific binding affinity to a
protease polypeptide of the invention may be used in methods for detecting the
presence and/or amount of protease polypeptide in a sample by probing the
sample
with the antibody under conditions suitable for protease-antibody
immunocomplex
formation and detecting the presence and/or amount of the antibody conjugated
to
the protease polypeptide. Diagnostic kits for performing such methods may be
constructed to include antibodies or antibody fragments specific for the
protease as
well as a conjugate of a binding partner of the antibodies or the antibodies
themselves.
An antibody or antibody fragment with specific binding affinity to a protease
polypeptide of the invention can be isolated, enriched, or purified from a
prokaryotic
or eukaryotic organism. Routine methods known to those skilled in the art
enable
production of antibodies or antibody fragments, in both prokaryotic and
eukaryotic
organisms. Purification, enrichment, and isolation of antibodies, which are
polypeptide molecules, are described above.
Antibodies having specific binding affinity to a protease polypeptide of the
invention may be used in methods for detecting the presence and/or amount of
protease polypeptide in a sample by contacting the sample with the antibody
under
conditions such that an immunocomplex forms and detecting the presence and/or
amount of the antibody conjugated to the protease polypeptide. Diagnostic
lcits for
performing such methods may be constructed to include a first container
containing
the antibody and a second container having a conjugate of a binding partner of
the
antibody and a label, such as, for example, a radioisotope. The diagnostic kit
may
also include notification of an FDA approved use and instructions therefor.
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In another aspect, the invention features a hybridoma which produces an
antibody having specific binding affinity to a protease polypeptide or a
protease
polypeptide domain, where the polypeptide is selected from the group
consisting of
those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39,
SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44,
SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54,
SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59,
SEQ m N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64,
SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69,
and SEQ 117 NO:70. By "hybridoma" is meant an immortalized cell line that is
capable of secreting an antibody, for example an antibody to a protease of the
invention. In preferred embodiments, the antibody to the protease comprises a
sequence of amino acids that is able to specifically bind a protease
polypeptide of
the invention.
In another aspect, the present invention is also directed to kits comprising
antibodies that bind to a polypeptide encoded by any of the nucleic acid
molecules
described above, and a negative control antibody.
The term "negative control antibody" refers to an antibody derived from
similar source as the antibody having specific binding affinity, but where it
displays
no binding affinity to a polypeptide of the invention.
In another aspect, the invention features a protease polypeptide binding agent
able to bind to a protease polypeptide selected from the group having an amino
acid
sequence selected from the group consisting of those set forth in SEQ ID
N0:36,
SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41,
SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46,
SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID N0:51,
SEQ ID N0:52, SEQ m N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56,
SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61,
SEQ ID N0:62, SEQ m N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66,
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SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70. The binding
agent is preferably a purified antibody that recognizes an epitope present on
a
protease polypeptide of the invention. Other binding agents include molecules
that
bind to protease polypeptides and analogous molecules that bind to a protease
polypeptide. Such binding agents may be identified by using assays that
measure
protease binding partner activity, or they may be identified using assays that
measure protease activity, such as the release of a fluorogenic or radioactive
marker
attached to a substrate molecule.
Screening Methods to Detect Protease Polypeptides
The invention also features a method for screening for human cells
containing a protease polypeptide of the invention or an equivalent sequence.
The
method involves identifying the novel polypeptide in human cells using
techniques
that are routine and standard in the art, such as those described herein for
identifying
the proteases of the invention (e.g., cloning, Southern or Northern blot
analysis, in
situ hybridization, PCR amplification, etc.).
Screenin;~ Methods to Identify Substances that Modulate Protease
Activi
In another aspect, the invention features methods for identifying a substance
that modulates protease activity comprising the steps of (a) contacting a
protease
polypeptide comprising an amino acid substantially identical to a sequence
selected
from the group consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37,
SEQ
ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ
ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ 117 N0:46, SEQ ID N0:47, SEQ
ID N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ 1D N0:51, SEQ ID N0:52, SEQ
ID N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID N0:56, SEQ ID N0:57, SEQ
117 N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ
ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ 1D N0:67, SEQ
ID N0:68, SEQ ID N0:69, and SEQ ID N0:70 with a test substance; (b) measuring
the activity of said polypeptide; and (c) determining whether said substance
modulates the activity of said polypeptide. More preferably the sequence is at
least
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about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
listed sequences.
The term "modulates" refers to the ability of a compound to alter the
function of a protease of the invention. A modulator preferably activates or
inhibits
the activity of a protease of the invention depending on the concentration of
the
compound exposed to the protease.
The term "modulates" also refers to altering the function of proteases of the
invention by increasing or decreasing the probability that a complex forms
between
the protease and a natural binding partner. A modulator preferably increases
the
probability that such a complex forms between the protease and the natural
binding
partner, more preferably increases or decreases the probability that a complex
forms
between the protease and the natural binding partner depending on the
concentration
of the compound exposed to the protease, and most preferably decreases the
probability that a complex forms between the protease and the natural binding
partner.
The term "activates" refers to increasing the cellular activity of the
protease.
The term "inhibits" refers to decreasing the cellular activity of the
protease.
The term "complex" refers to an assembly of at least two molecules bound to
one another. Signal transduction complexes often contain at least two protein
molecules bound to one another. For instance, a protein tyrosine receptor
protein
kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex
in response to a mitogenic ligand. Similarly, the proteases involved in blood
coagulation and their cofactors are known to form macromolecular complexes on
cellular membranes. Additionally, proteases involved in modification of the
extracellular matrix are known to form complexes with their inhibitors and
also with
components of the extracellular matrix.
The term "natural binding partner" refers to polypeptides, lipids, small
molecules, or nucleic acids that bind to proteases in cells. A change in the
interaction between a protease and a natural binding partner can manifest
itself as an
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increased or decreased probability that the interaction forms, or an increased
or
decreased concentration of protease/natural binding partner complex.
The term "contacting" as used herein refers to mixing a solution comprising
the test compound with a liquid medium bathing the cells of the methods. The
solution comprising the compound may also comprise another component, such as
dimethyl sulfoxide (DMSO), which facilitates the uptake of the test compound
or
compounds into the cells of the methods. The solution comprising the test
compound may be added to the medium bathing the cells by utilizing a delivery
apparatus, such as a pipette-based device or syringe-based device.
In another aspect, the invention features methods for identifying a substance
that modulates protease activity in a cell comprising the steps of: (a)
expressing a
protease polypeptide in a cell, wherein said polypeptide is selected from the
group
having an amino acid sequence selected from the group consisting of those set
forth
in SEQ ID NO:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID
N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID NO:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID
NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID N0:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID NO:59, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID
N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ
ID N0:70; (b) adding a test substance to said cell; and (c) monitoring a
change in
cell phenotype or the interaction between said polypeptide and a natural
binding
partner.
The term "expressing" as used herein refers to the production of proteases of
the invention from a nucleic acid vector containing protease genes within a
cell. The
nucleic acid vector is transfected into cells using well known techniques in
the art as
described herein.
Another aspect of the instant invention is directed to methods of identifying
compounds that bind to protease polypeptides of the present invention,
comprising
contacting the protease polypeptides with a compound, and determining whether
the
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compound binds the protease polypeptides. Binding can be determined by binding
assays which are well known to the skilled artisan, including, but not limited
to, gel-
shift assays, Western blots, radiolabeled competition assay, phage-based
expression
cloning, co-fractionation by chromatography, co-precipitation, cross linking,
interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the
like,
which are described in, for example, Current Protocols in Molecular Biology,
1999,
John Wiley & Sons, NY, which is incorporated herein by reference in its
entirety.
The compounds to be screened include, but are not limited to, compounds of
extracellular, intracellular, biological or chemical origin.
The methods of the invention also embrace compounds that are attached to a
label, such as a radiolabel (e.g., lzsh 3sS, 3aP~ 33P~ 3H)~ a fluorescence
label, a
chemiluminescent label, an enzymic label and an immunogenic label. The
protease
polypeptides employed in such a test may either be free in solution, attached
to a
solid support, borne on a cell surface, located intracellularly or associated
with a
portion of a cell. One skilled in the art can, for example, measure the
formation of
complexes between a protease polypeptide and the compound being tested.
Alternatively, one skilled in the art can examine the diminution in complex
formation between a protease polypeptide and its substrate caused by the
compound
being tested.
Other assays can be used to examine enzymatic activity including, but not
limited to, photometric, radiometric, HPLC, electrochemical, and the like,
which are
described in, for example, Enzyme Assays: A Practical Approach, eds. R.
Eisenthal
and M. J. Danson, 1992, Oxford University Press, which is incorporated herein
by
reference in its entirety.
Another aspect of the present invention is directed to methods of identifying
compounds which modulate (i. e., increase or decrease) activity of a protease
polypeptide comprising contacting the protease polypeptide with a compound,
and
determining whether the compound modifies activity of the protease
polypeptide.
These compounds are also referred to as "modulators of proteases." The
activity in
the presence of the test compound is measured to the activity in the absence
of the
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test compound. Where the activity of a sample containing the test compound is
higher than the activity in a sample lacking the test compound, the compound
will
' have increased the activity. Similarly, where the activity of a sample
containing the
test compound is lower than the activity in the sample lacking the test
compound,
the compound will have inhibited the activity.
The present invention is particularly useful for screening compounds by
using a protease polypeptide in any of a variety of drug screening techniques.
The
compounds to be screened include, but are not limited to, extracellular,
intracellular,
biological or chemical origin. The protease polypeptide employed in such a
test
may be in any form, preferably, free in solution, attached to a solid support,
borne on
a cell surface or located intracellularly. One skilled in the art can measure
the
change in rate that a protease of the invention cleaves a substrate
polypeptide. One
skilled in the art can also, for example, measure the formation of complexes
between
a protease polypeptide and the compound being tested. Alternatively, one
skilled in
the art can examine the diminution in complex formation between a protease
polypeptide and its substrate caused by the compound being tested.
The activity of protease polypeptides of the invention can be determined by,
for example, examining the ability to bind or be activated by chemically
synthesised
peptide ligands. Alternatively, the activity of the protease polypeptides can
be
assayed by examining their ability to bind metal ions such as calcimn,
hormones,
chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants,
and
photons. Thus, modulators of the protease polypeptide's activity may alter a
protease function, such as a binding property of a protease or an activity
such as
cleaving protein substrates or polypeptide substrates, or membrane
localization.
In various embodiments of the method, the assay may take the form of a
yeast growth assay, an Aequorin assay, a Luciferase assay, a mitogenesis
assay, a
MAP Kinase activity assay, as well as other binding or function-based assays
of
protease activity that are generally known in the art. In several of these
embodiments, the invention includes any of the serine proteases, cysteine
proteases,
aspartyl proteases, metalloproteases, threonine proteases, and other
proteases.
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Biological activities of proteases according to the invention include, but are
not
limited to, the binding of a natural or a synthetic ligand, as well as any one
of the
functional activities of proteases known in the art. Non-limiting examples of
protease activities include cleavage of polypeptide chains, processing the pro-
form
of a polypeptide chain to the active product, transmembrane signaling of
various
forms, and/or the modification of the extraceullar matrix.
The modulators of the invention exhibit a variety of chemical structures,
which can be generally grouped into mimetics of natural protease ligands, and
peptide and non-peptide allosteric effectors of proteases. The invention does
not
restrict the sources for suitable modulators, which may be obtained from
natural
sources such as plant, animal or mineral extracts, or non-natural sources such
as
small molecule libraries, including the products of combinatorial chemical
approaches to library construction, and peptide libraries.
The use of cDNAs encoding proteins in drug discovery programs is well-
known; assays capable of testing thousands of unknown compounds per day in
high-
throughput screens (HTSs) are thoroughly documented. The literature is replete
with examples of the use of radiolabelled ligands in HTS binding assays for
drug
discovery (see, Williams, Medicinal Research Reviews, 1991, 11:147-184.;
Sweetnam, et al., J. Natural Products, 1993, 56:441-455 for review).
Recombinant
proteins are preferred for binding assay HTS because they allow for better
specificity (higher relative purity), provide the ability to generate large
amounts of
receptor material, and can be used in a broad variety of formats (see Hodgson,
BiolTechnology, 1992, 10:973-980 which is incorporated herein by reference in
its
entirety). A variety of heterologous systems is available for functional
expression of
recombinant proteins that are well known to those skilled in the art. Such
systems
include bacteria (Strosberg, et al., Trends in Pharmacological Sciences, 1992,
13:95-98), yeast (Pausch, Trends in Biotec7Zyaology, 1997, 15:487-494),
several kinds
of insect cells (Vanden Broeck, Int. Rev. Cytology, 1996, 164:189-268),
amphibian
cells (Jayawickreme et al., Current Opinion irz Biotechnology, 1997, 8:629-
634) and
several mammalian cell lines (CHO, HEK293, COS, etc.; see, Gerhardt, et al.,
Eur.
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J. Pharmacology, 1997, 334:1-23). These examples do not preclude the use of
other
possible cell expression systems, including cell lines obtained from nematodes
(PCT
application WO 98/37177).
An expressed protease can be used for HTS binding assays in conjunction
with its defined ligand, in this case the corresponding peptide that activates
it. The
identified peptide is labeled with a suitable radioisotope, including, but not
limited
to, 12$I, 3H, 3sS or 32P, by methods that are well known to those skilled in
the art.
Alternatively, the peptides may be labeled by well-known methods with a
suitable
fluorescent derivative (Baindur, et al., Drug Dev. Res., 1994, 33:373-398;
Rogers,
Drug Discovery Today, 1997, 2:156-160). Radioactive ligand specifically bound
to
the receptor in membrane preparations made from the cell line expressing the
recombinant protein can be detected in HTS assays in one of several standard
ways,
including filtration of the receptor-ligand complex to separate bound ligand
from
unbound ligand (Williams, Med. Res. Rev., 1991, 11:147-184.; Sweetnam, et al.,
J.
Natural Products, 1993, 56:441-455). Alternative methods include a
scintillation
proximity assay (SPA) ox a FlashPlate format in which such separation is
unnecessary (Nakayama, Cur. Opinion Drug Disc. Dev., 1998, 1:85-91 Bosse, et
al.,
J. Biomolecular Screening, 1998, 3:285-292.). Binding of fluorescent ligands
can
be detected in various ways, including fluorescence energy transfer (FRET),
direct
spectrophotofluorometric analysis of bound ligand, or fluorescence
polarization
(Rogers, Drug Discovery Today, 1997, 2:156-160; Hill, Cur. Opinion Drug Disc.
Dev., 1998, 1:92-97).
The proteases and natural binding partners required for functional expression
of heterologous protease polypeptides can be native constituents of the host
cell or
can be introduced through well-known recombinant technology. The protease
polypeptides can be intact or chimeric. The protease activation may result in
the
stimulation or inhibition of other native proteins, events that can be linked
to a
measurable response.
Examples of such biological responses include, but are not limited to, the
following: the ability to survive in the absence of a limiting nutrient in
specifically
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engineered yeast cells (Pausch, Trends in Biotechnology, 1997, 15:487-494);
changes in intracellular Ca2+ concentration as measured by fluorescent dyes
(Murphy, et al., Cur. Opinion Drug Disc. Dev., 1998, 1:192-199). Fluorescence
changes can also be used to monitor ligand-induced changes in membrane
potential
or intracellular pH; an automated system suitable for HTS has been described
for
these purposes (Schroeder, et al., J. Biomolecular Screening, 1996, 1:75-80).
Assays are also available for the measurement of common second but these are
not
generally preferred for HTS.
The invention contemplates a multitude of assays to screen and identify
inhibitors of ligand binding to protease polypeptides or of substrate cleavage
by
protease polypeptides. In one example, the protease polypeptide is immobilized
and
interaction with a binding partner or substrate is assessed in the presence
and
absence of a candidate modulator such as an inhibitor compound. In another
example, interaction between the protease polypeptide and its binding partner
or a
substrate is assessed in a solution assay, both in the presence and absence of
a
candidate inhibitor compound. In either assay, an inhibitor is identified as a
compound that decreases binding between the protease polypeptide and its
natural
binding partner or the activity of a protease polypeptide in cleaving a
substrate
molecule. Another contemplated assay involves a variation of the di-hybrid
assay
wherein an inhibitor of protein/protein interactions is identified by
detection of a
positive signal in a transformed or transfected host cell, as described in PCT
publication number WO 95/20652, published August 3, 1995 and is included by
reference herein including any figures, tables, or drawings.
Candidate modulators contemplated by the invention include compounds
selected from libraries of either potential activators or potential
inhibitors. There are
a number of different libraries used for the identification of small molecule
modulators, including: (1) chemical libraries, (2) natural product libraries,
and (3)
combinatorial libraries comprised of random peptides, oligonucleotides or
organic
molecules. Chemical libraries consist of random chemical structures, some of
which
are analogs of known compounds or analogs of compounds that have been
identified
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as "hits" or "leads" in other drug discovery screens, while others are derived
from
natural products, and still others arise from non-directed synthetic organic
chemistry. Natural product libraries are collections of microorganisms,
animals,
plants, or marine organisms which are used to create mixtures for screening
by: (1)
fermentation and extraction of broths from soil, plant or marine
microorganisms or
(2) extraction of plants or marine organisms. Natural product libraries
include
polyketides, non-ribosomal peptides, and variants (non-naturally occurring)
thereof.
For a review, see, Science 282:63-68 (1998). Combinatorial libraries are
composed
of large numbers of peptides, oligonucleotides, or organic compounds as a
mixture.
These libraries are relatively easy to prepare by traditional automated
synthesis
methods, PCR, cloning, or proprietary synthetic methods. Of particular
interest are
non-peptide combinatorial libraries. Still other libraries of interest include
peptide,
protein, peptidomimetic, multiparallel synthetic collection, recombinatorial,
and
polypeptide libraries. For a review of combinatorial chemistry and libraries
created
therefrom, see, Myers, Cur. ~pin. Biotechnol. 8:701-707 (1997). Identification
of
modulators through use of the various libraries described herein permits
modification of the candidate "hit" (or "lead") to optimize the capacity of
the "hit"
to modulate activity.
Still other candidate inhibitors contemplated by the invention can be
designed and include soluble forms of binding partners, as well as such
binding
partners as chimeric, or fusion, proteins. A "binding partner" as used herein
broadly
encompasses both natural binding partners as described above as well as
chimeric
polypeptides, peptide modulators other than natural ligands, antibodies,
antibody
fragments, and modified compounds comprising antibody domains that are
immunospecific for the expression product of the identified protease gene.
Other assays may be used to identify specific peptide ligands of a protease
polypeptide, including assays that identify ligands of the target protein
through
measuring direct binding of test ligands to the target protein, as well as
assays that
identify ligands of target proteins through affinity ultrafiltration with ion
spray mass
spectroscopy/HPLC methods or other physical and analytical methods.
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Alternatively, such binding interactions are evaluated indirectly using the
yeast two-
hybrid system described in Fields et al., Nature, 340:245-246 (1989), and
Fields et
al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated
herein by
reference. The two-hybrid system is a genetic assay for detecting interactions
between two proteins or polypeptides. It can be used to identify proteins that
bind to
a known protein of interest, or to delineate domains or residues critical for
an
interaction. Variations on this methodology have been developed to clone genes
that
encode DNA binding proteins, to identify peptides that bind to a protein, and
to
screen for drugs. The two-hybrid system exploits the ability of a pair of
interacting
proteins to bring a transcription activation domain into close proximity with
a DNA
binding domain that binds to an upstream activation sequence (UAS) of a
reporter
gene, arid is generally performed in yeast. The assay requires the
construction of
two hybrid genes encoding (1) a DNA-binding domain that is fused to a first
protein
and (2) an activation domain fused to a second protein. The DNA-binding domain
targets the first hybrid protein to the UAS of the reporter gene; however,
because
most proteins lack an activation domain, this DNA-binding hybrid protein does
not
activate transcription of the reporter gene. The second hybrid protein, which
contains the activation domain, cannot by itself activate expression of the
reporter
gene because it does not bind the UAS. However, when both hybrid proteins are
present, the noncovalent interaction of the first and second proteins tethers
the
activation domain to the UAS, activating transcription of the reporter gene.
For
example, when the first protein is a protease gene product, or fragment
thereof, that
is known to interact with another protein or nucleic acid, this assay can be
used to
detect agents that interfere with the binding interaction. Expression of the
reporter
gene is monitored as different test agents are added to the system. The
presence of
an inhibitory agent results in lack of a reporter signal.
When the function of the protease polypeptide gene product is unknown and
no ligands are known to bind the gene product, the yeast two-hybrid assay can
also
be used to identify proteins that bind to the gene product. In an assay to
identify
proteins that bind to a protease polypeptide, or fragment thereof, a fusion
38
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polynucleotide encoding both a protease polypeptide (or fragment) and a UAS
binding domain (i. e., a first protein) may be used. In addition, a large
number of
hybrid genes each encoding a different second protein fused to an activation
domain
are produced and screened in the assay. Typically, the second protein is
encoded by
one or more members of a total cDNA or genomic DNA fusion library, with each
second protein coding region being fused to the activation domain. This system
is
applicable to a wide variety of proteins, and it is not even necessary to know
the
identity or function of the second binding protein. The system is highly
sensitive
and can detect interactions not revealed by other methods; even transient
interactions
may trigger transcription to produce a stable mRNA that can be repeatedly
translated
to yield the reporter protein.
Other assays may be used to search for agents that bind to the target protein.
One such screening method to identify direct binding of test ligands to a
target
protein is described in U.S. Patent No. 5,585,277, incorporated herein by
reference.
This method relies on the principle that proteins generally exist as a mixture
of
folded and unfolded states, and continually alternate between the two states.
When a
test ligand binds to the folded form of a target protein (i. e., when the test
ligand is a
ligand of the target protein), the target protein molecule bound by the ligand
remains
in its folded state. Thus, the folded target protein is present to a greater
extent in the
presence of a test ligand which binds the target protein, than in the absence
of a
ligand. Binding of the ligand to the target protein can be determined by any
method
which distinguishes between the folded and unfolded states of the target
protein.
The function of the target protein need not be known in order for this assay
to be
performed. Virtually any agent can be assessed by this method as a test
ligand,
including, but not limited to, metals, polypeptides, proteins, lipids,
polysaccharides,
polynucleotides and small organic molecules.
Another method for identifying ligands of a target protein is described in
Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporated herein by
reference. This technique screens combinatorial libraries of 20-30 agents at a
time
in solution phase for binding to the target protein. Agents that bind to the
target
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protein are separated from other library components by simple membrane
washing.
The specifically selected molecules that are retained on the filter are
subsequently
liberated from the target protein and analyzed by HPLC and pneumatically
assisted
electrospray (ion spray) ionization mass spectroscopy. This procedure selects
library components with the greatest affinity for the target protein, and is
particularly
useful for small molecule libraries.
In preferred embodiments of the invention, methods of screening for
compounds which modulate protease activity comprise contacting test compounds
with protease polypeptides and assaying for the presence of a complex between
the
compound and the protease polypeptide. In such assays, the ligand is typically
labelled. After suitable incubation, free ligand is separated from that
present in
bound form, and the amount of free or uncomplexed label is a measure of the
ability
of the particular compound to bind to the protease polypeptide.
W another embodiment of the invention, high throughput screening for
compounds having suitable binding affinity to protease polypeptides is
employed.
Briefly, large numbers of different small peptide test compounds are
synthesised on
a solid substrate. The peptide test compounds are contacted with the protease
polypeptide and washed. Bound protease polypeptide is then detected by methods
well known in the art. Purified polypeptides of the invention can also be
coated
directly onto plates for use in the aforementioned drug screening techniques.
In
addition, non-neutralizing antibodies can be used to capture the protein and
immobilize it on the solid support.
Other embodiments of the invention comprise using competitive screening
assays in which neutralizing antibodies capable of binding a polypeptide of
the
invention specifically compete with a test compound for binding to the
polypeptide.
In this manner, the antibodies can be used to detect the presence of any
peptide that
shares one or more antigenic determinants with a protease polypeptide.
Radiolabeled competitive binding studies are described in A.H. Lin et al.
An.timicrobial Agents and Chemotherapy, 1997, vol. 41, no. 10. pp. 2127-2131,
the
disclosure of which is incorporated herein by reference in its entirety.
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Therapeutic Methods
The invention includes methods for treating a disease or disorder by
administering to a patient in need of such treatment a protease polypeptide
substantially identical to an amino acid sequence selected from the group
consisting
of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID
N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID
N0:44, SEQ ID NO:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID
NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID
N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID
N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID NO:63, SEQ ID
N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID
N0:69, SEQ ID N0:70, and any other protease polypeptide of the present
invention.
As discussed in the section "Gene Therapy," a protease polypeptide of the
invention
may also be administered indirectly by via administration of suitable
polynucleotide
means for ih vivo expression of the protease polypeptide. Preferably the
protease
polypeptide will have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%
identity to one of the aforementioned sequences.
In another aspect, the invention provides methods for treating a disease or
disorder by administering to a patient in need of such treatment a substance
that
modulates the activity of a protease substantially identical to a sequence
selected
from the group consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37,
SEQ
ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ
ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID NO:46, SEQ ID N0:47, SEQ
ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ
ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ
ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ
ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ
ID N0:68, SEQ ID N0:69, and SEQ ID N0:70.
Preferably the disease is selected from the group consisting of cancers,
immune-related diseases and disorders, cardiovascular disease, brain or
neuronal-
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associated diseases, and metabolic disorders. More specifically these diseases
include cancer of tissues, blood, or hematopoietic origin, particularly those
involving
breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney;
central or
peripheral nervous system diseases and conditions including migraine, pain,
sexual
dysfunction, mood disorders, attention disorders, cognition disorders,
hypotension,
and hypertension; psychotic and neurological disorders, including anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Tourette's Syndrome;
neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple
sclerosis,
and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-
1,
HIV-2 or other viral- or prion-agents or fwgal- or bacterial- organisms;
metabolic
disorders including Diabetes and obesity and their related syndromes, among
others;
cardiovascular disorders including reperfusion restenosis, coronary
thrombosis,
clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular
disease
including glaucoma, retinopathy, and macular degeneration; inflammatory
disorders
including rheumatoid arthritis, chronic inflammatory bowel disease, chronic
inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis,
psoriasis,
atherosclerosis, rhinitis, autoimmunity, and organ transplant rej ection.
In preferred embodiments, the invention provides methods for treating or
preventing a disease or disorder by administering to a patient in need of such
treatment a substance that modulates the activity of a protease polypeptide
having an
amino acid sequence selected from the group consisting of those sat forth in
SEQ ID
N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID
N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID
N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID
NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID
N0:56, SEQ ID NO:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID
N0:61, SEQ 117 N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID
N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70.
Preferably the disease is selected from the group consisting of cancers,
immune-
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related diseases and disorders, cardiovascular disease, brain or neuronal-
associated
diseases, and metabolic disorders. More specifically these diseases include
cancer
of tissues, blood, or hematopoietic origin, particularly those involving
breast, colon,
lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or
peripheral
nervous system diseases and conditions including migraine, pain, sexual
dysfunction, mood disorders, attention disorders, cognition disorders,
hypotension,
and hypertension; psychotic and neurological disorders, including anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Tourette's Syndrome;
neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple
sclerosis,
and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-
1,
HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms;
metabolic
disorders including Diabetes and obesity and their related syndromes, among
others;
cardiovascular disorders including reperfusion restenosis, coronary
thrombosis,
clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular
disease
including glaucoma, retinopathy, and macular degeneration; inflammatory
disorders
including rheumatoid arthritis, chronic inflammatory bowel disease, chronic
inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis,
psoriasis,
atherosclerosis, rhinitis, autoimmunity, and organ transplant rejection.
The invention also features methods of treating or preventing a disease or
disorder by administering to a patient in need of such treatment a substance
that
modulates the activity of a protease polypeptide having an amino acid sequence
selected from the group consisting those set forth in SEQ ID N0:36, SEQ ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID
N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ m NO:50, SEQ ID NO:S1, SEQ ID
N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID
N0:57, SEQ ID N0:58, SEQ m N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID
N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70. Preferably the disease
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is selected from the group consisting of immune-related diseases and
disorders,
cardiovascular disease, and cancer. Most preferably, the immune-related
diseases
and disorders are selected from the group consisting of rheumatoid arthritis,
chronic
inflammatory bowel disease, chronic inflammatory pelvic disease, multiple
sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis,
autoimmunity,
and organ transplantation.
Substances useful for treatment of protease-related disorders or diseases
preferably show positive results in one or more ih vitro assays for an
activity
corresponding to treatment of the disease or disorder in question (Examples of
such
assays are provided herein, including Example 7). Examples of substances that
can
be screened for favorable activity are provided and referenced throughout the
specification, including this section (Screenin'g Methods to
Identif~Substances
that Modulate Protease Activity). The substances that modulate the activity of
the
proteases preferably include, but are not limited to, antisense
oligonucleotides,
ribozymes, and other inhibitors of proteases, as determined by methods and
screens
referenced this section and in Example 7, below, and any other suitable
methods.
The use of antisense oligonucleotides and ribozymes are discussed more fully
in the
Section "Gene Therapy," below.
The term "preventing" refers to decreasing the probability that an organism
contracts or develops an abnormal condition.
The term "treating" refers to having a therapeutic effect and at least
partially
alleviating or abrogating an abnormal condition in the organism.
The term "therapeutic effect" refers to the inhibition or activation factors
causing or contributing to the abnormal condition. A therapeutic effect
relieves to
some extent one or more of the symptoms of the abnormal condition. In
reference to
the treatment of abnormal conditions, a therapeutic effect can refer to one or
more of
the following: (a) an increase or decrease in the proliferation, growth,
and/or
differentiation of cells; (b) activation or inhibition (i.e., slowing or
stopping) of cell
death; (c) inhibition of degeneration; (d) relieving to some extent one or
more of the
symptoms associated with the abnormal condition; and (e) enhancing the
function of
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the affected population of cells. Compounds demonstrating efficacy against
abnormal conditions can be identified as described herein.
The term "abnormal condition" refers to a function in the cells or tissues of
an organism that deviates from their normal functions in that organism. An
abnormal condition can relate to cell proliferation, cell differentiation, or
cell
survival.
Abnormal cell proliferative conditions include cancers such as fibrotic and
mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing,
psoriasis, diabetes mellitus, and inflammation.
Abnormal differentiation conditions include, but are not limited to
neurodegenerative disorders, slow wound healing rates, and slow tissue
grafting
healing rates.
Abnormal cell survival conditions relate to conditions in which programmed
cell death (apoptosis) pathways are activated or abrogated. A number of
proteases
are associated with the apoptosis pathways. Aberrations in the function of any
one
of the proteases could lead to cell immortality or premature cell death.
The term "aberration", in conjunction with the function of a protease in a
signal transduction process, refers to a protease that is over- or under-
expressed in
an organism, mutated such that its catalytic activity is lower or higher than
wild-type
protease activity, mutated such that it can no longer interact with a natural
binding
partner, is no longer modified by another protein, or no longer interacts with
a
natural binding partner.
The term "administering" relates to a method of incorporating a compound
into cells or tissues of an organism. The abnormal condition can be prevented
or
treated when the cells or tissues of the organism exist within the organism or
outside
of the organism. Cells existing outside the organism can be maintained or
grown in
cell culture dishes. For cells harbored within the organism, many techniques
exist in
the art to administer compounds, including (but not limited to) oral,
parenteral,
dermal, injection, and aerosol applications. For cells outside of the
organism,
multiple techniques exist in the art to administer the compounds, including
(but not
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limited to) cell microinjection techniques, transformation techniques, and
carrier
techniques.
The abnormal condition can also be prevented or treated by administering a
compound to a group of cells having an aberration in a signal transduction
pathway
S to an organism. The effect of administering a compound on organism function
can
then be monitored. The organism is preferably a mouse, rat, rabbit, guinea
pig, or
goat, more preferably a monkey or ape, and most preferably a human.
In another aspect, the invention features methods for detection of a protease
polypeptide in a sample as a diagnostic tool for diseases or disorders,
wherein the
method comprises the steps of: (a) contacting the sample with a nucleic acid
probe
which hybridizes under hybridization assay conditions to a nucleic acid target
region
of a protease polypeptide having an amino acid sequence selected from the
group
consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0:39, SEQ ID NO:40, SEQ ID N0:41, SEQ ID NO:42, SEQ ID N0:43,
SEQ ID NO:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID NO:48,
SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53,
SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID NO:58,
SEQ m N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID NO:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID N0:69, and SEQ ID N0:70, said probe comprising the nucleic acid
sequence encoding the polypeptide, fragments thereof, and the complements of
the
sequences and fragments; and (b) detecting the presence or amount of the
probeaarget region hybrid as an indication of the disease.
In preferred embodiments of the invention, the disease or disorder is selected
from the group consisting of rheumatoid arthritis, arteriosclerosis,
autoimmune
disorders, organ transplantation, myocardial infarction, cardiomyopathies,
stroke,
renal failure, oxidative stress-related neurodegenerative disorders, and
cancer.
Preferably the disease is selected from the group consisting of cancers,
immune-
related diseases and disorders, cardiovascular disease, brain or neuronal-
associated
diseases, and metabolic disorders. More specifically these diseases include
cancer
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of tissues, blood, or hematopoietic origin, particularly those involving
breast, colon,
lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or
peripheral
nervous system diseases and conditions including migraine, pain, sexual
dysfunction, mood disorders, attention disorders, cognition disorders,
hypotension,
and hypertension; psychotic and neurological disorders, including anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Tourette's Syndrome;
neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple
sclerosis,
and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-
1,
HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms;
metabolic
disorders including Diabetes and obesity and their related syndromes, among
others;
cardiovascular disorders including reperfusion restenosis, coronary
thrombosis,
clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular
disease
including glaucoma, retinopathy, and macular degeneration; inflammatory
disorders
including rheumatoid arthritis, chronic inflammatory bowel disease, chronic
inflammatory pelvic disease, multiple sclerosis, astluna, osteoarthritis,
psoriasis,
atherosclerosis, rhinitis, autoimmunity, and organ transplant rejection.
The protease "target region" is the nucleotide base sequence selected from
the group consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID
N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8,
SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID NO:13,
SEQ ID N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18,
SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23,
SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28,
SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33,
SEQ ID N0:34, and SEQ ID N0:35, or the corresponding full-length sequences, a
functional derivative thereof, or a fragment thereof or a domain thereof to
which the
nucleic acid probe will specifically hybridize. Specific hybridization
indicates that
in the presence of other nucleic acids the probe only hybridizes detectably
with the
nucleic acid target region of the protease of the invention. Putative target
regions
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can be identified by methods well known in the art consisting of alignment and
comparison of the most closely related sequences in the database.
In preferred embodiments the nucleic acid probe hybridizes to a protease
target region encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or
350
contiguous amino acids of a sequence selected from the group consisting of
those set
forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID
N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID NO:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID
NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID
NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID
N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ
ID N0:70, or the corresponding full-length amino acid sequence, or a
functional
derivative thereof. Hybridization conditions should be such that hybridization
occurs only with the protease genes in the presence of other nucleic acid
molecules.
Under stringent hybridization conditions only highly complementary nucleic
acid
sequences hybridize. Preferably, such conditions prevent hybridization of
nucleic
acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides.
Such
conditions are defined in Berger et al. (1987) (Guide to Molecular Cloning
Techniques pg 421, hereby incorporated by reference herein in its entirety
including
any figures,.tables, or drawings.).
The diseases for which detection of protease genes in a sample could be
diagnostic include diseases in which protease nucleic acid (DNA and/or RNA) is
amplified in comparison to normal cells. By "amplification" is meant increased
numbers of protease DNA or RNA in a cell compared with normal cells. In normal
cells, proteases may be found as single copy genes. In selected diseases, the
chromosomal location of the protease genes may be amplified, resulting in
multiple
copies of the gene, or amplification. Gene amplification can lead to
amplification of
protease RNA, or protease RNA can be amplified in the absence of protease DNA
amplification.
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"Amplification" as it refers to RNA can be the detectable presence of
protease RNA in cells, since in some normal cells there is no basal expression
of
protease RNA. In other normal cells, a basal level of expression of protease
exists,
therefore in these cases amplification is the detection of at least 1-2-fold,
and
S preferably more, protease RNA, compared to the basal level.
The diseases that could be diagnosed by detection of protease nucleic acid in
a sample preferably include cancers. The test samples suitable for nucleic
acid
probing methods of the present invention include, for example, cells or
nucleic acid
extracts of cells, or biological fluids. The samples used in the above-
described
methods will vary based on the assay format, the detection method and the
nature of
the tissues, cells or extracts to be assayed. Methods for preparing nucleic
acid
extracts of cells are well known in the art and can be readily adapted in
order to
obtain a sample that is compatible with the method utilized.
In a final aspect, the invention features a method for detection of a protease
1S polypeptide in a sample as a diagnostic tool for a disease or disorder,
wherein the
method comprises: (a) comparing a nucleic acid target region encoding the
protease
polypeptide in a sample, where the protease polypeptide has an amino acid
sequence
selected from the group consisting those set forth in SEQ ID N0:36, SEQ ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:4S, SEQ ID N0:46, SEQ ID
NO:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:SO, SEQ ID NO:S1, SEQ ID
NO:S2, SEQ ID NO:S3, SEQ ID NO:S4, SEQ ID NO:SS, SEQ ID NO:S6, SEQ ID
NO:S7, SEQ ID NO:SB, SEQ ID NO:S9, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:6S, SEQ ID N0:66, SEQ ID
2S N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70, or one or more
fragments thereof, with a control nucleic acid target region encoding the
protease
polypeptide, or one or more fragments thereof; and (b) detecting differences
in
sequence or amount between the target region and the control target region, as
an
indication of the disease or disorder. Preferably the disease is selected from
the
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group consisting of cancers, immune-related diseases and disorders,
cardiovascular
disease, brain or neuronal-associated diseases, and metabolic disorders.
More specifically these diseases include cancer of tissues, blood, or
hematopoietic origin, particularly those involving breast, colon, lung,
prostate,
cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous
system
diseases and conditions including migraine, pain, sexual dysfunction, mood
disorders, attention disorders, cognition disorders, hypotension, and
hypertension;
psychotic and neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, severe mental retardation and dyskinesias,
such as
Huntington's disease or Tourette's Syndrome; neurodegenerative diseases
including
Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral
sclerosis;
viral or non-viral infections caused by HIV-1, HIV-2 or other viral- or prion-
agents
or fiulgal- or bacterial- organisms; metabolic disorders including Diabetes
and
obesity and their related syndromes, among others; cardiovascular disorders
including reperfusion restenosis, coronary thrombosis, clotting disorders,
unregulated cell growth disorders, atherosclerosis; ocular disease including
glaucoma, retinopathy, and macular degeneration; inflammatory disorders
including
rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory
pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis,
atherosclerosis,
rhinitis, autoimmunity, and organ transplant rejection.
The term "comparing" as used herein refers to identifying discrepancies
between the nucleic acid target region isolated from a sample, and the control
nucleic acid target region. The discrepancies can be in the nucleotide
sequences,
e.g. insertions, deletions, or point mutations, or in the amount of a given
nucleotide
sequence. Methods to determine these discrepancies in sequences are well-known
to
one of ordinary skill in the art. The "control" nucleic acid target region
refers to the
sequence or amount of the sequence found in normal cells, e.g. cells that are
not
diseased as discussed previously.
The term "domain" refers to a region of a polypeptide which serves a
particular function. For instance, N-terminal or C-ternlinal domains of signal
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transduction proteins can serve functions including, but not limited to,
binding
molecules that localize the signal transduction molecule to different regions
of the
cell or binding other signaling molecules directly responsible for propagating
a
particular cellular signal. Some domains can be expressed separately from the
rest
of the protein and function by themselves, while others must remain part of
the
intact protein to retain function. The latter are termed functional regions of
proteins
and also relate to domains.
The expression of proteases can be modulated by signal transduction
pathways such as the Ras/MAP kinase signaling pathways. Additionally, the
activity of proteases can modulate the activity of the MAP kinase signal
transduction
pathway. Furthermore, proteases can be shown to be instrumental in the
communication between disparate signal transduction pathways.
The term "signal transduction pathway" refers to the molecules that
propagate an extracellular signal through the cell membrane to become an
intracellular signal. This signal can then stimulate a cellular response. The
polypeptide molecules involved in signal transduction processes are typically
receptor and non-receptor protein tyrosine kinases, receptor and non-receptor
protein
phosphatases, polypeptides containing SRC homology 2 and 3 domains,
phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine
binding (PTB and PH) domain containing proteins), proline-rich binding
proteins
(SH3 domain containing proteins), GTPases, phosphodiesterases, phospholipases,
prolyl isomerases, proteases, Ca2+ binding proteins, cAMP binding proteins,
guanyl
cyclases, adenylyl cyclases, NO generating proteins, nucleotide exchange
factors,
and transcription factors.
The summary of the invention described above is not limiting and other
features and advantages of the invention will be apparent from the following
detailed description of the invention, and from the claims.
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BRIEF DESCRIPTION OF THE FIGURES
Figures lA-W shows the partial nucleotide sequences for human proteases
oriented in a 5' to 3' direction (SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ
ID
N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14,
SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19,
SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24,
SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID NO:28, SEQ ID N0:29,
SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34,
and SEQ ID N0:35). In the sequences, N means any nucleotide.
Figure 2A-I shows the partial amino acid sequences for the human proteases
encoded by SEQ TD No. 1-35 in the direction of translation (SEQ ID N0:36, SEQ
ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ
ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ 117 N0:45, SEQ ID N0:46, SEQ
ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ
ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ
ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ
ID N0:62, SEQ ID N0:63, SEQ ID NO:64, SEQ ID N0:65, SEQ ID N0:66, SEQ
ID NO:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70). In the sequences,
X means any amino acid.
DETAILED DESCRIPTION OF THE INVENTION
The following description of the background of the invention is provided to
aid in understanding the invention, but is not admitted to be or to describe
prior art
to the invention.
Proteases are enzymes capable of severing the amino acid backbone of other
proteins, and are involved in a large number of diverse processes within the
body.
Their normal functions include modulation of apoptosis (caspases) (Salvesen
and
Dixon, Cell, 1997, 91:443-46), control of blood pressure (renin, angiotensin-
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converting enzymes) (van Hooft et al., 1991, N Engl J Med. 324(19):1305-11,
and chapters 254 and 359 in Barrett et al., Handbook of Proteolytic Enzymes,
1998,
Academic Press, San Diego), tissue remodeling and tumor invasion (collagenase)
(Vu et al., 1998, Cell 93:411-22, Werb, 1997, Cell, 91:439-442), development
of
Alzheimer's Disease ((3-secretase) (De Strooper et al., 1999, Nature 398:518-
22),
protein turnover and cell-cycle regulation~(proteosome) (Bastians et al.,
1999, Mol.
Biol. Cell. 10:3927-41, Gottesman, et al., 1997, Cell, 91:435-38, Larsen et
al., 1997,
Cell, 91:431-34), inflammation (TNF-oc convertase) (Black et al., Nature,
1997,
385:729-33), and protein turnover (Bochtler et al., 1999, Ahnu. Rev. Biophys
Biomol
Struct.28:295-317). Proteases may be classified into several major groups
including
serine proteases, cysteine proteases, aspartyl proteases, metalloproteases,
threonine
proteases, and other proteases.
1. Asuart~proteases (Al; Prosite number PS00141):
Aspartate proteases of eukaryotes are monomeric enzymes which consist of
two domains. Each domain contains an active site centered on a catalytic
aspartyl
residue. Examples of aspartyl protease polypeptides according to the invention
include SGPr140, r197, r005 and r078 (SEQ ID NOS:l, 2, 3, and 4,
respectively).
These polypeptides may have one or more of the following activites.
Cathepsins
Cathepsin E is an immunologically discrete aspartic protease found in the
gastrointestinal tract (Azuma et al., 1992, J. Biol. Chem., 267:1609-1614).
Cathepsin E is an intracellular proteinase that does not appear to be involved
in the
digestion of dietary protein. It is found in highest concentration in the
surface of
epithelial mucus-producing cells of the stomach. It is the first aspartic
proteinase
expressed in the fetal stomach and is found in more than half of gastric
cancers. It
appears, therefore, to be an 'oncofetal' antigen. Its association with stomach
cancers
suggests it may play a role in the development of this disease.
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Cathepsin D, a lyosomal aspartyl protease, is being studied as a prognostic
marker in various cancers, in particular, breast cancer. (Rochefort et al.,
Cliya. Chim.
Acta, 2000, 291:157-170).
Renin
Released by the juxtaglomerular cells of the kidney, renin catalyzes the first
step in the activation pathway of angiotensinogen--a cascade that can result
in
aldosterone release, vasoconstriction, and increase in blood pressure. Renin
cleaves
angiotensinogen to form angiotensin I, which is converted to angiotensin II by
angiotensin I converting enzyme, an important regulator of blood pressure and
electrolyte balance. Renin occurs in other organs than the kidney, e.g., in
the brain,
where it is implicated in the regulation of numerous activities.
Presenilin proteins
Alzheimer's disease (AD) patients with an inherited form of the disease carry
mutations in the presenilin proteins (PSENl; PSEN2) or the amyloid precursor
protein (APP). These disease-linked mutations result in increased production
of the
longer form of amyloid-beta (main component of amyloid deposits found in AD
brains) (Saftig et al., Eur. Arch. Psychia~tfy Cliya. Neurosci., 1999, 249:271-
79).
Presenilins are postulated to regulate APP processing through their effects on
y-
secretase, an enzyme that cleaves APP (Cruts et al., 1998, Hum. Mutat., 11:183-
190,
Haass et al., Science, 1999, 286:916-19). Also, it is thought that the
presenilins are
involved in the cleavage of the Notch receptor, such that that they either
directly
regulate y-secretase activity or themselves are protease enzymes (De Strooper
et al.,
Natuy~e, 1999, 398:518-22). Two alternative transcripts of PSEN2 have been
identified (Sato et al., 1999, JNeurochem. 72(6):2498-505). Point mutations in
the
PS 1 gene result in a selective increase in the production of the
amyloidogenic
peptide amyloid-beta (1-42) by proteolytic processing of the amyloid precursor
protein (APP)(Lemere et al., 1996, Nat. Med. 2(10):1146-50). The possible role
of
PS I in normal APP processing was studied by De Strooper et al. (Nature 391:
387-
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390, 1998) in neuronal cultures derived from PS 1-deficient mouse embryos.
They
found that cleavage by a,- and [3-secretase of the extracellular domain of APP
was
not affected by the absence of PS1, whereas cleavage by y-secretase of the
transmembrane domain of APP was prevented, causing C-terminal fragments of
APP to accumulate and a 5-fold drop in the production of amyloid peptide.
Pulse-
chase experiments indicated that PSl deficiency specifically decreased the
turnover
of the membrane-associated fragments of APP. Thus, PS 1 appears to facilitate
a
proteolytic activity that cleaves the integral membrane domain of APP. The
results
indicated to the authors that mutations in PS 1 that manifest clinically cause
a gain of
function, and that inhibition of PS 1 activity is a potential target for anti-
amyloidogenic therapy in Alzheimer disease.
-secretase
(3-secretase, expressed specifically in the brain, is responsible for the
proteolytic processing of the amyloid precursor protein (APP) associated with
Alzheimer's disease (Potter et al., 2000, Nat. Biotechhol. 18(2):125-26). It
cleaves
at the amino terminus of the (3-peptide sequence, between residues 671 and 672
of
APP, leading to the generation and extracellular release of (3-cleaved soluble
APP,
and a carboxyterminal fragment that is later released by y-secretase (Kimberly
et al.,
2000, J. Biol. Chem. 275(5):3173-78). Yan et al. (Nature, 1999 402:533-37)
identified a new membrane-bound aspartyl protease (Asp2) with (3-secretase
activity. The Asp2 gene is expressed widely in brain and other tissues.
Decreasing
the expression of Asp2 in cells reduces amyloid (3-peptide production and
blocks the
accumulation of the carboxy-terminal APP fragment that is created by (3-
secretase
cleavage. Asp2 is a new protein target for drugs that are designed to block
the
production of amyloid (3-peptide peptide and the consequent formation of
amyloid
plaque in' Alzheimer's disease.
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Two aspartyl proteases involved in human placentation have recently been
isolated: decidual aspartyl protease (DAP-1), and DAP-2. (Moses et al., Mol.
Hum.
Rep~od., 1999, 5:983-89)
Another member of the aspartyl peptidase family is HIV-1 retropepsin, from
the human immunodeficiency virus type 1. This enzyme is vital for processing
of
the viral polyprotein and maturation of the mature virion.
2. Cysteine nroteases
Another class of proteases which perform a wide variety of functions within
the body are the cysteine proteases. Among their roles are the processing of
precursor proteins, and intracellular degradation of proteins marked for
disposal via
the ubiquitin pathway. Catalysis proceeds through a thioester intermediate and
is
facilitated by a nearby histidine side chain; an asparagine completes the
essential
catalytic triad. Peptidases in this family with important roles in disease
include
calpain, the caspases, hedgehog, papain, and Ubiquitin hydrolases. Examples of
cysteine protease polypeptides of the present invention include SGPr084, r009,
r286,
r008, r198, r210, r290, r116, r003, r016 (SEQ ID NOS:S, 6, 7, 8, 9, 10, 11,
12, and
13, respectively). These polypeptides may have one or more of the following
activities.
Cysteine proteases are produced by a large number of cells including those
of the immune system (macrophages, monocytes, etc.). These immune cells
exercise their protective role in the body, in part, by migrating to sites of
inflammation and secreting molecules, among the secreted molecules are
cysteine
proteases.
Under some conditions, the inappropriate regulation of cysteine proteases of
the immune system can lead to autoimmune diseases such as rheumatoid
arthritis.
For example, the over-secretion of the cysteine protease cathepsin C causes
the
degradation of elastin, collagen, laminin and other structural proteins found
in
bones. Bone subjected to this inappropriate digestion is more susceptible to
metastasis.
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Cysteine proteases may also influence vascular permeability through their
effect on the kallikrein/kinin pathway, their ability to form complexes with
hemagglutinins, their effect in activation of complement components and their
ability to destroy serpins.
Caspase (C141- apopotosis
A cascade of protease reactions is believed to be responsible for the
apoptotic changes observed in mammalian cells undergoing programmed cell
death.
This cascade involves many members of the aspartate-specific cysteine
proteases of
the caspase family, including Caspases 2, 3, 6, 7, 8, and 10 ((Salvesen and
Dixit,
Cell, 1997, 91:443-446). Cancer cells that escape apoptotic signals, generated
by
cytotoxic chemotherapeutics or loss of normal cellular survival signals (as in
metastatic cells), can go on to develop palpable tumors.
Other caspases are also involved in the activation of pro-inflammatory
cytokines. Caspase 1 specifically processes the precursors of IL-1 [3, and IL-
18
(interferon-y-inducing factor) (Salvesen and Dixit, Cell, 1997).
Calpain (C2) - axonal death, dystrophies
Calcium-dependent cysteine proteases, collectively called calpain, axe widely
distributed in mammalian cells (Wang, 2000, T~ehds Neu~osci. 23(1):20-26). The
calpains are nonlysosomal intracellular cysteine proteases. The mammalian
calpains
include 2 ubiquitous proteins, CAPNI and CAPN2, as well as 2 stomach-specific
proteins, and CAPN3, which is muscle-specific (Herasse et al., 1999, Mol.
Cell.
Biol. 19(6):4047-55). The ubiquitous enzymes consist of heterodimers with
distinct
large subunits associated with a common small subunit, all of wl>ich are
encoded by
different genes. The large subunits of calpains can be subdivided into 4
domains;
domains I and III, whose functions remain unknown, show no homology with
known proteins. The former, however, may be important for the regulation of
the
proteolytic activity. Domain II shows similarity with other cysteine
proteases,
which share histidine, cysteine, and asparagine residues at their active
sites. Domain
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IV is calinodulin-like. CAPNS and CAPN6 differ from previously identified
vertebrate calpains in that they lack a calinodulin-like domain IV (Ohno et
al., 1990,
Cytogenet. Cell Genet. 53(4):225-29).
Mutations in the CAPN3 gene have been associated with limb-girdle
muscular dystrophy, type 2A (LGMD2A) (Allamand et al., 1995, Hum. Molec.
Genet. 4:459-463). The slowly progressive muscle weakness associated with this
disease is usually first evident in the pelvic girdle and then spreads to the
upper
limbs while sparing facial muscles. Calpain has also been implicated in the
development of hyperactive CdkS leading to neuronal cell death associated with
Alzheimer's disease (Patrick et al., 1999, Nature 402:615-622).
Hed~-ehog~C46 - Cancer
The organization and morphology of the developing embryo are established
through a series of inductive interactions. One family of vertebrate genes has
been
described related to the Drosophila gene 'hedgehog' (hh) that encodes
inductive
signals during embryogenesis (Johnson and Tabin, 1997, Cell 90:979-990).
'Hedgehog' encodes a secreted protein that is involved in establishing cell
fates at
several points during Drosophila development (Marigo et al., 1995, Genomics
28:44-51). There are 3 known mammalian homologs of hh: Sonic hedgehog (Shh),
Indian hedgehog (Ihh), and desert hedgehog (Dhh) (Johnson and Tabin, 1997,
Cell
90:979-990). Like its Drosophila cognate, Shh encodes a signal that is
instrumental
in patterning the early embryo. It is expressed in Hensen's node, the
floorplate of
the neural tube, the early gut endoderm, the posterior of the limb buds, and
throughout the notochord (Chiang et al., 1996, Nature 383:407-413). It has
been
implicated as the key inductive signal in patterning of the ventral neural
tube, the
anterior-posterior limb axis, and the ventral somites. Oro et al. ("Basal cell
carcinomas in mice overexpressing sonic hedgehog." Science 276: 817-821, 1997)
showed that transgenic mice overexpressing SHH in the skin developed many
features of the basal cell nevus syndrome, demonstrating that SHH is
sufficient to
induce basal cell carcinomas (BCCs) in mice. The data suggested that SHH may
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have a role in human tumorigenesis. Activating mutations of SHH or another
'hedgehog' gene may be an alternative pathway for BCC formation in humans. The
human mutation his133tyr (his134tyr in mouse) is a candidate. It is distinct
from
loss-of function mutations reported for individuals with holoprosencephaly
(Oro et
al., 1997, Science 276:817-821). Hisl33 lies adjacent in the catalytic site to
his134,
one of the conserved residues thought to be necessary for catalysis. SHH may
be a
dominant oncogene in multiple human tumors, a mirror of the tumor suppressor
activity of the opposing 'patched' (PTCH) gene (Aszterbaum et al., 1998, J.
Invest.
Deem. 110:885-888). The rapid and frequent appearance of Shh-induced tumors in
the mice suggested that disruption of the SHH-PTC pathway is sufficient to
create
BCCs.
Members of the vertebrate hedgehog family (Sonic, Indian, and Desert) have
been shown to be essential for the development of various organ systems,
including
neural, somite, limb, skeletal, and for male gonad morphogenesis. Desert
hedgehog
is expressed in the developing retina, whereas Indian hedgehog (Thh) is
expressed in
the developing and mature retinal pigmented epithelium beginning at embryonic
day
13 (Levine et al., J.Neu~osci., 1997, 17(16):6277-88). Dhh has also been
implicated
in having a role in the regulation of spermatogenesis. Sertoli cell precursors
express
Sry, sex determining gene, which leads to testis development in mammals. Dhh
expression is initiated in Sertoli cell precursors shortly after the
activation of Sry and
persists in the testis into the adult. Bitgood et al. (Curs. Biol., 1996,
6(3):298-304)
disclose that female mice homozygous for a Dhh-null mutation show no obvious
phenotype, whereas males are viable but infertile having a complete absence of
mature sperm, demonstrating that Dhh signaling plays an essential role in the
regulation of mammalian spermatogenesis. Dhh has also been found to have a
role
in the and maintenance of protective nerve sheaths endo-, peri- and
epineurium. In
Dhh knockout mice, the connective tissue sheaths in adult nerves appear highly
abnormal by electron microscopy. Mirsky et al., (Ann. N. Y. Acad. Sci., 1999,
883:196-202) demonstrate that Dhh signaling from Schwann cells to the
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mesenchyme is involved in the formation of a morphologically and functionally
normal perineurium.
Recent advances in developmental and molecular biology during
embryogenesis and organogenesis have provided new insights into the mechanism
of bone formation. Iwasaki et al., (J. Bone .Ioirct Sing. B~., 1999,
81(6):1076-82)
demonstrate that Indian Hedgehog (Ihh) is expressed in cartilage cell
precursors and
later in mature and hypertrophic chondrocytes. Ihh plays a critical role in
the
morphogenesis of the vertebrate skeleton. Becker et al. (Dev. Biol., 1997,
187(2):298-310) provide data which suggests that Ihh is also involved in
mediating
differentiation of extraembryonic endoderm during early mouse embryogenesis.
Short limbed dwarfism, with decreased chondrocyte proliferation and extensive
hypertrophy are the results of targeted deletion of Ihh (Karp et al., 2000,
Development 127(3):543-48). The expression of Ihh mRNA and protein is
unregulated dramatically as F9 cells differentiate in response to retinoic
acid, into
either parietal endoderm or embryoid bodies, containing an outer visceral
endoderm
layer. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that
whereas
little or no Ihh message is present in blastocysts, significant levels appear
upon
subsequent days of culture, coincident with the emergence of parietal endoderm
cells.
Ubiquitin hydrolases (Cl2Lpoptosis, checkpoint integrity
Ubiquitin carboxyl-terminal hydrolases (3.1.2.15) (deubiquitinating
enzymes) are thiol proteases that recognize and hydrolyze the peptide bond at
the C-
terminal glycine of ubiquitin. These enzymes are involved in the processing of
poly-ubiquitin precursors as well as that of ubiquinated proteins. In
eukaryotic cells,
the covalent attachment of ubiquitin to proteins plays a role in a variety of
cellular
processes. In many cases, ubiquitination leads to protein degradation by the
26S
proteasome. Protein ubiquitination is reversible, and the removal of ubiquitin
is
catalyzed by deubiquitinating enzymes, or DUBs. A defect in these enzymes,
catalyzing the removal of ubiquitin from ubiquinated proteins, may be
characteristic
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of neurodegenerative diseases such as Alzheimer's, Parkinson's, progressive
supranuclear palsy, and Pick's and Kuf's disease.
Papain (C1) - cathepsins K, S and B,- bone resorbtion, Ag_processin~(Prosite
PS00139
Cathepsin K, a member of the papain family of peptidases, is involved in
osteoclastic resorption. It plays an important role in extracellular
degradation and
may have a role in disorders of bone remodeling, such as pyncodysostosis, an
autosomal recessive osteochondrodysplasia characterized by osteosclerosis and
short
stature. Antigen presentation by major histocompatibility complex (MHC) class
II
molecules requires the participation of different proteases in the endocytic
route to
degrade endocytosed antigens as well as the MHC class II-associated invariant
chain. Only cathepsin S, a member of the papain family, appears to be
essential for
complete destruction of the invariant chain. Cathepsin B is overexpressed in
tumors
of the lung, prostate, colon, breast, and stomach. Hughes et al. (PYOG. Nat.
Acad.
Sci. 95: 12410-12415, 1998) found an amplicon at 8p23-p22 that resulted in
cathepsin B overexpression in esophageal adenocarcinoma. Abundant
extracellular
expression of CTSB protein was found in 29 of 40 (72.5%) of esophageal
adenocarcinoma specimens by use of immunohistochemical analysis. The findings
were thought to support an important role for CTSB in esophageal
adenocarcinoma
and possibly in other tumors.
Cathepsin B, a lyosomal protease, is being studied as a prognostic marker in
various cancers (breast, pulmonary adenocarcinomas).
Cysteine Protease AEP
The cysteine protease AEP plays another role in the immune functions. It
has been implicated in the protease step required for antigen processing in B
cells.
(Manoury et al. Nature 396:695-699 (1998))
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Hepatitis A viral protease (C3E)
The Hepatitis A genome encodes a cysteine protease required for enzymatic
cleavages in vivo to yield mature proteins (Wang, 1999, Prog. Drug Res. 52:197-
219). This enzyme and its homologs in other viruses (such as hepatitis E
virus) are
potential targets for chemotherapeutic intervention.
3. Metalloproteases
Examples of metalloprotease protease polypeptides according to the
invention include SGPr016, r352, r050, r282, r046, r060, r068, r096, r119,
r143,
r164, r281, r075, r292, r069, r212, r049, r026, r203, r157, r154, r088 (SEQ ID
NOS:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34,
and 35 respectively). These polypeptides may have one or more of the following
activities.
Colla_ e~ n~(M10) - invasion
Matrix degradation is an essential step in the spread of cancer. The 72- and
92-kD type IV collagenases are members of a group of secreted zinc
metalloproteases which, in mammals, degrade the collagens of the extracellular
matrix. Other members of this group include interstitial collagenase and
stromelysin
(Nagase et al., 1992, Matrix Suppl. 1:421-424). By targeted disruption in
embryonic
stem cells, Vu et al. (Cell, 1998, 934:11-22) created homozygous mice with a
null
mutation in the MMP9/gelatinase B gene. These mice exhibited an abnormal
pattern
of skeletal growth plate vascularization and ossification. Growth plates from
MMP9-null mice in culture showed a delayed release of an angiogenic activator,
establishing a role for this proteinase in controlling angiogenesis.
MMP2 (gelatinase A) have been associated with the aggressiveness of
human cancers (Chenard et al., 1999, Irat. J. Cancer, 82:208-12). In a study
comparing basal cell carcinomas (BCC) with the more aggressive squamous cell
carcinomas (SCC), both MMP2 and MMP9 were expressed at a higher level in SCC
(pumas et al., 1999, Anticancer Res., 19(4B):2929-38). Additionally,
expression of
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MMP2 and MMP9 in T lymphocytes has recently been shown to be modulated by
the Ras/MAP kinase signaling pathways (Esparza et al., 1999, Blood, 94:2754-
66)
(see also, Li et al., 1998, Biochim. Biophys. Acta, 1405:110-20).
ADAMS (M12) - TNF, inflammation, growth factor processing
The ADAM peptidases are a family of proteins containing a disintegrin and
metalloproteinase (ADAM) domain (Werb and Yan, Science, 1998, 282:1279-1280).
Members of this family are cell surface proteins with a unique structure
possessing
both potential adhesion and protease domains (Primakoff and Myles, T~ehds ifZ
Genet., 2000, 16:83-87). Activity of these proteases can be linked to TNF,
inflammation, and/or growth factor processing.
ADAM proteases have also been characterized as having a pro- and
metalloproteinase domain, a disintegrin domain, a cysteine-rich region and an
EGF
repeat (Blobel, 1997, Cell, 90:589-592 which is hereby incorporated herein by
reference in its entirety including any figures, tables, or drawings). They
have been
associated with the release from the plasma membrane of numerous proteins
including Tumor Necrosis Factor-a (TNF-a), kit-ligand, TGFa, Fas-ligand,
cytokine receptors such as the Il-6 receptor and the NGF receptor, as well as
adhesion proteins such as L-selectin, and the b amyloid precursor proteins
(Blobel,
1997, Cell, 90:589-592).
Tumor necrosis factor-a is synthesized as a proinflammatory cytokine from a
233-amino acid precursor. Conversion of the membrane-bound precursor to a
secreted mature protein is mediated by a protease termed TNF-a convertase. TNF-
a
is involved in a variety of diseases. ADAM17, which contains a disintegrin and
metalloproteinase domains, is also called 'tumor necrosis factor-a converting
enzyme' (TALE) (Black et al., Nature, 1997, 385:729-33). The gene encodes an
824-amino acid polypeptide containing the features of the ADAM family: a
secretory signal sequence, a disintegrin domain, and a metalloprotease domain.
Expression studies showed that the encoded protein cleaves precursor tumor
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necrosis factor-a to its mature form. This enzyme may also play a role in the
processing of Transforming Growth Factor-a (TGF-a), as mice which lack the
gene
are similar in phenotype to those that lack TGF-a (Peschon et al., Science,
282:1281-1284).
Neprylisin 13) - Endothelin-converting enz~
Neprylisin, a metallopeptidase active in degradation of enkephalins and other
bioactive peptides, is a drug target in hypertension and renal disease
(Oefner, et al.,
J. Mol. Biol., 2000, 296:341-49).
Carboxypeptidase (M14) - Neurotransmitter processing
Caxboxypeptidases specifically remove COOH-terminal basic amino
acids(arginine or lysine) (Nesheim, 1998, Curs. Opin. Hematol. 5(5):309-13).
They
have important functions in many biologic processes, including activation,
inactivation, or modulation of peptide hormone activity, neurotransmitter
processing, and alteration of physical properties of proteins and enzymes
(Ostrowska
et al., 1998, Rocz. Akad. Med. Bialymst. 43:39-55).
Dipeptidase (M2) - ACE
Angiotensin I converting enzyme (EC 3.4.15.1 ), or kininase II, is
adipeptidyl carboxypeptidase that plays an important role in blood pressure
regulation and electrolyte balance by hydrolyzing angiotensin I into
angiotensin II, a
potent vasopressor, andaldosterone-stimulating peptide. The enzyme is also
able to
inactivate bradykinin, a potent vasodilator. Although angiotensin-converting
enzyme has been studied primarily in the context of its role in blood pressure
regulation, this widely distributed enzyme has many other physiologic
functions.
There are two forms of ACE: a testis-specific isozyme and a somatic isozyme
which
has two active centers.
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Matrix metalloproteases (M10B) - tissue remodeling and inflammation
The matrix metalloproteases (MMPs) are a family of related matrix-
degrading enzymes that are important in tissue remodeling and repair during
development and inflammation (Belotti et al., 1999, Int. J. Biol. MarkeYS
14(4):232-
38). Abnormal expression is associated with various diseases such as tumor
invasiveness (Johansson and Kahari, 2000, Histol. Histopathol. 15(1):225-37),
arthritis (Malemud et al., 1999, Front. Biosci. 4:D762-71), and
atherosclerosis
(Nagase, 1997, Biol. Chem. 378(3-4):151-60). MMP activity may also be related
to
tobacco-induced pulmonary emphysema (Dhami et al., Am. J. Respi~. Cell Mol.
Biol., 2000, 22:244-52).
SREBP Protease (M50)
The sterol regulatory element-binding proteins protease functions in the
infra-membrane proteolysis and release of sterol-regulatory binding proteins
(SREBPs) (Duncan et al., 1997, J. Biol. Chem. 272:12778-85). SREBPs activate
genes of cholesterol and fatty acid metabolism, making the SREBP protease an
attractive target for therapeutic modulation (Brown et al., 1997, Cell 89:331-
340).
Metalloprotease processin og~f .rowth factors
In addition to the processing of TGF-a described above, metalloproteases
have been directly demonstrated to be active in the processing of the
precursor of
other growth factors such as heparin-binding EGF (proHB-EFG) (Izumi et al.,
EMBO J, 1998,17:7260-72), and amphiregulin (Brown et al., 1998, J. Biol.
Chem.,
27:17258-68).
Additionally, metalloproteases have recently been shown to be instrumental
in the communication whereby stimulation of a GPCR pathway results in
stimulation of the MAP kinase pathway (Prenzel et al., 1999, Nature, 402:884-
888).
The growth factor intermediate in the pathway, HB-EGF is released by the cell
in a
proteolytic step regulated by the GPCR pathway involving an uncharacterized
metalloprotease. After release, the HB-EGF is bound by the extracellular
matrix and
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then presented to the EGF receptors on the surface, resulting in the
activation of the
MAP kinase pathway (Prenzel et al., 1999, Nature, 402:884-888).
A recent study by Gallea-Robache et al. (1997) has also implicated a
metalloprotease family displaying different substrate specificites in the
shedding of
other growth factors including macrophage colony-stimulating factor (M-CSF)
and
stem cell factor (SCF) (Gallea-Robache et al., 1997, Cytokihe 9:340-46). The
shedding of M-CSF (also known as CSF-1) has been linked to activation of
Protein
Kinase C by phorbol esters (Stein et al., 1991, Ohcogehe, 6:601-OS).
4. Serine Proteases
The serine proteases are a class which includes trypsin, kallikrein,
chymotrypsin, elastase, thrombin, tissue plasminogen activator (tPA),
urokinase
plasminogen activator (uPA), plasmin (Werb, Cell, 1997, 91:439-442),
kallikrein
(Clements, Biol. Res., 1998, 31151-59), and cathepsin G (Shamamian et al.,
SuYge~y, 2000, 127:142-47). These proteases have in common a well-conserved
catalytic triad of amino acid residues in their active site consisting of
histidine-57,
aspartic acid-102, and serine-195 (using the chymotrypsin numbering system).
Serine protease activity has been linked to coagulation and they may have use
as
tumor markers.
Serine proteases can be further subclassified by their specificity in
substrates.
The elastases prefer to cleave substrates adjacent to small aliphatic residues
such as
valine, chymases prefer to cleave near large aromatic hydrophobic residures,
and
tryptases prefer positively charged residues. One additional class of serine
protease
has been described recently which prefers to cleave adjacent to a proline.
This
prolyl endopeptidase has been implicated in the progression of memory loss in
Alzheimer's patients (Toide et al., 1998, Rev. Neurosci. 9(1):17-29).
A partial list of proteases known to belong to this large and important family
include: blood coagulation factors VII, IX, X, XI and XII; thrombin;
plasminogen;
complement components Clr, Cls, C2; complement factors B, D and I;
complement-activating component of RA-reactive factor; elastases 1, 2, 3A, 3B
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(protease E); hepatocyte growth factor activator; glandular (tissue)
kallikreins
including EGF-binding protein types A, B, and C; NGF-y chain, y-renin, and
prostate specific antigen (PSA); plasma kallikrein; mast cell proteases;
myeloblastin
(proteinase 3) (Wegener's autoantigen); plasminogen activators (urokinase-
type, and
tissue-type); and the trypsins I, II, III, and IV. These peptidases play key
roles in
coagulation, tumorigenesis, control of blood pressure, release of growth
factors, and
other roles. (http://www.babraham.co.uk/Merops/Merops.htm).
5. Threonine peptidases (T1) - (Prosite PDOC003261PDOC00668~
Proteasomal subunits (T1A)
The proteasome is a multicatalytic threonine proteinase complex involved in
ATP/ubiquitin dependent non-lysosomal proteolysis of cellular substrates. It
is
responsible for selective elimination of proteins with aberrant structures, as
well as
naturally occurring short-lived proteins related to metabolic regulation and
cell-cycle
progression (Momand et al., 2000, Gene 242(1-2):15-29, Bochtler et al., 1999,
Ahuu. Rev. Biophys Biomol St~uct.28:295-317). The proteasome inhibitor
lactacystin reversibly inhibits proliferation of human endothelial cells,
suggesting a
role for proteasomes in angiogenesis (Kumeda, et al., Ahticance~ Res. 1999 Sep-
Oct;19(SB):3961-8). Another important function of the proteasome in higher
vertebrates is to generate the peptides presented on MHC-class 1 molecules to
circulating lymphocytes (Castelli et al., 1997, Iht. J. Clih. Lab. Res.
27(2):103-10).
The proteasome has a sedimentation coefficient of 26S and is composed of a 20S
catalytic core and a 22S regulatory complex. Eukaryotic 20S proteasomes have a
molecular mass of 700 to 800 kD and consist of a set of over 15 kinds of
polypeptides of 21 to 32 kD. All eukaryotic 20S proteasome subunits can be
classified grossly into 2 subfamilies, a and [3, by their high similarity with
either the
a or (3 subunits of the archaebacterium Thermoplasma acidophilum (Mayr et al.,
1999, Biol. Chefya. 380(10):1183-92). Several of the components have been
identified as threonine peptidases, suggesting that this class of peptidases
plays a key
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role in regulating metabolic pathways and cell-cycle progression, among other
functions (Yorgin et al., 2000, J. Immuhol. 164(6):2915-23).
6. Peptidases of Unknown Catalytic Mechanism
The prenyl-protein specific protease responsible for post-translational
processing of the Ras proto-oncogene and other prenylated proteins falls into
this
class. This class also includes several viral peptidases that may play a role
in
mammalian infection, including cardiovirus endopeptidase 2A
(encephalomyocarditis virus) (Molla et al., 1993, J. Virol. 67(8):4688-95),
NS2-3
protease (hepatitis C virus) (Blight et al., 1998, Ahtivir. Ther. 3(Suppl
3):71-81),
endopeptidase (infectious pancreatic necrosis virus) (Lejal et al., J. Geh.
Virol.,
2000, 81:983-992), and the Npro endopeptidase (hog cholera virus) (Tratschin
et al.,
1998, J. Tirol. 72(9):7681-84).
Nucleic Acid Probes, Methods, and Kits for Detection of Proteases
A nucleic acid probe of the present invention may be used to probe an
appropriate chromosomal or cDNA library by usual hybridization methods to
obtain
other nucleic acid molecules of the present invention. A chromosomal DNA or
cDNA library may be prepared from appropriate cells according to recognized
methods in the art (cf. "Molecular Cloning: A Laboratory Manual", second
edition,
Cold Spring Harbor Laboratory, Sambrook, Fritsch, & Maniatis, eds., 1989).
In the alternative, chemical synthesis can be carried out in order to obtain
nucleic acid probes having nucleotide sequences which correspond to N-terminal
and C-terminal portions of the amino acid sequence of the polypeptide of
interest.
The synthesized nucleic acid probes may be used as primers in a polymerase
chain
reaction (PCR) carried out in accordance with recognized PCR techniques,
essentially according to PCR Protocols, "A Guide to Methods and Applications",
Academic Press, Michael, et al., eds., 1990, utilizing the appropriate
chromosomal
or cDNA library to obtain the fragment of the present invention.
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One skilled in the art can readily design such probes based on the sequence
disclosed herein using methods of computer alignment and sequence analysis
known
in the art ("Molecular Cloning: A Laboratory Manual", 1989, supra). The
hybridization probes of the present invention can be labeled by standard
labeling
techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-
avidin
Iabel, chemiluminescence, and the Like. After hybridization, the probes may be
visualized using known methods.
The nucleic acid probes of the present invention include RNA, as well as
DNA probes, such probes being generated using techniques known in the art. The
nucleic acid probe may be immobilized on a solid support. Examples of such
solid
supports include, but are not limited to, plastics such as polycarbonate,
complex
carbohydrates such as agarose and sepharose, and acrylic resins, such as
polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to
such solid supports are well known in the art.
The test samples suitable for nucleic acid probing methods of the present
invention include, for example, cells or nucleic acid extracts of cells, or
biological
fluids. The samples used in the above-described methods will vary based on the
assay format, the detection method and the nature of the tissues, cells or
extracts to
be assayed. Methods for preparing nucleic acid extracts of cells are well
known in
the art and can be readily adapted in order to obtain a sample which is
compatible
with the method utilized.
One method of detecting the presence of nucleic acids of the invention in a
sample comprises (a) contacting said sample with the above-described nucleic
acid
probe under conditions such that hybridization occurs, and (b) detecting the
presence
of said probe bound to said nucleic acid molecule. One skilled in the art
would
select the nucleic acid probe according to techniques known in the art as
described
above. Samples to be tested include but should not be limited to RNA samples
of
human tissue.
A kit for detecting the presence of nucleic acids of the invention in a sample
comprises at least one container means having disposed therein the above-
described
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nucleic acid probe. The kit may further comprise other containers comprising
one or
more of the following: wash reagents and reagents capable of detecting the
presence
of bound nucleic acid probe. Examples of detection reagents include, but are
not
limited to radiolabelled probes, enzymatic labeled probes (horseradish
peroxidase,
alkaline phosphatase), and affinity labeled probes (biotin, avidin, or
steptavidin).
Preferably, the kit fiuther comprises instructions for use.
In detail, a compartmentalized kit includes any kit in which reagents are
contained in separate containers. Such containers include small glass
containers,
plastic containers or strips of plastic or paper. Such containers allow the
efficient
transfer of reagents from one compartment to another compartment such that the
samples and reagents are not cross-contaminated and the agents or solutions of
each
container can be added in a quantitative fashion from one compartment to
another.
Such containers will include a container which will accept the test sample, a
container which contains the probe or primers used in the assay, containers
which
' contain wash reagents (such as phosphate buffered saline, Tris-buffers, and
the like),
and containers which contain the reagents used to detect the hybridized probe,
bound
antibody, amplified product, or the like. One skilled in the art will readily
recognize
that the nucleic acid probes described in the present invention can readily be
incorporated into one of the established kit formats which are well known in
the art.
DNA Constructs Comprising a Protease Nucleic Acid Molecule and Cells
Containing These Constructs.
The present invention also relates to a recombinant DNA molecule
comprising, 5' to 3', a promoter effective to initiate transcription in a host
cell and
the above-described nucleic acid molecules. In addition, the present invention
relates to a recombinant DNA molecule comprising a vector and an above-
described
nucleic acid molecule. The present invention also relates to a nucleic acid
molecule
comprising a transcriptional region functional in a cell, a sequence
complementary
to an RNA sequence encoding an amino acid sequence corresponding to the above-
described polypeptide, and a transcriptional termination region functional in
said
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cell. The above-described molecules may be isolated andlor purified DNA
molecules.
The present invention also relates to a cell or organism that contains an
above-described nucleic acid molecule and thereby is capable of expressing a
polypeptide. The polypeptide may be purified from cells which have been
altered to
express the polypeptide. A cell is said to be "altered to express a desired
polypeptide" when the cell, through genetic manipulation, is made to produce a
protein which it normally does not produce or which the cell normally produces
at
lower levels. One skilled in the art can readily adapt procedures for
introducing and
expressing either genomic, cDNA, or synthetic sequences into either eukaryotic
or
prokaryotic cells.
A nucleic acid molecule, such as DNA, is said to be "capable of expressing"
a polypeptide if it contains nucleotide sequences which contain
transcriptional and
translational regulatory information and such sequences are "operably linked"
to
nucleotide sequences which encode the polypeptide. An operable linkage is a
linkage in which the regulatory DNA sequences and the DNA sequence sought to
be
expressed are connected in such a way as to permit gene sequence expression.
The
precise nature of the regulatory regions needed for gene sequence expression
may
vary from organism to organism, but shall in general include a promoter region
which, in prokaryotes, contains both the promoter (which directs the
initiation of
RNA transcription) as well as the DNA sequences which, when transcribed into
RNA, will signal synthesis initiation. Such regions will normally include
those 5'-
non-coding sequences involved with initiation of transcription and
translation, such
as the TATA box, capping sequence, CART sequence, and the like.
If desired, the non-coding region 3' to the sequence encoding a protease of
the invention may be obtained by the above-described methods. This region may
be
retained for its transcriptional termination regulatory sequences, such as
termination
and polyadenylation. Thus, by retaining the 3'-region naturally contiguous to
the
DNA sequence encoding a protease of the invention, the transcriptional
termination
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signals may be provided. Where the transcriptional termination signals are not
satisfactorily functional in the expression host cell, then a 3' region
functional in the
host cell may be substituted.
Two DNA sequences (such as a promoter region sequence and a sequence
encoding a protease of the invention) are said to be operably linked if the
nature of
the linkage between the two DNA sequences allows the protease sequence to be
transcribed, i.e., where the linkage does not (1) result in the introduction
of a frame-
shift mutation, (2) interfere with the ability of the promoter region sequence
to direct
the transcription of a gene sequence encoding a protease of the invention, or
(3) interfere with the ability of the gene sequence of a protease of the
invention to be
transcribed by the promoter region sequence. Thus, a promoter region would be
operably linked to a DNA sequence if the promoter were capable of effecting
transcription of that DNA sequence. Thus, to express a gene encoding a
protease of
the invention, transcriptional and translational signals recognized by an
appropriate
host are necessary.
The present invention encompasses the expression of a gene encoding a
protease of the invention (or a functional derivative thereof) in either
prokaryotic or
eukaryotic cells. Prokaryotic hosts are, generally, very efficient and
convenient for
the production of recombinant proteins and are, therefore, one type of
preferred
expression system for proteases of the invention. Prokaryotes most frequently
are
represented by various strains of E. coli. However, other microbial strains
may also
be used, including other bacterial strains.
In prokaryotic systems, plasmid vectors that contain replication sites and
control sequences derived from a species compatible with the host may be used.
Examples of suitable plasmid vectors may include pBR322, pUC118, pUC119 and
the like; suitable phage or bacteriophage vectors may include ~,gtl0, ~,gtl l
and the
like; and suitable virus vectors may include pMAM-neo, pI~RC and the like.
Preferably, the selected vector of the present invention has the capacity to
replicate
in the selected host cell.
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Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus,
St~eptomyces, Pseudomonas, Salmonella, S'e~~atia, and the like. However, under
such conditions, the polypeptide will not be glycosylated. The prokaryotic
host
must be compatible with the replicon and control sequences in the expression
plasmid.
To express a protease of the invention (or a functional derivative thereof) in
a
prokaryotic cell, it is necessary to operably link the sequence encoding the
protease
of the invention to a functional prokaryotic promoter. Such promoters may be
either
constitutive or, more preferably, regulatable (i.e., inducible or
derepressible).
Examples of constitutive promoters include the int promoter of bacteriophage
~,, the
bla promoter of the (3-lactamase gene sequence of pBR322, and the cat promoter
of
the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
Examples of inducible prokaryotic promoters include the major right and left
promoters of bacteriophage ~, (PL and PR), the tip, recA, ~l.acZ, ~l,acl, and
gal
promoters of E. coli, the a-amylase (LJlmanen et al., J. Bacte~iol. 162:176-
182,
1985) and the S-28-specific promoters of B. subtilis (Gilman et al., Gene
Sequence
32:11-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan, in:
The
Molecular Biolo~y of the Bacilli, Academic Press, Inc., NY, 1982), and
St~eptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478, 1986).
Prokaryotic promoters are reviewed by Glick (Ind. Mic~obiot. 1:277-282, 1987),
Cenatiempo (Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet.
18:415-442, 1984).
Proper expression in a prokaryotic cell may also require the presence of a
ribosome-binding site upstream of the gene sequence-encoding sequence. Such
ribosome-binding sites are disclosed, for example, by Gold et al. (An32. Rev.
Mic~obiol. 35:365-404, 1981). The selection of control sequences, expression
vectors, transformation methods, and the like, are dependent on the type of
host cell
used to express the gene. As used herein, "cell", "cell line", and "cell
culture" may
be used interchangeably and all such designations include progeny. Thus, the
words
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"transformants" or "transformed cells" include the primary subject cell and
cultures
derived therefrom, without regard to the number of transfers. It is also
understood
that all progeny may not be precisely identical in DNA content, due to
deliberate or
inadvertent mutations. However, as defined, mutant progeny have the same
functionality as that of the originally transformed cell.
Host cells which may be used in the expression systems of the present
invention are not strictly limited, provided that they are suitable for use in
the
expression of the protease polypeptide of interest. Suitable hosts may often
include
eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast,
fungi,
insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian
cells
which may be useful as hosts include HeLa cells, cells of fibroblast origin
such as
VERO or CHO-K1, or cells of lymphoid origin and their derivatives. Preferred
mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell
lines
such as IMR 332, which may provide better capacities for correct post-
translational
processing.
In addition, plant cells are also available as hosts, and control sequences
compatible with plant cells are available, such as the cauliflower mosaic
virus 35S
and 195, and nopaline synthase promoter and polyadenylation signal sequences.
Another preferred host is an insect cell, for example the D~osophila larvae.
Using
insect cells as hosts, the D~osophila alcohol dehydrogenase promoter can be
used
(Rubin, Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can
be
engineered to express large amounts of proteases of the invention in insect
cells
(Jasny, Science 238:1653, 1987; Miller et al., in: Genetic Erzginee~i~cg, Vol.
8,
Plenum, Setlow et al., eds., pp. 277-297, 1986).
Any of a series of yeast expression systems can be utilized which incorporate
promoter and termination elements from the actively expressed sequences coding
for
glycolytic enzymes that are produced in large quantities when yeast are grown
in
mediums rich in glucose. Known glycolytic gene sequences can also provide very
efficient transcriptional control signals. Yeast provides substantial
advantages in
that it can also carry out post-translational modifications. A number of
recombinant
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DNA strategies exist utilizing strong promoter sequences and high copy number
plasmids which can be utilized for production of the desired proteins in
yeast. Yeast
recognizes leader sequences on cloned mammalian genes and secretes peptides
bearing leader sequences (i.e., pre-peptides). Several possible vector systems
are
available for the expression of proteases of the invention in a mammalian
host.
A wide variety of transcriptional and translational regulatory sequences may
be employed, depending upon the nature of the host. The transcriptional and
translational regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the
like,
where the regulatory signals are associated with a particular gene sequence
which
has a high level of expression. Alternatively, promoters from mammalian
expression products, such as actin, collagen, myosin, and the like, may be
employed.
Transcriptional initiation regulatory signals may be selected which allow for
repression or activation, so that expression of the gene sequences can be
modulated.
Of interest are regulatory signals which are temperature-sensitive so that by
varying
the temperature, expression can be repressed or initiated, or are subject to
chemical
(such as metabolite) regulation.
Expression of proteases of the invention in eukaryotic hosts requires the use
of eukaryotic regulatory regions. Such regions will, in general, include a
promoter
region sufficient to direct the initiation of RNA synthesis. Preferred
eukaryotic
promoters include, for example, the promoter of the mouse metallothionein I
gene
sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter
of
Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter
(Benoist
et al., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence
promoter (Johnston et al., P~oc. Natl. Acad. Sci. (LJSA) 79:6971-6975, 1982;
Silver
et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).
Translation of eukaryotic mRNA is initiated at the codon which encodes the
first methionine. For this reason, it is preferable to ensure that the linkage
between a
eukaryotic promoter and a DNA sequence which encodes a protease of the
invention
(or a functional derivative thereof) does not contain any intervening codons
which
CA 02408105 2002-11-O1
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are capable of encoding a methionine (i.e., AUG). The presence of such codons
results either in the formation of a fusion protein (if the AUG codon is in
the same
reading frame as the protease of the invention coding sequence) or a frame-
shift
mutation (if the AUG codon is not in the same reading frame as the protease of
the
invention coding sequence).
A nucleic acid molecule encoding a protease of the invention and an
operably linked promoter may be introduced into a recipient prokaryotic or
eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may
either
be a linear molecule or, more preferably, a closed covalent circular molecule.
Since
such molecules are incapable of autonomous replication, the expression of the
gene
may occur through the transient expression of the introduced sequence.
Alternatively, permanent expression may occur through the integration of the
introduced DNA sequence into the host chromosome.
A vector may be employed which is capable of integrating the desired gene
sequences into the host cell chromosome. Cells which have stably integrated
the
introduced DNA into their chromosomes can be selected by also introducing one
or
more markers which allow for selection of host cells which contain the
expression
vector. The marker may provide for prototrophy to an auxotrophic host, biocide
resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
The
selectable marker gene sequence can either be directly linked to the DNA gene
sequences to be expressed, or introduced into the same cell by co-
transfection.
Additional elements may also be needed for optimal synthesis of mRNA. These
elements may include splice signals, as well as transcription promoters,
enhancers,
and termination signals. cDNA expression vectors incorporating such elements
include those described by Okayama (Mol. Cell. Biol. 3:280-289, 1983).
The introduced nucleic acid molecule can be incorporated into a plasmid or
viral vector capable of autonomous replication in the recipient host. Any of a
wide
variety of vectors may be employed for this purpose. Factors of importance in
selecting a particular plasmid or viral vector include: the ease with which
recipient
cells that contain the vector may be recognized and selected from those
recipient
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cells which do not contain the vector; the number of copies of the vector
which are
desired in a particular host; and whether it is desirable to be able to
"shuttle" the
vector between host cells of different species.
Preferred prokaryotic vectors include plasmids such as those capable of
replication in E. coli (such as, for example, pBR322, ColEl, pSC101, pACYC
184,
~VX; "Molecular Cloning: A Laboratory Manual", 1989, supra). Bacillus plasmids
include pC194, pC221, pT127, and the like (Gryczan, In: The Molecular
Biolo~~of
the Bacilli, Academic Press, NY, pp. 307-329, 1982). Suitable St~eptomyces
plasmids include p1J101 (Kendall et al., J. Baete~iol. 169:4177-4183, 1987),
and
streptomyces bacteriophages such as ~C31 (Chater et al., In: Sixth
International
Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary,
pp. 45-54, 1986). Pseudomonas plasmids are reviewed by John et al. (Rev.
Infect.
Dis. 8:693-704, 1986), and Izaki (Jph. J. Bacte~iol. 33:729-742, 1978).
Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-
micron circle, and the like, or their derivatives. Such plasmids are well
known in the
art (Botstein et al., Miami Wutr. Symp. 19:265-274, 1982; Broach, In: The
Molecular Biolo~y of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach,
Cell
28:203-204, 1982; Bolton et al., J. Clin. Hematol. Ohcol. 10:39-48, 1980;
Maniatis,
In: Cell Biolo~y: A Combrehensive Treatise, Voh. 3, Gene Sequence Expression,
Academic Press, NY, pp. 563-608, 1980).
Once the vector or nucleic acid molecule containing the constructs) has been
prepared for expression, the DNA constructs) may be introduced into an
appropriate host cell by any of a variety of suitable means, i.e.,
transformation,
transfection, conjugation, protoplast fusion, ehectroporation, particle gun
technology,
calcium phosphate-precipitation, direct microinjection, and the hike. After
the
introduction of the vector, recipient cells are grown in a selective medium,
which
selects for the growth of vector-containing cells. Expression of the cloned
genes)
results in the production of a protease of the invention, or fragments
thereof. This
can take place in the transformed cells as such, or following the induction of
these
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cells to differentiate (for example, by administration of bromodeoxyuracil to
neuroblastoma cells or the like). A variety of incubation conditions can be
used to
form the peptide of the present invention. The most preferred conditions are
those
which mimic physiological conditions.
Antibodies, Hybridomas, Methods of Use and Fits for Detection of Proteases
The present invention relates to an antibody having binding affinity to a
protease of the invention. The protease polypeptide may have the amino acid
sequence selected from the group consisting of those set forth in SEQ ID
N0:36,
SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41,
SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46,
SEQ ID NO:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID NO:56,
SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61,
SEQ ID NO:62, SEQ ID N0:63, SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66,
SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and SEQ ID N0:70, or a
functional derivative thereof, or at least 9 contiguous amino acids thereof
(preferably, at least 20, 30, 35, or 40 contiguous amino acids thereof).
The present invention also relates to an antibody having specific binding
affinity to a protease of the invention. Such an antibody may be isolated by
comparing its binding affinity to a protease of the invention with its binding
affinity
to other polypeptides. Those which bind selectively to a protease of the
invention
would be chosen for use in methods requiring a distinction between a protease
of the
invention and other polypeptides. Such methods could include, but should not
be
limited to, the analysis of altered protease expression in tissue containing
other
polypeptides.
The proteases of the present invention can be used in a variety of procedures
and methods, such as for the generation of antibodies, for use in identifying
pharmaceutical compositions, and for studying DNA/protein interaction.
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The proteases of the present invention can be used to produce antibodies or
hybridomas. One skilled in the art will recognize that if an antibody is
desired, such
a peptide could be generated as described herein and used as an immunogen. The
antibodies of the present invention include monoclonal and polyclonal
antibodies, as
well fragments of these antibodies, and humanized forms. Humanized forms of
the
antibodies of the present invention may be generated using one of the
procedures
known in the art such as chimerization or CDR grafting.
The present invention also relates to a hybridoma which produces the above-
described monoclonal antibody, or binding fragment thereof. A hybridoma is an
i0 immortalized cell line which is capable of secreting a specific monoclonal
antibody.
In general, techniques for preparing monoclonal antibodies and hybridomas
are well known in the art (Campbell, Monoclonal Antibody Technolo~y:
Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers,
Amsterdam, The Netherlands, 1984; St. Groth et al., J. Immunol. Methods 35:1-
21,
1980). A~Zy animal (mouse, rabbit, and the like) which is known to produce
antibodies can be immunized with the selected polypeptide. Methods fox
immunization are well known in the art. Such methods include subcutaneous or
intraperitoneal injection of the polypeptide. One skilled in the art will
recognize that
the amount of polypeptide used for immunization will vary based on the animal
which is immunized, the antigenicity of the polypeptide and the site of
injection.
The polypeptide may be modified or administered in an adjuvant in order to
increase the peptide antigenicity. Methods of increasing the antigenicity of a
polypeptide are well known in the art. Such procedures include coupling the
antigen
with a heterologous protein (such as globulin or (3-galactosidase) or through
the
inclusion of an adjuvant during immunization.
For monoclonal antibodies, spleen cells from the immunized animals are
removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and
allowed to become monoclonal antibody producing hybridoma cells. Any one of a
number of methods well known in the art can be used to identify the hybridoma
cell
which produces an antibody with the desired characteristics. These include
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screening the hybridomas with an ELISA assay, western blot analysis, or
radioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124, 1988). Hybridomas
secreting the desired antibodies are cloned and the class and subclass are
determined
using procedures known in the art (Campbell, "Monoclonal Antibody Technolog_~
Laboratory Techniques in Biochemistry and Molecular Biolo~y", supra, 1984).
For polyclonal antibodies, antibody-containing antisera is isolated from the
immunized animal and is screened for the presence of antibodies with the
desired
specificity using one of the above-described procedures. The above-described
antibodies may be detectably labeled. Antibodies can be detectably labeled
through
the use of radioisotopes, affinity labels (such as biotin, avidin, and the
like),
enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and
the like)
fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic
atoms,
and the like. Procedures for accomplishing such labeling are well-known in the
art,
for example, see Stemberger et al., J. Histochem. CytocIZem. 18:315, 1970;
Bayer et
al., Metla. Enzym. 62:308, 1979; Engval et al., Immunol. 109:129, 1972;
Coding, J.
Immuhol. Meth. 13:215, 1976. The antibodies of the present invention may be
indirectly labelled by the use of secondary labelled antibodies, such as
labelled anti-
rabbit antibodies.The labeled antibodies of the present invention can be used
for in
vitro, in vivo, and in situ assays to identify cells or tissues which express
a specific
peptide.
The above-described antibodies may also be immobilized on a solid support.
Examples of such solid supports include plastics such as polycarbonate,
complex
carbohydrates such as agarose and sepharose, acrylic resins such as
polyacrylamide
and latex beads. Techniques for coupling antibodies to such solid supports are
well
known in the art (Weir et al., "Handbook of Experimental Immunolo~y" 4th Ed.,
Blackwell Scientific Publications, Oxford, England, Chapter 10, 1986; Jacoby
et al.,
Meth. Ehzym. 34, Academic Press, N.Y., 1974). The immobilized antibodies of
the
present invention can be used for in vitro, in vivo, and ih situ assays as
well as in
immunochromotography.
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Furthermore, one skilled in the art can readily adapt currently available
procedures, as well as the techniques, methods and kits disclosed herein with
regard
to antibodies, to generate peptides capable of binding to a specific peptide
sequence
in order to generate rationally designed antipeptide peptides (Hurby et al.,
"Apphication of Synthetic Peptides: Antisense Peptides", In Synthetic
Peptides, A
User's Guide, W.H. Freeman, NY, pp. 289-307, 1992; Kaspczak et al.,
Biochemistry
28:9230-9238, 1989).
Anti-peptide peptides can be generated by replacing the basic amino acid
residues found in the peptide sequences of the proteases of the invention with
acidic
residues, while maintaining hydrophobic and uncharged polar groups. For
example,
lysine, arginine, and/or histidine residues are replaced with aspartic acid or
glutamic
acid and glutamic acid residues are replaced by lysine, arginine or histidine.
The present invention also encompasses a method of detecting a protease
polypeptide in a sample, comprising: (a) contacting the sample with an above-
described antibody, under conditions such that immunocomplexes form, and (b)
detecting the presence of said antibody bound to the polypeptide. In detail,
the
methods comprise incubating a test sample with one or more of the antibodies
of the
present invention and assaying whether the antibody binds to the test sample.
Altered levels of a protease of the invention in a sample as compared to
normal
levels may indicate disease.
Conditions for incubating an antibody with a test sample vary. Incubation
conditions depend on the format employed in the assay, the detection methods
employed, and the type and nature of the antibody used in the assay. One
skilled in
the art will recognize that any one of the commonly available immunological
assay
formats (such as radioimmunoassays, enzyme-linked immunosorbent assays,
diffusion-based Ouchterlony, or rocket immunofluorescent assays) can readily
be
adapted to employ the antibodies of the present invention. Examples of such
assays
can be found in Chard ("An Introduction to Radioimmunoassay and Related
Techniques" Elsevier Science Publishers, Amsterdam, The Netherlands, 1986),
Bullock et al. ("Techniques in Irmnunocytochemistry," Academic Press, Orlando,
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FL Vol. 1, 1982; Vol. 2, 1983; Vol. 3, 1985), Tijssen ("Practice and Theory of
Enzyme Immunoassays: Laboratory Technidues in Biochemistry and Molecular
Biolo~y," Elsevier Science Publishers, Amsterdam, The Netherlands, 1985).
The immunological assay test samples of the present invention include cells,
protein or membrane extracts of cells, or biological fluids such as blood,
serum,
plasma, or urine. The test samples used in the above-described method will
vary
based on the assay format, nature of the detection method and the tissues,
cells or
extracts used as the sample to be assayed. Methods for preparing protein
extracts or
membrane extracts of cells are well known in the art and can readily be
adapted in
order to obtain a sample which is testable with the system utilized.
A kit contains all the necessary reagents to carry out the previously
described
methods of detection. The kit may comprise: (i) a first container means
containing
an above-described antibody, and (ii) second container means containing a
conjugate
comprising a binding partner of the antibody and a label. In another preferred
embodiment, the kit fixrther comprises one or more other containers comprising
one
or more of the following: wash reagents and reagents capable of detecting the
presence of bound antibodies.
Examples of detection reagents include, but are not limited to, labeled
secondary antibodies, or in the alternative, if the primary antibody is
labeled, the
chromophoric, enzymatic, or antibody binding reagents which are capable of
reacting with the labeled antibody. The compartmentalized kit may be as
described
above for nucleic acid probe kits. One skilled in the art will readily
recognize that
the antibodies described in the present invention can readily be incorporated
into one
of the established kit formats which are well known in the art.
Isolation of Compounds Which Interact with Proteases
The present invention also relates to a method of detecting a compound
capable of binding to a protease of the invention comprising incubating the
compound with a protease of the invention and detecting the presence of the
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compound bound to the protease. The compound may be present within a complex
mixture, for example, serum, body fluid, or cell extracts.
The present invention also relates to a method of detecting an agonist or
antagonist of protease activity or protease binding partner activity
comprising
incubating cells that produce a protease of the invention in the presence of a
compound and detecting changes in the level of protease activity or protease
binding
partner activity. The compounds thus identified would produce a change in
activity
indicative of the presence of the compound. The compound may be present within
a
complex mixture, for example, serum, body fluid, or cell extracts. Once the
compound is identified it can be isolated using techniques well known in the
art.
The present invention also encompasses a method modulating protease
associated activity in a mammal comprising administering to said mammal an
agonist or antagonist to a protease of the invention in an amount sufficient
to effect
said modulation. A method of treating diseases in a mammal with an agonist or
antagonist of the activity of one of the proteases of the invention comprising
administering the agonist or antagonist to a mammal in an amount sufficient to
agonize or antagonize protease-associated functions is also encompassed in the
present application.
In an effort to discover novel treatments for diseases, biomedical researchers
and chemists have designed, synthesized, and tested molecules that inhibit the
function of proteases. Some small organic molecules form a class of compounds
that modulate the function of protein proteases.
Examples of molecules that have been reported to inhibit the function of
protein proteases include, but are not limited to, phenylmethylsulfonyl
fluoride
(PMSF), diisopropylfluorophosphate (DFP) (chapter 3, Barrett et al., Handbook
of
Proteolytic Enzymes, 1998, Academic Press, San Diego), 3,4-dichloroisocoumarin
(DCI) (Id., chapter 16), serpins (Id., chapter 37), E-64 (tratas-epoxysuccinyl
L-
leucylamido-(4-guanidino) butane) (Id., chapter 188), peptidyl-diazomethanes,
peptidyl-O-acyl-hydroxamates, epoxysuccinyl-peptides (Id., chapter 210), DAN,
EPNP (1,2-epoxy-3(p-nitrophenoxy)propane) (Id., chapter 298), thiorphan (dl-3-
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Mercapto-2-benzylpropanoyl-glycine) (Id., chapter 362), CGS 26303, PD 069185
(Id., chapter 363), and COT989-00 (N-4-hydroxy-Nl-[1-(s)-(4-
aminosulfonyl)phenylethyl-aminocarboxyl-2-cyclohexylethyl)-2R-[4-
methyl)phenylpropyl]succinamide) (Id., chapter 401). Other protease inhibitors
include, but are not limited to, aprotinin, amastatin, antipain, calcineurin
autoinhibitory fragment, and histatin 5 (Id.). Preferably, these inhibitors
will have
molecular weights from 100 to 200 daltons, from 200 to 300 daltons, from 300
to
400 daltons, from 400 to 600 daltons, from 600 to 1000 daltons, from 1000 to
2000
daltons, from 2000 to 4000 daltons, and from 4000 to 8000 daltons.
Compounds that can traverse cell membranes and are resistant to acid
hydrolysis are potentially advantageous as therapeutics as they can become
highly
bioavailable after being administered orally to patients. However, many of
these
protease inhibitors only weakly inhibit the function of proteases. In
addition, many
inhibit a variety of proteases and will therefore cause multiple side-effects
as
therapeutics for diseases.
Trans~enic Animals.
A variety of methods are available for the production of transgenic animals
associated with this invention. DNA can be injected into the pronucleus of a
fertilized egg before fusion of the male and female pronuclei, or injected
into the
nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo)
following the
initiation of cell division (Brinster et al., P~oc. Nat. Acad. Sci. USA
82:4438-4442,
1985). Embryos can be infected with viruses, especially retroviruses, modified
to
carry inorganic-ion receptor nucleotide sequences of the invention.
Pluripotent stem cells derived from the inner cell mass of the embryo and
stabilized in culture can be manipulated in culture to incorporate nucleotide
sequences of the invention. A transgenic animal can be produced from such
cells
through implantation into a blastocyst that is implanted into a foster mother
and
allowed to come to term. Animals suitable for transgenic experiments can be
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obtained from standard commercial sources such as Charles River (Wilmington,
MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, III, etc.
The procedures for manipulation of the rodent embryo and for microinjection
of DNA into the pronucleus of the zygote are well known to those of ordinary
skill
in the art (Hogan et al., supra). Microinjection procedures for fish,
amphibian eggs
and birds axe detailed in Houdebine and Chourrout (Experieutia 47:897-905,
1991).
Other procedures for introduction of DNA into tissues of animals are described
in
U.S. Patent No. 4,945,050 (Sanford et al., July 30, 1990).
By way of example only, to prepare a transgenic mouse, female mice are
induced to superovulate. Females are placed with males, and the mated females
are
sacrificed by C02 asphyxiation or cervical dislocation and embryos are
recovered
from excised oviducts. Surrounding cumulus cells are removed. Pronuclear
embryos are then washed and stored until the time of injection. Randomly
cycling
adult female mice are paired with vasectomized males. Recipient females are
mated
at the same time as donor females. Embryos then are transferred surgically.
The
procedure for generating transgenic rats is similar to that of mice (Hammer et
al.,
Cell 63:1099-1112, 1990).
Methods for the culturing of embryonic stem (ES) cells and the subsequent
production of transgenic animals by the introduction of DNA into ES cells
using
methods such as electroporation, calcium phosphate/DNA precipitation and
direct
injection also are well known to those of ordinary skill in the art
(Teratocarcinomas
and Embryonic Stem Cells, A Practical Approach, E.J. Robertson, ed., IRL
Press,
1987).
In cases involving random gene integration, a clone containing the
sequences) of the invention is co-transfected with a gene encoding resistance.
Alternatively, the gene encoding neomycin resistance is physically linked to
the
sequences) of the invention. Transfection and isolation of desired clones are
carried
out by any one of several methods well known to those of ordinary skill in the
art
(E.J. Robertson, supra).
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DNA molecules introduced into ES cells can also be integrated into the
chromosome through the process of homologous recombina-tion (Capecchi, Science
244:1288-1292, 1989). Methods for positive selection of the recombination
event
(i.e., neo resistance) and dual positive-negative selection (i.e., neo
resistance and
gancyclovir resistance) and the subsequent identification of the desired
clones by
PCR have been described by Capecchi, supra and Joyner et al. (Nature 338:153-
156, 1989), the teachings of which are incorporated herein in their entirety
including
any drawings. The final phase of the procedure is to inject targeted ES cells
into
blastocysts and to transfer the blastocysts into pseudopregnant females. The
resulting chimeric animals are bred and the offspring are analyzed by Southern
blotting to identify individuals that carry the transgene. Procedures for the
production of non-rodent mammals and other animals have been discussed by
others
(Houdebine and Chourrout, supra; Pursel et al., Science 244:1281-1288, 1989;
and
Simms et al., BiolTechnology 6:179-183, 1988).
Thus, the invention provides transgenic, nonhuman mammals containing a
transgene encoding a protease of the invention or a gene affecting the
expression of
the protease. Such transgenic nonhuman mammals are particularly useful as an
in
vivo test system for studying the effects of introduction of a protease, or
regulating
the expression of a protease (i.e., through the introduction of additional
genes,
antisense nucleic acids, or ribozymes).
A "transgenic animal" is an animal having cells that contain DNA which has
been artificially inserted into a cell, which DNA becomes part of the genome
of the
animal which develops from that cell. Preferred transgenic animals are
primates,
mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. The transgenic
DNA may
encode human proteases. Native expression in an animal may be reduced by
providing an amount of antisense RNA or DNA effective to reduce expression of
the
receptor.
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Gene Theraby
Proteases or their genetic sequences will also be useful in gene therapy
(reviewed in Miller, Nature 357:455-460, 1992). Miller states that advances
have
resulted in practical approaches to human gene therapy that have demonstrated
positive initial results. The basic science of gene therapy is described in
Mulligan
(Sciehce 260:926-931, 1993).
In one preferred embodiment, an expression vector containing a protease
coding sequence is inserted into cells, the cells are grown ih vitro and then
infused in
large numbers into patients. In another preferred embodiment, a DNA segment
containing a promoter of choice (for example a strong promoter) is transferred
into
cells containing an endogenous gene encoding proteases of the invention in
such a
manner that the promoter segment enhances expression of the endogenous
protease
gene (for example, the promoter segment is transferred to the cell such that
it
becomes directly linked to the endogenous protease gene).
The gene therapy may involve the use of an adenovirus containing protease
cDNA targeted to a tumor, systemic protease increase by implantation of
engineered
cells, injection with protease-encoding virus, or injection of naked protease
DNA
into appropriate tissues.
Target cell populations may be modified by introducing altered forms of one
or more components of the protein complexes in order to modulate the activity
of
such complexes. For example, by reducing or inhibiting a complex component
activity within target cells, an abnormal signal transduction events) leading
to a
condition may be decreased, inhibited, or reversed. Deletion or missense
mutants of
a component, that retain the ability to interact with other components of the
protein
complexes but cannot function in signal transduction, may be used to inhibit
an
abnormal, deleterious signal transduction event.
Expression vectors derived from viruses such as retroviruses, vaccinia virus,
adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or
bovine
papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA)
encod-ing recombinant protease of the invention protein into the targeted cell
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population (e.g., tumor cells). Methods which are well known to those skilled
in the
art can be used to construct recombinant viral vectors containing coding
sequences
(Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, N.Y., 1989; Ausubel et al., Current Protocols in Molecular
Biolo~y,
Greene Publishing Associates and Wiley Interscience, N.Y., 1989).
Alternatively,
recombinant nucleic acid molecules encoding protein sequences can be used as
naked DNA or in a reconstituted system e.g., liposomes or other lipid systems
for
delivery to target cells (e.g., Felgner et al., NatuYe 337:387-8, 1989).
Several other
methods for the direct transfer of plasmid DNA into cells exist for use in
human
gene therapy and involve targeting the DNA to receptors on cells by complexing
the
plasmid DNA to proteins (Miller, supra).
In its simplest form, gene transfer can be performed by simply injecting
minute amounts of DNA into the nucleus of a cell, through a process of
microinjection (Capecchi, Cell 22:479-88, 1980). Once recombinant genes are
introduced into a cell, they can be recognized by the cell's normal mechanisms
for
transcription and translation, and a gene product will be expressed. Other
methods
have also been attempted for introducing DNA into larger numbers of cells.
These
methods include: transfection, wherein DNA is precipitated with calcium
phosphate
and taken into cells by pinocytosis (Chen et al., Mol. Cell Biol. 7:2745-52,
1987);
electroporation, wherein cells are exposed to large voltage pulses to
introduce holes
into the membrane (Chu et al., Nucleic Acids Res. 15:1311-26, 1987);
lipofection/liposome fusion, wherein DNA is packaged into lipophilic vesicles
which fuse with a target cell (Felgner et al., Proc. Natl. Acad. Sci. USA.
84:7413-
7417, 1987); and particle bombardment using DNA bound to small projectiles
(Yang et al., P~oc. Natl. Acad. Sci. 87:9568-9572, 1990). Another method for
introducing DNA into cells is to couple the DNA to chemically modified
proteins.
It has also been shown that adenovirus proteins are capable of destabilizing
endosomes and enhancing the uptake of DNA into cells. The admixture of
adenovirus to solutions containing DNA complexes, or the binding of DNA to
polylysine covalently attached to adenovirus using protein crosslinking agents
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substantially improves the uptake and expression of the recombinant gene
(Curiel et
al., Am. J. Respir. Cell. Mol. Biol., 6:247-52, 1992).
As used herein "gene transfer" means the process of introducing a foreign
nucleic acid molecule into a cell. Gene transfer is commonly performed to
enable
the expression of a particular product encoded by the gene. The product may
include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active
RNA. Gene transfer can be performed in cultured cells or by direct
administration
into animals. Generally gene transfer involves the process of nucleic acid
contact
with a target cell by non-specific or receptor mediated interactions, uptake
of nucleic
acid into the cell through the membrane or by endocytosis, and release of
nucleic
acid into the cyto-plasm from the plasma membrane or endosome. Expression may
require, in addition, movement of the nucleic acid into the nucleus of the
cell and
binding to appropriate nuclear factors for transcription.
As used herein "gene therapy" is a form of gene transfer and is included
within the defiiution of gene transfer as used herein and specifically refers
to gene
transfer to express a therapeutic product from a cell ih vivo or ih vitro.
Gene transfer
can be performed ex vivo on cells which are then transplanted into a patient,
or can
be performed by direct administration of the nucleic acid or nucleic acid-
protein
complex into the patient.
In another preferred embodiment, a vector having nucleic acid sequences
encoding a protease polypeptide is provided in which the nucleic acid sequence
is
expressed only in specific tissue. Methods of achieving tissue-specific gene
expression are set forth in International Publication No. WO 93/09236, filed
November 3, 1992 and published May 13, 1993.
In all of the preceding vectors set forth above, a further aspect of the
invention is that the nucleic acid sequence contained in the vector may
include
additions, deletions or modifications to some or all of the sequence of the
nucleic
acid, as defined above.
Expression, including over-expression, of a protease polypeptide of the
invention can be inhibited by administration of an antisense molecule that
binds to and
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inhibits expression of the mRNA encoding the polypeptide. Alternatively,
expression
can be inhibited in an analogous manner using a ribozyme that cleaves the
mRNA.
General methods of using antisense and ribozyme technology to control gene
expression, or of gene therapy methods for expression of an exogenous gene in
this
manner are well known in the art. Each of these methods utilizes a system,
such as a
vector, encoding either an antisense or ribozyme transcript of a protease
polypeptide of
the invention.
The term "ribozyme" refers to an RNA structure of one or more RNAs
having catalytic properties. Ribozymes generally exhibit endonuclease, ligase
or
polymerase activity. Ribozyrnes are structural RNA molecules which mediate a
number of RNA self cleavage reactions. Various types of trans-acting
ribozymes,
including "hammerhead" and "hairpin" types, which have different secondary
structures, have been identified. A variety of ribozymes have been
characterized.
See, for example, U.S. Pat. Nos. 5,246,921, 5,225,347, 5,225,337 and
5,149,796.
Mixed ribozymes comprising deoxyribo and ribooligonucleotides with catalytic
activity have been described. Perreault, et al., Nature, 344:565-567 (1990).
As used herein, "antisense" refers of nucleic acid molecules or their
derivatives which specifically hybridize, e.g., bind, under cellular
conditions, with
the genomic DNA and/or cellular mRNA encoding a protease polypeptide of the
invention, so as to inlubit expression of that protein, for example, by
inhibiting
transcription and/or translation. The binding may be by conventional base pair
complementarity, or, for example, in the case of binding to DNA duplexes,
through
specific interactions in the major groove of the double helix.
In one aspect, the antisense construct is an nucleic acid which is generated
ex
vivo and that, when introduced into the cell, can inhibit gene expression by,
without
limitation, hybridizing with the mRNA and/or genomic sequences of a protease
polynucleotide of the invention.
Antisense approaches can involve the design of oligonucleotides (either
DNA or RNA) that are complementary to protease polypeptide mRNA and are
based on the protease polynucleotides of the invention, including SEQ m NO:1,
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SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID NO:6, SEQ
ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID
N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID
N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID NO:25, SEQ ID N0:26, SEQ ID
N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID NO:31, SEQ ID
N0:32, SEQ ID N0:33, SEQ ID N0:34, and SEQ ID N0:35. The antisense
oligonucleotides will bind to the protease polypeptide mRNA transcripts and
prevent
translation.
Although absolute complementarity is preferred, it is not required. A
sequence "complementary" to a portion of an RNA, as referred to herein, means
a
sequence having sufficient complementarity to be able to hybridize with the
RNA,
forming a stable duplex; in the case of double-stranded antisense nucleic
acids, a
single strand of the duplex DNA may thus be tested, or triplex formation may
be
assayed. The ability to hybridize will depend on both the degree of
complementarity
and the length of the antisense nucleic acid. Generally, the longer the
hybridizing
nucleic acid, the more base mismatches with an RNA it may contain and still
form a
stable duplex (or triplex, as the case may be). One skilled in the art can
ascertain a
tolerable degree of mismatch by use of standard procedures to determine the
melting
point of the hybridized complex.
In general, oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the AUG
initiation
codon, should work most efficiently at inhibiting translation. However,
sequences
complementary to the 3' untranslated sequences of mRNAs have been shown to be
effective at inhibiting translation of mRNAs as well. (Wagner, R. (1994)
Nature
372:333). Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could be used in accordance with
the
invention. Whether designed to hybridize to the 5', 3' or coding region of the
protease polypeptide mRNA, antisense nucleic acids should be at least six
nucleotides in length, and are preferably less than about 100 and more
preferably
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less than about 50 or 30 nucleotides in length. Typically they should be
between 10
and 25 nucleotides in length. Such principles will inform the practitioner in
selecting the appropriate oligonucleotides In preferred embodiments, the
antisense
sequence is selected from an oligonucleotide sequence that comprises, consists
of, or
consists essentially of about 10-30, and more preferably 15-25, contiguous
nucleotide bases of a nucleic acid sequence selected from the group consisting
of
SEQ ID NO:l, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ
ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID
NO:16, SEQ ID N0:17, SEQ ID NO:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID
N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID
N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, and SEQ ID N0:35 or
domains thereof.
In another preferred embodiment, the invention includes an isolated,
enriched or purified nucleic acid molecule comprising, consisting of or
consisting
essentially of about 10-30, and more preferably 15-25 contiguous nucleotide
bases
of a nucleic acid sequence that encodes a polypeptide that selected from the
group
consisting of SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ
ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ
ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ
ID N0:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ
ID N0:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ
ID N0:60, SEQ 117 N0:61, SEQ ID NO:62, SEQ ID N0:63, SEQ ID N0:64, SEQ
ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, and
SEQ ID NO:70.
Using the sequences of the present invention, antisense oligonucleotides can
be designed. Such antisense oligonucleotides would be administered to cells
expressing the target protease and the levels of the target RNA or protein
with that
of an internal control RNA or protein would be compared. Results obtained
using
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the antisense oligonucleotide would also be compared with those Qbtained using
a
suitable control oligonucleotide. A preferred control oligonucleotide is an
oligonucleotide of approximately the same length as the test oligonucleotide.
Those
antisense oligonucleotides resulting in a reduction in levels of target RNA or
protein
would be selected.
The oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or double-stranded.
The
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate
backbone, for example, to improve stability of the molecule, hybridization,
etc. The
oligonucleotide may include other appended groups such as peptides (e.g., for
targeting host cell receptors ih vivo), or agents facilitating transport
across the cell
membrane (see, e.g., Letsinger et al. (1989) P~oc. Natl. Acad. Sci. U.S.A.
86:6553-
6556; Lemaitre et al. (1987) P~oc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO 88/09810, published Dec. 15, 1988) or the blood-brain
barrier
(see, e.g., PCT Publication No. WO 89/10134, published Apr. 25, 1988),
hybridization-triggered cleavage agents. (See, e.g., Krol et al. (1988)
BioTechniques
6:958-976) or intercalating agents. (See, e.g, Zon (1988) Pha~m. Res. 5:539-
549).
To this end, the oligonucleotide may be conjugated to another molecule, e.g.,
a
peptide, hybridization triggered cross-linking agent, transport agent,
hybridization
triggered cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base
moiety which is selected from moieties such as 5-fluorouracil, 5-bromouracil,
5-
chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, and 5-
(carboxyhydroxyethyl) uracil. The antisense oligonucleotide may also comprise
at
least one modified sugar moiety selected from the group including but not
limited to
arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least
one modified phosphate backbone selected from the group consisting of a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
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phosphotriester, and a formacetal or analog thereof. (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775)
In yet a further embodiment, the antisense oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual (3-units, the
strands run parallel to each other (Gautier et al. (1987) Nucl. Acids Res.
15:6625-
6641). The oligonucleotide is a 2'-0-methylribonucleotide (moue et al. (1987)
Nucl.
Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (moue et al. (1987)
FEBS Lett. 215:327-330).
Also suitable are peptidyl nucleic acids, which are polypeptides such as
polyserine, polythreonine, etc. including copolymers containing various amino
acids, which are substituted at side-chain positions with nucleic acids
(T,A,G,C,U).
Chains of such polymers are able to hybridize through complementary bases in
the
same manner as natural DNA/RNA.. Alternatively, an antisense construct of the
present invention can be delivered, for example, as an expression plasmid or
vector
that, when transcribed in the cell, produces RNA complementary to at least a
unique
portion of the cellular mRNA which encodes a protease polypeptide of the
invention.
While antisense nucleotides complementary to the protease polypeptide
coding region sequence can be used, those complementary to the transcribed
untranslated region are most preferred.
In another preferred embodiment, a method of gene replacement is set forth.
"Gene replacement" as used herein means supplying a nucleic acid sequence
which
is capable of being expressed in vivo in an animal and thereby providing or
augmenting the function of an endogenous gene which is missing or defective in
the
animal.
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Pharmaceutical Formulations And Routes Of Administration
The compounds described herein, including protease polypeptides of the
invention, antisense molecules, ribozymes, and any other compound that
modulates
the activity of a protease polypeptide of the invention, can be administered
to a
human patient peg se, or in pharmaceutical compositions where it is mixed with
other active ingredients, as in combination therapy, or suitable carriers or
excipient(s). Techniques for formulation and administration of the compounds
of
the instant application may be found in "Remington's Pharmaceutical Sciences,"
Mack Publishing Co., Easton, PA, latest edition.
A. Routes Of Administration
Suitable routes of administration may, for example, include oral, rectal,
transmucosal, or intestinal administration; parenteral delivery, including
intramuscular, subcutaneous, intravenous, intramedullary injections, as well
as
intrathecal, direct intraventricular, intraperitoneal, intranasal, or
intraocular
inj ections.
Alternately, one may administer the compound. in a local rather than
systemic manner, for example, via injection of the compound directly into a
solid
tumor, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system,
for example, in a liposome coated with tumor-specific antibody. The liposomes
will
be targeted to and taken up selectively by the tumor.
B. Composition/Formulation
The pharmaceutical compositions of the present invention may be
manufactured in a manner that is itself known, e.g., by means of conventional
mixing, dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present
invention thus may be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and auxiliaries
which
facilitate processing of the active compounds into preparations which can be
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pharmaceutically. Proper formulation is dependent upon the route of
administration
chosen.
For injection, the agents of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hanks's
solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation.
Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by
combining the active compounds with pharmaceutically acceptable carriers well
known in the art. Such carriers enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries,
suspensions and the like, for oral ingestion by a patient to be treated.
Suitable
Garners include excipients such as, fillers such as sugars, including lactose,
sucrose,
mamutol, or sorbitol; cellulose preparations such as, for example, maize
starch,
wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose,
hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added,
such as
the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as
sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally contain gum
arabic,
talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium
dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active compound
doses.
Pharmaceutical preparations which can be used orally include push-fit
capsules made of gelatin, as well as soft, sealed capsules made of gelatin and
a
plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain
the active
ingredients in admixture with filler such as lactose, binders such as
starches, and/or
lubricants such as talc or magnesium stearate and, optionally, stabilizers. In
soft
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capsules, the active compounds may be dissolved or suspended in suitable
liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition,
stabilizers may be added. All formulations for oral administration should be
in
dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present invention are conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a
pressurized aerosol the dosage unit may be determined by providing a valve to
deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in
an
inhaler or insufflator may be formulated containing a powder mix of the
compound
and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion. Formulations for
injection
may be presented in unit dosage form, e.g., in ampoules or in mufti-dose
containers,
with an added preservative. The compositions may take such forms as
suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form. Additionally,
suspensions
of the active compounds may be prepared as appropriate oily injection
suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as sesame
oil, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides, or
liposomes.
Aqueous injection suspensions may contain substances which increase the
viscosity
of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or
dextran.
Optionally, the suspension may also contain suitable stabilizers or agents
which
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increase the solubility of the compounds to allow for the preparation of
highly
concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., contaiiung conventional suppository
bases
such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may
also be formulated as a depot preparation. Such long acting formulations may
be
administered by implantation (for example subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds may be formulated
with
suitable polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble derivatives,
for
example, as a sparingly soluble salt.
A pharmaceutical Garner for the hydrophobic compounds of the invention is
a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-
miscible organic polymer, and an aqueous phase. The cosolvent system may be
the
VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of
the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made
up
to volume in absolute ethanol. The VPD co-solvent system (VPD:DSVV~ consists
of
VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system
dissolves hydrophobic compounds well, and itself produces low toxicity upon
systemic administration. Naturally, the proportions of a co-solvent system may
be
varied considerably without destroying its solubility and toxicity
characteristics.
Furthermore, the identity of the co-solvent components may be varied: for
example,
other low-toxicity nonpolar surfactants may be used instead of polysorbate 80;
the
fraction size of polyethylene glycol may be varied; other biocompatible
polymers
may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars
or
polysaccharides may substitute for dextrose.
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Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds may be employed. Liposomes and emulsions are well known examples
of delivery vehicles or carriers for hydrophobic drugs. Certain organic
solvents such
as dimethylsulfoxide also may be employed, although usually at the cost of
greater
toxicity. Additionally, the compounds may be delivered using a sustained-
release
system, such as semipermeable matrices of solid hydrophobic polymers
containing
the therapeutic agent. Various sustained-release materials have been
established and
are well known by those skilled in the art. Sustained-release capsules may,
depending on their chemical nature, release the compounds for a few weeks up
to
over 100 days. Depending on the chemical nature and the biological stability
of the
therapeutic reagent, additional strategies for protein stabilization may be
employed.
The pharmaceutical compositions also may comprise suitable solid or gel
phase carriers or excipients. Examples of such carriers or excipients include
but are
not limited to calcium carbonate, calcium phosphate, various sugars, starches,
cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the protease modulating compounds of the invention may be
provided as salts with pharmaceutically compatible counterions.
Pharmaceutically
compatible salts may be formed with many acids, including but not limited to
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts
tend to be
more soluble in aqueous or other protonic solvents that are the corresponding
free
base forms.
C. Effective Dosage
Pharmaceutical compositions suitable for use in the present invention include
compositions where the active ingredients are contained in an amount effective
to
achieve its intended purpose. More specifically, a therapeutically effective
amount
means an amount of compound effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subj ect being treated.
Determination of a therapeutically effective amount is well within the
capability of
those skilled in the art, especially in light of the detailed disclosure
provided herein.
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For any compound used in the methods of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays. For
example, a
dose can be formulated in animal models to achieve a circulating concentration
range that includes the ICSO as determined in cell culture (i.e., the
concentration of
the test compound which achieves a half maximal inhibition of the protease
activity). Such information can be used to more accurately determine useful
doses
in humans.
Toxicity and therapeutic efficacy of the compounds described herein can be
determined by standard pharmaceutical procedures in cell cultures or
experimental
animals, e.g., for determining the LDSO (the dose lethal to 50% of the
population)
and the EDSO (the dose therapeutically effective in 50% of the population).
The dose
ratio between toxic and therapeutic effects is the therapeutic index and it
can be
expressed as the ratio between LDso and EDso. Compounds which exhibit high
therapeutic indices are preferred. The data obtained from these cell culture
assays
and animal studies can be used in formulating a range of dosage for use in
human.
The dosage of such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity. The dosage
may vary
within this range depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration and
dosage
can be chosen by the individual physician in view of the patient's condition.
(See
e.g., Fingl et al., 1975, in The Pharmacological Basis of Therapeutics, Ch. 1
p.1).
Dosage amount and interval may be adjusted individually to provide plasma
levels of the active moietywhich are sufficient to maintain the protease
modulating
effects, or minimal effective concentration (MEC). The MEC will vary for each
compound but can be estimated from in vitro data; e.g., the concentration
necessary
to achieve 50-90% inhibition of the protease using the assays described
herein.
Dosages necessary to achieve the MEC will depend on individual characteristics
and
route of administration. However, HPLC assays or bioassays can be used to
determine plasma concentrations.
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Dosage intervals can also be determined using MEC value. Compounds
should be administered using a regimen which maintains plasma levels above the
MEC for 10-90% of the time, preferably between 30-90% and most preferably
between 50-90%.
In cases of local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the
subject being treated, on the subject's weight, the severity of the
affliction, the
manner of administration and the judgment of the prescribing physician.
D. Packaging
The compositions may, if desired, be presented in a pack or dispenser device
which may contain one or more unit dosage forms containing the active
ingredient.
The pack may for example comprise metal or plastic foil, such as a blister
pack. The
pack or dispenser device may be accompanied by instructions for
administration.
The pack or dispenser may also be accompanied with a notice associated with
the
container in form prescribed by a governmental agency regulating the
manufacture,
use, or sale of pharmaceuticals, which notice is reflective of approval by the
agency
of the form of the polynucleotide for human or veterinary administration. Such
notice, for example, may be the labeling approved by the U.S. Food and Drug
Administration for prescription drugs, or the approved product insert.
Compositions
comprising a compound of the invention formulated in a compatible
pharmaceutical
carrier may also be prepared, placed in an appropriate container, and labeled
for
treatment of an indicated condition. Suitable conditions indicated on the
label may
include treatment of a tumor, inhibition of angiogenesis, treatment of
fibrosis,
diabetes, and the like.
Functional Derivatives
Also provided herein axe functional derivatives of a polypeptide or nucleic
acid of the invention. By "functional derivative" is meant a "chemical
derivative,"
"fragment," or "variant," of the polypeptide or nucleic acid of the invention,
which
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terms are defined below. A functional derivative retains at least a portion of
the
function of the protein, for example reactivity with an antibody specific for
the
protein, enzymatic activity or binding activity mediated through noncatalytic
domains, which permits its utility in accordance with the present invention.
It is
well known in the art that due to the degeneracy of the genetic code numerous
different nucleic acid sequences can code for the same amino acid sequence.
Equally, it is also well known in the art that conservative changes in amino
acid can
be made to arrive at a protein or polypeptide that retains the functionality
of the
original. In both cases, all permutations are intended to be covered by this
disclosure.
Included within the scope of this invention are the functional equivalents of
the herein-described isolated nucleic acid molecules. The degeneracy of the
genetic
code permits substitution of certain codons by other codons that specify the
same
amino acid and hence would give rise to the same protein. The nucleic acid
sequence can vary substantially since, with the exception of methionine and
tryptophan, the known amino acids can be coded for by more than one codon.
Thus,
portions or all of the genes of the invention could be synthesized to give a
nucleic
acid sequence significantly different from one selected from the group
consisting of
those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ
ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID
NO:10, SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID
NO:15, SEQ ID N0:16, SEQ 117 N0:17, SEQ ID NO:18, SEQ ID N0:19, SEQ ID
N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID
N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID
N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, and SEQ
ID N0:35. The encoded amino acid sequence thereof would, however, be
preserved.
In addition, the nucleic acid sequence may comprise a nucleotide sequence
which results from the addition, deletion or substitution of at least one
nucleotide to
the 5'-end and/or the 3'-end of the nucleic acid formula selected from the
group
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consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ
ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID
N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID
N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID
N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID
N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:34, and SEQ ID N0:35, or a derivative thereof. Any nucleotide or
polynucleotide may be used in this regard, provided that its addition,
deletion or
substitution does not alter the amino acid sequence selected from the group
consisting of those set forth in SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38,
SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID NO:43,
SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48,
SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ m N0:53,
SEQ ID N0:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID N0:57, SEQ ID NO:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63,
SEQ ID N0:64, SEQ ID N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68,
SEQ ID N0:69, and SEQ ID N0:70 which is encoded by the nucleotide sequence.
For example, the present invention is intended to include any nucleic acid
sequence
resulting from the addition of ATG as an initiation codon at the 5'-end of the
inventive nucleic acid sequence or its derivative, or from the addition of
TTA, TAG
or TGA as a termination codon at the 3'-end of the inventive nucleotide
sequence or
its derivative. Moreover, the nucleic acid molecule of the present invention
may, as
necessary, have restriction endonuclease recognition sites added to its 5'-end
and/or
3'-end.
Such functional alterations of a given nucleic acid sequence afford an
opportunity to promote secretion and/or processing of heterologous proteins
encoded
by foreign nucleic acid sequences fused thereto. All variations of the
nucleotide
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sequence of the protease genes of the invention and fragments thereof
permitted by
the genetic code are, therefore, included in this invention.
Further, it is possible to delete colons or to substitute one or more colons
with colons other than degenerate colons to produce a structurally modified
polypeptide, but one which has substantially the same utility or activity as
the
polypeptide produced by the unmodified nucleic acid molecule. As recognized in
the art, the two polypeptides are functionally equivalent, as are the two
nucleic acid
molecules that give rise to their production, even though the differences
between the
nucleic acid molecules are not related to the degeneracy of the genetic code.
A "chemical derivative" of the complex contains additional chemical
moieties not normally a part of the protein. Covalent modifications of the
protein or
peptides are included within the scope of this invention. Such modifications
may be
introduced into the molecule by reacting targeted amino acid residues of the
peptide
with an organic derivatizing agent that is capable of reacting with selected
side
chains or terminal residues, as described below.
Cysteinyl residues most commonly are reacted with a-haloacetates (and
corresponding amines), such as chloroacetic acid or chloroacetamide, to give
carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone, chloroacetyl phosphate, N-
alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-
chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-
oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylprocarbonate at pH
5.5-7.0 because this agent is relatively specific for the histidyl side chain.
Para-
bromophenacyl bromide also is useful; the reaction is preferably performed in
0.1 M
sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues are reacted with succinic or other
carboxylic acid anhydrides. Derivatization with these agents has the effect of
reversing the charge of the lysinyl residues. Other suitable reagents for
derivatizing
primary amine containing residues include imidoesters such as methyl
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picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and
trausaminase-
catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional
reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and
ninhydrin. Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pKa of the guanidine
functional
group. Furthermore, these reagents may react with the groups of lysine as well
as
the arginine a-amino group.
Tyrosyl residues are well-known targets of modification for introduction of
spectral labels by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are
used to form O-acetyl tyrosyl species and 3-vitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by
reaction with carbodiimide (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-
morpholinyl(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)
carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to
asparaginyl and glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues are frequently deamidated to the
corresponding glutamyl and aspartyl residues. Alternatively, these residues
are
deamidated under mildly acidic conditions. Either form of these residues falls
within the scope of this invention.
Derivatization with bifunctional agents is useful, for example, for cross-
linking the component peptides of the protein to each other or to other
proteins in a
complex to a water-insoluble support matrix or to other macromolecular
Garners.
Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-
2-
phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters
with 4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl
esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional
maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[p-
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azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that
are
capable of forming crosslinks in the presence of light. Alternatively,
reactive water-
insoluble matrices such as cyanogen bromide-activated carbohydrates and the
reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016;
4,195,128;
4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Other modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of Beryl or threonyl residues, methylation
of the
a-amino groups of lysine, arginine, and histidine side chains (Creighton,
T.E.,
Proteins: Structure and Molecular Pro erties, W.H. Freeman & Co., San
Francisco,
pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some
instances,
amidation of the C-terminal carboxyl groups.
Such derivatized moieties may improve the stability, solubility, absorption,
biological half life, and the like. The moieties may alternatively eliminate
or
attenuate any undesirable side effect of the protein complex and the like.
Moieties
capable of mediating such effects are disclosed, for example, in Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).
The term "fragment" is used to indicate a polypeptide derived from the
amino acid sequence of the proteins, of the complexes having a length less
than the
full-length polypeptide from which it has been derived. Such a fragment may,
for
example, be produced by proteolytic cleavage of the full-length protein.
Preferably,
the fragment is obtained recombinantly by appropriately modifying the DNA
sequence encoding the proteins to delete one or more amino acids at one or
more
sites of the C-terminus, N-terminus, and/or within the native sequence.
Fragments
of a protein are useful for screening for substances that act to modulate
signal
transduction, as described herein. It is understood that such fragments may
retain
one or more characterizing portions of the native complex. Examples of such
retained characteristics include: catalytic activity; substrate specificity;
interaction
with other molecules in the intact cell; regulatory functions; or binding with
an
antibody specific for the native complex, or an epitope thereof.
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Another functional derivative intended to be within the scope of the present
invention is a "variant" polypeptide which either lacks one or more amino
acids or
contains additional or substituted amino acids relative to the native
polypeptide. The
variant may be derived from a naturally occurring complex component by
appropriately modifying the protein DNA coding sequence to add, remove, and/or
to
modify codons for one or more amino acids at one or more sites of the C-
terminus,
N-terminus, and/or within the native sequence. It is understood that such
variants
having added, substituted and/or additional amino acids retain one or more
characterizing portions of the native protein, as described above.
A functional derivative of a protein with deleted, inserted and/or substituted
amino acid residues may be prepared using standard techniques well-known to
those
of ordinary skill in the art. For example, the modified components of the
functional
derivatives may be produced using site-directed mutagenesis techniques (as
exemplified by Adelman et al., 1983, DN~12:183) wherein nucleotides in the DNA
coding the sequence are modified such that a modified coding sequence is
modified,
and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic
host
cell, using techniques such as those described above. Alternatively, proteins
with
amino acid deletions, insertions and/or substitutions may be conveniently
prepared
by direct chemical synthesis, using methods well-known in the art. The
functional
derivatives of the proteins typically exhibit the same qualitative biological
activity as
the native proteins.
TABLES AND DESCRIPTION THEROF
This patent describes novel protease identified in databases of genomic
sequence. The results are summarized in four tables, which are described
below.
Table 1 documents the name of each gene, the classification of each gene,
the positions of the open reading frames within the sequence, and the length
of the
corresponding peptide. From left to right the data presented is as follows:
"Gene
Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family",
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"NA length", "ORF Start", "ORF End", "ORF Length", and "AA length". "Gene
name" refers to name given the sequence encoding the protease enzyme. Each
gene
is represented by "SGPr" designation followed by an arbitrary number. The SGPr
name usually represents multiple overlapping sequences built into a single
contiguous sequence (a "contig"). The "ID#na" and "ID#aa" refer to the
identification numbers given each nucleic acid and amino acid sequence in this
patent application. "FLICat" refers to the length of the gene, with FL
indicating full
length, and "Cat' indicating that only the catalytic domain is presented.
"Partial" in
this column indicates that the sequence encodes a partial catalytic domain.
"Superfamily" identifies whether the gene is a protease. "Group" and "Family"
refer to the protease classification defined by sequence homology. "NA length"
refers to the length in nucleotides of the corresponding nucleic acid
sequence. "ORF
start" refers to the beginning nucleotide of the open reading frame. "ORF end"
refers to the last nucleotide of the open reading frame, including the stop
codon.
"ORF length" refers to the length in nucleotides of the open reading frame
(including the stop codon). "AA length" refers to the length in amino acids of
the
peptide encoded in the corresponding nucleic acid sequence.
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CA 02408105 2002-11-O1
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109
CA 02408105 2002-11-O1
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i
Table 2 lists the following features of the genes described in this patent
application: chromosomal localization, single nucleotide polymorphisms (SNPs),
representation in dbEST, and repeat regions. From left to right the data
presented is
as follows: "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group",
"Family", "Chromosome", "SNPs", "dbEST hits", & "Repeats". The contents of
the first 7 columns (i.e.,. "Gene Name", "ID#na", "ID#aa", "FL/Cat",
"Superfamily", "Group", "Family") are as described above for Table 1.
"Chromosome" refers to the cytogenetic localization of the gene. Information
in the
"SNPs" column describes the nucleic acid position and degenerate nature of
candidate single nucleotide polymorphisms (SNPs; please see table of
polymorphism below). These SNPs were identified by blastn of the DNA sequence
against the database of single nucleotide polymorphisms maintained at NCBI
http://www.ncbi.nlm.nih.~ov/SNP/snpblastByChr.htmll. "dbEST hits" lists
accession numbers of entries in the public database of ESTs (dbEST,
http://www.ncbi.nhn.nih.gov/dbEST/index.html) that contain at least 150 by of
100% identity to the corresponding gene. These ESTs were identified by blastn
of
dbEST. "Repeats" contains information about the location of short sequences,
approximately 20 by in length, that are of low complexity and that are present
in
several distinct genes.
110
CA 02408105 2002-11-O1
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N NNN
CA 02408105 2002-11-O1
WO 01/83782 PCT/USO1/14431
Table 3 lists the extent and the boundaries of the protease catalytic domains,
and
other protein domains. The column headings are: "Gene Name", "ID#na", "ID#aa",
"FL/Cat" "Profile start" "Profile end" "Protease start" "Protease end"
_ ~ _
"Profile", and "Other Domains". The contents of the first 7 columns (i.e.,.
"Gene
Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family") are as
described above for Table 1. "Profile Start", "Profile End", "Protease Start"
and
"Protease End" refer to data obtained using a Hidden-Markov Model to define
catalytic range boundaries. The boundaries of the catalytic domain within the
overall protein are noted in the "Protease Start" and "Protease End" columns.
"Profile" indicates whether the I~VIMR search was done with a complete
("Global")
or Smith Waterman ("Local") model, as described below. Starting from a
multiple
sequence alignment of catalytic domains, two hidden Markov models were built.
One of them allows for partial matches to the catalytic domain; this is a
"local"
HMM, similar to Smith-Waterman alignments in sequence matching. The other
model allows matches only to the complete catalytic domain; this is a "global"
HMM similar to Needleman-Wunsch alignments in sequence matching. The Smith
Waterman local model is more specific, allowing for fragmentary matches to the
catalytic domain whereas the global "complete" model is more sensitive,
allowing
for remote homologue identification. The "Other domains" column lists the
names
and positions of domains within the protein sequence in addition to the
protein
protease domain. These domains were identified using PFAM
(http://pfam.wiistl.edu/hnunsearch.shtml) models, a large collection of
multiple
sequence alignments and hidden Markov models covering many common protein
domains. Version 5.5 of Pfam (Sept 2000) contains alignments and models for
2478
protein families (http://pfam.wustl.edu/faq.shtml). The PFAM alignments were
downloaded from http://pfam.wustl.edu/h~nmsearch.shtml and the HIVIMr searches
were run locally on a Timelogic computer (TimeLogic Corporation, Incline
Village,
NV).
112
CA 02408105 2002-11-O1
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113
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Table 4 describes the results of Smith Waterman similarity searches (Matrix:
Pam100; gap open/extension penalties 12/2) of the amino acid sequences against
the
NCBI database of non-redundant protein sequences
(ht~p://wyvw.ncbi.nlm.nih.~ov/Entrez/protein.html). The column headings are:
"Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family",
"Pscore", "aa length", "aa ID match", "%Identity", "%Similar",
"ACC# nraa match", and "Description". The contents of the first 7 columns
(i.e.,.
"Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family") are
as described above for Table 1. "Pscore" refers to the Smith Waterman
probability
score. This number approximates the chance that the alignment occurred by
chance.
Thus, a very low number, such as 2.10E-64, indicates that there is a very
significant
match between the query and the database target. "aa length" refers to the
length of
the protein in amino acids. "aa ID match" indicates the number of amino acids
that
were identical in the alignment. "% Identity" lists the percent of amino acids
that
were identical over the aligned region. "% Similarity" lists the percent of
amino
acids that were similar over the alignment. "ACC#nraa match" lists the
accession
number of the most similar protein in the NCBI database of non-redundant
proteins.
"Description" contains the name of the most similar protein in the NCBI
database of
non-redundant proteins.
114
CA 02408105 2002-11-O1
WO 01/83782 PCT/USO1/14431
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I I
CA 02408105 2002-11-O1
WO 01/83782 PCT/USO1/14431
EXAMPLES
The examples below are not limiting and are merely representative of various
aspects and features of the present invention. The examples below demonstrate
the
isolation and characterization of the proteases of the invention.
EXAMPLE 1: Identification of Genomic Fragments Encoding Proteases
Novel proteases were identified from the Celera human genomic sequence
databases, and from the public Human Genome Sequencing project
(http:l/www.ncbi.nlm.uh.~ov~ using hidden Markov models (HIV~VIR). The
genomic database entries were translated in six open reading frames and
searched
against the model using a Timelogic Decypher box with a Field programmable
array
(FPGA) accelerated version of HMMR2.1. The DNA sequences encoding the
predicted protein sequences aligning to the HMMR profile were extracted from
the
original genomic database. The nucleic acid sequences were then clustered
using
the Pangea Clustering tool to eliminate repetitive entries. The putative
protease
sequences were then sequentially run through a series of queries and filters
to
identify novel protease sequences. Specifically, the HM1VIR identified
sequences
were searched using BLASTN and BLASTX against a nucleotide and amino acid
repository containing known human proteases and all subsequent new protease
sequences as they are identified. The output was parsed into a spreadsheet to
facilitate elimination of known genes by manual inspection. Two models were
used,
a "complete" model and a "partial" or Smith Waterman model. The partial model
was used to identify sub-catalytic domains, whereas the complete model was
used to
identify complete catalytic domains. The selected hits were then queried using
BLASTN against the public NRNA and EST databases to confirm they are indeed
unique.
Extension of partial DNA sequences to encompass the longer sequences,
including full-length open-reading frame, was carried out by several methods.
116
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Iterative blastn searching of the cDNA databases listed in Table 5 was used to
find
cDNAs that extended the genomic sequences. "LifeGold" databases are from
Incyte
Genomics, Inc (http://www.incyte.com~. NCBI databases are from the National
Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ ). All
blastn
searches were conducted using a blosum62 matrix, a penalty for a nucleotide
mismatch of -3 and reward for a nucleotide match of 1. The gapped blast
algorithm
is described in: Altschul, Stephen F., Thomas L. Madden, Alejandro A.
Schaffer,
Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped
BLAST and PSI-BLAST: a new generation of protein database search programs",
Nucleic Acids Res. 25:3389-3402).
Extension of partial DNA sequences to encompass the full-length open-reading
frame was also carried out by iterative searches of genomic databases. The
first
method made use of the Smith-Waterman algorithm to carry out protein-protein
searches of the closest homologue or orthologue to the partial. The target
databases
consisted of Genscan [Chris Burge and Sam Karlin "Prediction of Complete Gene
Structures in Human Genomic DNA", JMB (1997) 268(1):78-94)] and open-reading
frame (ORF) predictions of all human genomic sequence derived from the human
genome project (HGP) as well as from Celera. The complete set of genomic
databases searched is shown in Table 6 below. Genomic sequences encoding
potential extensions were further assessed by blastp analysis against the NCBI
nonredundant to confirm the novelty of the hit. The extending genomic
sequences
were incorporated into the cDNA sequence after removal of potential introns
using
the Seqman program from DNAStar. The default parameters used for Smith-
Waterman searches were as shown next. Matrix: PAM100; gap-opening penalty: 12;
gap extension penalty: 2. Genscan predictions were made using the Genscan
program as detailed in Chris Burge and Sam Karlin "Prediction of Complete Gene
Structures in Human Genomic DNA", JMB (1997) 268(1):78-94). ORF predictions
from genomic DNA were made using a standard 6-frame translation.
Another method for defining DNA extensions from genomic sequence used
iterative searches of genomic databases through the Genscan program to predict
117
CA 02408105 2002-11-O1
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exon splicing [Barge and Karlin, JMB (1997) 268(1):78-94)]. These predicted
genes were then assessed to see if they represented "real" extensions of the
partial
genes based on homology to related proteases.
Another method involved using the Genewise program
(http://www.sanger.ac.uk/Software/Wise2/ ) to predict potential ORFs based on
homology to the closest orthologue/homologue. Genewise requires two inputs,
the
homologous protein, and genomic DNA containing the gene of interest. The
genomic DNA was identified by blastn searches of Celera and Human Genome
Project databases. The orthologs were identified by blastp searches of the
NCBI
non-redundant protein database (NRAA). Genewise compares the protein sequence
to a genomic DNA sequence, allowing for introns and frameshifting errors.
Table 5. Databases used for cDNA-based sequence extensions
Database Database Date
LifeGold templates March 2001
LifeGold compseqs March 2001
LifeGold compseqs March 2001
LifeGold compseqs March 2001
LifeGold fl March 2001
LifeGold flft March 2001
NCBI human Ests March 2001
NCBI marine Ests March 2001
NCBI nonredundant March 2001
118
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I. TABLE 6. DATABASES USED FOR GENOMIC-BASED SEQUENCE
EXTENSIONS
Database Number of Database
entries Date
Celera v. 1-5 5,306,158 Jan 2000
Celera v. 6-10 4,209,980 March 2000
Celera v. 11-14 7,222,425 April 2000
Celera v. 15 243,044 April 2000
Celera v. 16-17 25,885 April 2000
Celera Assembly 5 (release479,986 March 2001
25h)
HGP Phase 0 3,189 Nov 1100
HGP Phase 1 20,447 Jan 1101
HGP Phase 2 1,619 Jan 1101
HGP Phase 3 9,224 March 2001
HGP Chromosomal 2759 March 2001
assemblies
Results:
The sources for the sequence information used to identify genes are listed
below. For genes that were extended using Genewise, the accession numbers of
the
protein ortholog and the genomic DNA are given. (Genewise uses the ortholog to
assemble the coding sequence of the target gene from the genomic sequence).
The
amino acid sequences for the orthologs were obtained from the NCBI non-
redundant
database of proteins .(http://www.ncbi.nlin.nih.gov/Entrez/protein.html). The
genomic DNA came from two sources: Celera and NCBI-NRNA, as indicated
below. cDNA sources are also listed below. All of the genomic sequences were
used as input for Genscan predictions to predict splice sites [Barge and
Karlin, JMB
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(1997) 268(1):78-94)]. Abbreviations: HGP: Human Genome Project; NCBI,
National Center for Biotechnology Information.
SGPr140, SEQ ID NOS:1, 36
Genomic DNA source: Cetera Assembly Sh contig 90000642234645
Homologs used for Genewise: gi_5822085, gi_11265696, gi 2136604
SGPrl97, SEQ ID NOS:2, 37
Genomic DNA source: Cetera Assembly Sh contig 90000640151915
Homologs used for Genewise: gi_12731929, gb_AAA60062.1, gi 999902
SGPr005, SEQ ID NOS:3, 38
Genomic DNA source: Cetera Assembly Sh contig 90000642234645
Homologs used for Genewise: gi_11265695, gi_12731929, dbj BAB 11754.1
The genomic sequence containing the original HMM hit was blast against
Cetera AsmSh where it aligned with contig 90000642234645 (4157978 bp) in the
anti-sense orientation. 200 kb of the contig was used for genewise/genscan/
sym4
predictions. Genewise was run with human pepsinogen C (gi~ 12731929) as the
model and the result extended the original HMM tut to 370 aa. The genewise
prediction was then blastx against NCBI nonredundant to find that it shared
strongest homology (64% identity over 372 aa) with pepsinogen C from
Rhiholophus fer~umequinumxx. The extended sequence also shares homology (74%
over 324 aa) with the profiled Pfam Eukaryotic aspartyl protease. All
overlapping
Genscan predictions were blastx vs NCBI nonredundant. Only one prediction (id
83280) contained sequence with homology to pepsinogen C. The genewise
prediction was then blastn vs all EST and cDNA databases. Several hits were
found:
1.) LGTemplatesMAR2001: AAA41827.1 g206083 pepsinogen 0
2.) LGcompseqsMAR2001: 7477287CB1
3.) LGcompseqsMAR2001: 825016H1
4.) LGflMAR2001: 7477287CB 1
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l
5.) LGfIMAR2001n: g8546678 edit 02
6.) LGfIMAR2001n: 825016H1 edit 1
7.) LGflftAPR2001n: 7477287CB1
The LGcompseqsMAR2001 EST 7477287CB 1 contains an ORF of 1173 by
or 390 aa. When blastx against NCBI nonredundant 7477287CB1 shares 62%
identity over 372 as to pepsinogen C of Rhinolophus fe~~umequinum. When
aligned
with the SGPr005 genewise prediction, 7477287CB 1 has 3 conflicts and 4
inserts/deletions.
Conflict #1
The first conflict occurs at nucleotide 189 of 7477287CB 1. In the
7477287CB1 sequence nucleotide 189 is a "T" while in the SGPr005 genewise
prediction the corresponding nucleotide is a "C". The nucleotide conflict is
silent
and does not give rise to an amino acid change.
At conflict #1 both the SGPr005 genewise sequence and the 7477287CB 1
sequence are supported by genomic data.
SGPr005 genewise sequence: Celera AsmSh contig 90000642234645
7477287CB1: HGP_s contig gi~9213869 5
Conflict #2
The second conflict occurs at nucleotide 379 of 7477287CB 1. In the
7477287CB 1 sequence nucleotide 379 is a "G" while in the SGPr005 genewise
prediction the corresponding nucleotide is a "A". The nucleotide conflict
gives rise
to an as change of D (7477287CB1) to N (SGPr005).
At conflict #2 both the SGPr005 genewise sequence and the 7477287CB 1
sequence are supported by genomic data.
SGPr005 genewise sequence: Celera AsmSh contig 90000642234645
7477287CB1: HGP s contig gi~9213869 5
Conflict #3
The third conflict occurs at nucleotide 745 of 7477287CB 1. In the
7477287CB 1 sequence nucleotide 745 is a "G" while in the SGPr005 genewise
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prediction the corresponding nucleotide is a "T". The nucleotide conflict
gives rise
to an as acid conflict of E (7477287CB1) to STOP (SGPr005).
At conflict #3 the only sequence supported by genomic data is the SGPr005
genewise sequence which gives rise to the stop codon.
SGPr005 genewise sequence: Celera AsmSh contig 90000642234645 and
HGP_s contig gi~9213869 5
Inserts #1 and #2
The first two inserts occurs at nucleotide 214 of the SGPr005 genewise
predicted sequence and nucleotide 297 of 7477287CB1.
7477287CB 1: TTCCTAGTC
TCTTTGATACGGGTTCCTCCAATCTGTAGCCTGCCCTC
SGPr005gw:
TTCCTAGTCCTCTTTGATACGGGTTCCTCCAATCTGTAG CTGCCCTC
Because one insert occurs on the genewise prediction while the other occurs
on the EST the two sequences are only frameshifted for 31 nucleotides. When
this
stretch of sequence is blastx vs NCBI nonredundant, it is clear that the
SGPr005
genewise predicted sequence contains the correct reading frame in order to
maintain
homology to pepsinogen C.
The genomic data from Celera AsmSh contig 90000642234645 supports the
SGPr005 genewise sequence while the HGP_s contig gi~9213869 5 supports the
7477287CB 1 sequence.
Insert #3
The third insert occurs at nucleotide 706 of 7477287CB1.
7477287CB 1:
ATCCTTGGAGGTGTGGACCCCAACCTTTATTCTGGTCAGATCATCTGGACC
SGPr005gw: ATCCTTGGAGGTGTGGACCCCAAC_
TTTATTCTGGTCAGATCATCTGGACC
When this stretch of sequence is translated and blastp vs ncbi redundant, it
is
clear that the 7477287CB 1 sequence contains the necessary reading frame to
maintain homology with pepsinogen C. However, both the Celera AsmSh and
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HGP s genomic hits (Cetera AsmSh contig 90000642234645 and HGP_s contig
gi~9213869 5) support the SGPr005 genewise predicted sequence.
Insert #4
The fourth insert occurs at nucleotide 873 of 7477287CB 1.
7477287CB 1:
GAGACCTTCCTGCTGGCAGTTCCTCAGCAGTACATGGCCTCCTTCCTGCA
G
SGPr005gw: GAGACCTTCCTGCTGGCAGTTCCTCAGCAGTACAT_
GCCTCCTTCCTGCAG
When this stretch of sequence is translated and blastp vs ncbi redundant, it
is
clear that the 7477287CB 1 sequence contains the necessary reading frame to
maintain homology with pepsinogen C. However, both the Cetera AsmSh and
HGP_s genomic hits (Cetera AsmSh contig 90000642234645 and HGP_s contig
gi~9213869 5) support the SGPr005 genewise predicted sequence.
SGPr078, SEQ ID NOS:4, 39
Genomic DNA source: Public genomic contig: gig 11560222, subfragment 11
Homologs used for Genewise: gi 5822085
SGPr084, SEQ ID NOS:S, 40
Genomic DNA source: Cetera Assembly Sh contig 90000636191372
Homologs used for Genewise: gb AAD31927.1, sp 043323, ref NP_031883.1
SGPr009, SEQ ID NOS:6, 41
Genomic DNA source: Cetera Assembly Sh contig 90000642045264
Homologs used for Genewise: gi_12736472, gb_AAC99852.1, gb AAC99854.1
The original HMM hit was blast against Cetera AsmSh where it aligned with
contig 90000642045264 (8,329,407bp) in the sense orientation. Nucleotides
14,659
to 111,952 of the contig were used for genewise/genscan/sym4 predictions.
Genewise was run with human caspase 4 (gi~12736472~gn1) as the model and the
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prediction extended SGPr009 through the 3' most 274aa (through the stop
codon).
The SGPr009 genewise prediction shares homology (62% identity over 274 aa)
with
human caspase 4 (gi~4502577). The genewise prediction also overlaps SGPrl l l,
merging these two fragments into one gene (SGPr009=SGPrl 11). However, the
genewise prediction does have one internal stop and one frame shift. The
internal
stop codon and the frame shift were corrected for through. analysis with other
genomic contigs and ESTs. One EST of importance was LGcompseqsMAR2001
7478251 CB 1 which overlaps with the SGPr009 genewise prediction and extends
the
prediction in the 5' direction through the start codon. To correct for
sequencing
errors in the extended 7478251 CB 1 sequence, the EST was blastn vs. genomic
databases and the following changes were made: nucleotide 391 and 393 were
changed from A to G based on HGP_s and Celera contigs, and nucleotide 1041 was
changed from A to T based on HGP s and Celera contigs.
SGPr286, SEQ ID NOS:7, 42
Genomic DNA source: Celera Assembly Sh contig 90000628729589
Homologs used for Genewise: ref NP 036246.1, gi_6753280
The genomic sequence containing the original HMM hit was blast against
Celera AsmSh where it aligned with contig 90000628729589 (1,488,284 bp) in the
anti-sense orientation. 200 kb of the contig was used for
genewise/genscan/syrn4
predictions. Genewise was run with human caspase 14 (gi~6912286) as the model
and the result extended the original I~VVIM hit to 233 aa. The genewise result
shares
good homology to caspase 14 (44% identity over 236aa) from amino acid 11
through the stop codon. The genewise result was then blastn vs. all EST and
cDNA
databases where it hit several ESTs: LGtemplatesMAR2001:
292606.4, LGflftAPR2001n: 7648238CB1, LGcompseqsMAR2001:
7648638J1, 7013516H1, NCBI Nonredundant NA: gi~3982609, mega_cdna:
cluster381375 2 incyte, cluster381375= 4 incyte. The overlapping EST data was
used to support the genewise prediction. ,
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SGPr008, SEQ ID NOS:B, 43
Genomic DNA source: Celera Assembly Sh contig 301714258
Homologs used for Genewise: emb_CAA86994.1, gb AAF57563.1, gb AAF57564.1
SGPr198, SEQ ID NOS:9, 44
Genomic DNA source: Celera Assembly Sh contigs: 9802310, 90000642810957
Homologs used for Genewise: gb A.AF99682.1, gb AAG22771.1, gi-12722673
SGPr210, SEQ ID NOS:10, 45
Genomic DNA source: Celera Assembly Sh contig 92000004252572
Homologs used for Genewise: emb CAC10067.1, emb_CAC10068:1, ref NP_068694.1
SGPr290, SEQ ID NOS:l 1, 46
Genomic DNA source: Celera Assembly Sh contig 301714258
Homologs used for Genewise: gb AAD34600.1, gb AAD51699.1, gb AAD56236.1
SGPrl 16, SEQ ID NOS:12, 47
Genomic DNA source: Celera Assembly Sh contig 90000627067487
Homologs used for Genewise: sp P00789, gi_12732105, ref NP_008989.1
SGPr003, SEQ ID NOS:13, 48
Genomic DNA source: 90000640081635
Homologs used for Genewise: gb AAH05681.1, ref NP_035926.1,
gb AAG17967.1
Notes: Recently published as ref~NP_075574.1 ~ calpain 10, isoform d; calcium-
activated neutral protease
SGPr016, SEQ ID NOS:14, 49
Genomic DNA source: Celera Assembly Sh contig 90000642821147
Homologs used for Genewise: gi_1079470, ref NP_055052.1
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Notes: Genomic region may be misassembled, predicted protein may have gaps in
the middle. Used incyte template 094916.1 to extend genewise prediction
SGPr352, SEQ ID NOS:15, 50
Genomic DNA source: Cetera Assembly Sh contig 90000628457498
Homologs used for Genewise: ref NP_055087.1, gb_AAG35563.1, gb_AF163762.1
SGPr050, SEQ ID NOS:16, 51
Genomic DNA source: Cetera Assembly Sh contig 90000626814267
Homologs used for Genewise: ref NP_055087.1, gb AAG35563.1
Used Incyte sequences to aid gene finding and show tissue expression:
333039.1,
333039.4, 1011933.1, 333039.3, 333039.2, 3533147CB1. Clones were expressed in
urinary tract (9), respiratory system (3), female genitalia (2), nervous
system (2) and
connective, exocrine, digestive and musculoskeletal systems (one each)
SGPr282, SEQ ID NOS:17, 52
Genomic DNA source: Cetera Assembly Sh contig 90000641115460
Homologs used for Genewise: gb AAC09475.1, pir ~I65253
SGPr046, SEQ ID NOS:18, 53
Genomic DNA source: Cetera Assembly Sh contig 92000004436076
Homologs used for Genewise: ref NP_055087.1, gb AAG35563.1
Also used Incyte sequences 207915.2 , 207915.5, 207915.11 , 207915.4,
7478405CB1, 9123702. Resolved differences between genomic and EST sequence
by blasting against Cetera raw reads, public and Incyte ESTs and HGP genomic
contigs.
SGPr060, SEQ ID NOS:19, 54
Genomic DNA source: Cetera Assembly Sh contig 90000642001297
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Homologs used for Genewise: gb AAG35563.1, ref NM 022122.1,
ref NP 112217.1
Incyte sequences 452273.1, 013006.4, 013006.3, 322264.1 and public ESTs
gi~7115818, gi~6837795 were used to extend and verify the genewise prediction.
SGPr068, SEQ ID NOS:20, 55
Genomic DNA source: Celera Assembly Sh contig 90000624770881
Homologs used for Genewise: sp_015072, gi_11417111, gi_12731510
Incyte sequence 7477386CB1, 1719204CB1 also used. Sequence from 3062-3172
in the mRNA is missing in incyte sequence 7477386CB1, leading to the
replacement
of the peptide "GNHQNSTVRADVWELGTPEGQWVPQSEPLHPINKISST" with
"A" in the predicted protein. In 7477386CB1 there are two ant inserts at
splice sites,
and a 5 nt insert followed shortly by a 1 nt insert, none of which are found
in any
genomic sequences, and so may be the result of atypical splicing. This
alternative
form would insert a V at position 291 of the protein, a Q at 318, a G at 386,
and
changes a LWS at 584-586 to a PAYGG. Incyte template 196583.5 uses an
alternative splice acceptor site in one intron, inserting the sequence
"CTCCCCATCTCCCCTCAG" at position 2420 of the mRNA and inserting the
sequence PISPQA into the protein.
SGPr096, SEQ ID NOS:21, 56
Genomic DNA source: Celera Assembly Sh contig 90000637859600
Homologs used for Genewise: dbj BAA92550.1, ref NP_064634.1
Partial fragments published in 2000 as NP_064634.1 and as I~IAA1312. 121 ESTs
from Incyte template 1501550.6, show broad expression, highest in female
genitalia
and nervous system.
SGPr119, SEQ ID NOS:22, 57
Genomic DNA source: Celera Assembly Sh contig 90000642194924
Homologs used for Genewise: dbj BAA92550.1, ref IvTP_064634.1
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Public sequence gi) 13376516~re~NM 025003.1 encodes an alternative splice form
which is missing 3586-3693 of the RNA sequence
SGPr143, SEQ m NOS:23, 58
Genomic DNA source: Celera Assembly Sh contig 90000641832427
Homologs used for Genewise: em ~CAC16509.2, gb_AAB51194.1,
gb AAK07852.1
SGPr164, SEQ ID NOS:24, 59
Genomic DNA source: Celera Assembly Sh contig 90000642493829
Homologs used for Genewise: sp P97857, ref NP_077376.1, dbj BAA11088.1
3 ESTs cover this gene. 2 are from brain tumors, 1 from testis. One EST has 1
AA
deletion. Start is probably at first Met in the AA sequence
SGPr281, SEQ ID NOS:25, 60
Genomic DNA source: Celera Assembly Sh contig 92000004763172
Homologs used for Genewise: emb AL523577.1
SGPr075, SEQ ID NOS:26, 61
Genomic DNA source: Assembly of Celera Assembly Sg contigs
165000100324361,165000102322372,165000101460952,165000102528372,
165000102358388,165000100557102,165000102544200,165000102496419,
165000101581219,165000100483148,165000100004880,165000102322372,
165000100324361
Homologs used for Genewise: emb CAC18729.1
The nnn in NA sequence and X in peptide sequence represents a probable missing
exon; the gene may also be incomplete at either end. Based on searches of all
human
DNA databases, this gene is likely to be a fragment of the ortholog of the rat
gene
used as genewise homolog.
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SGPr292, SEQ ID NOS:27, 62
Genomic DNA source: Cetera Assembly Sh contig 90000641768196
Homologs used for Genewise: gb AAH02631.1, ref NP_077278.1,
gb_AAC21447.1 '
The following polymorphisms are seen: C->T at 572, T->A at 591, T->A at 593, C-
>T at 981, deletion of A at 1720. The first is seen in some ESTs and public
genomic
sources and changes an A to a V in the protein; the second and third are seen
in
ESTs and a single public genomic sequence, and change a V to an E in the
protein.
The third is seen only in ESTs and is a synonymous substitution. The fourth is
seen
in ESTs and public genomic data and is in the 3' LTTR of the gene. In
addition, a ant
deletion at 701-703 is seen in Incyte template 1510368.1, resulting in
deletion of the
D at position 558 of the peptide.
SGPr069, SEQ ID NOS:28, 63
Genomic DNA source: Cetera Assembly Sh contig 90000624872437
Homologs used for Genewise: gb_AAG18446.1, gb AAG18448.1,
gb AAF69247.1
SGPr212, SEQ m NOS:29, 64
Genomic DNA source: Cetera Assembly Sh contig 90000640657088
Homologs used for Genewise: dbj BAB25647.1, pir A75464, sp_P91885
SGPr049, SEQ ID NOS:30, 65
Genomic DNA source: Cetera Assembly Sh contig 90000641091876
Homologs used for Genewise: dbj BAB29490.1, emb AL543134.1, sp P15145,
gb_AAC32807.1
An alternatively spliced form is predicted by public EST gi~3805192 in which
an
extra exon ("TCTTTTATTTACTTTTTTAACTACAGCCACACTTTGAGCAG") is
inserted at position 3335 of the mRNA. This has an in-frame stop codon at it's
end
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and so predicts a truncated protein, which has the first 918 AA of the
predicted
protein, followed by "SLLFTFLTTATL*". Also used Incyte sequences 231695.1,
231695.7, 231695.2 to aid the prediction
SGPr026, SEQ ID N0:31, SEQ ID N0:66
Genomic DNA source: Celera Assembly Sh contig 113000081526387 and public
genomic contig gig 12227482
Homologs used for Genewise: gi_12654473, gi_10933784, gi_10800858 (all parital
sags of this gene), gi_1754515 (rat ortholog)
gi~9368836 encodes an alternative splice form, missing one axon, and with
another
axon extended. It predicts a truncated protein product, with AA 1-230 of the
main
form, followed by EPGVG*.
SGPr203, SEQ ID N0:32, SEQ ID N0:67
Genomic DNA source: Cetera Assembly Sh contig 90000640081635
Homologs used for Genewise: ref NM 016552.1, emb_CAC14047.1,
gb_AAG22080.1
A splice variant is created by use of an alternative splice acceptor that
eliminates
from 1526-1558 in ESTs such as Incyte cDNA 1868183CA2, resulting in the
removal of the peptide LEFERWLNATG from the protein.An intron within the final
axon is seen in Incyte template 1398043.12, which eliminates sequence from
2027-
2079 in the mRNA, a region within the 3' UTR. There may also be another form
with a longer intron in the last axon, eliminating the sequence from 2050-
2584,
which would cause a shift in reading frame, and open the reading frame until
the end
of the mRNA.
SGPr157, SEQ ID N0:33, SEQ ID N0:68
Genomic DNA source: Cetera Assembly Sh contig 90000625988051
Homologs used for Genewise: gi_11427093, dbj BAB22991.1, ref NP_060705.1
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SGPr154, SEQ ID N0:34 SEQ ID N0:69
Genomic DNA source: Public genomic contigs: gi_9798229, gi 9798027
Homologs used for Genewise: gb AAK22721.1, pir T38349
SGPr088, SEQ ID N0:35, SEQ ID N0:70
Genomic DNA source: Celera Assembly Sh contig 90000625988051
Homologs used for Genewise: dbj BAB22991.1, ref NP_060705.1, gi_7023108
Notes: Alternative splice forms are predicted by Incyte EST template
997089.29,
public sequence gi_10440455, and Sugen-built clusters of public ESTs:
cluster2209_-11 ncbi, cluster2209_-15 ncbi and cluster2209_-14 ncbi. All
result
in truncated proteins with short unique C-termini.
DESCRIPTION OF NOVEL PROTEASE POLYNUCLEOTIDES
SGPr140, SEQ ID NO:l, SEQ ID N0:36 is 1140 nucleotides long. The open
reading frame starts at position 1 and ends at position 1140, giving an ORF
length of
1140 nucleotides. The predicted protein is 379 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Aspartyl, PepsinAl. This gene maps to chromosomal position 1p13-p33.
This nucleotide sequence contains the following single nucleotide
polymorphisms
(sequence preceding SNP is given, followed by identity of SNP, the accession
number of SNP, and the allele position of SNP in the reference
sequence):ctggtggggcctggy, ss2008313 allelePos=201 ; ctctgtctactgcaacagk,
ss703383 allelePos=201. SNP ss2008313 occurs at nucleotide 846 (aa 282) of the
ORF (C or T = Gly or Gly) (silent). SNP ss703383 occurs at nucleotide 321 (aa
107) of the ORF (G or T = Arg or Ser). This sequence is represented in the
database
of public ESTs (dbEST) by the following ESTs:A969042, AA411567. The nucleic
acid contains short repetitive sequence (the position and sequence of the
repeat): 295
tgggtgccctctgtctactgc 315.
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SGPr197, SEQ ID N0:2, SEQ ID N0:37 is 1500 nucleotides long. The open
reading frame starts at position 1 and ends at position 1500, giving an ORF
length of
1500 nucleotides. The predicted protein is 499 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Aspartyl, PepsinAl. This gene maps to chromosomal position
6p21.1.This
sequence is represented in the database of public ESTs (dbEST) by the
following
ESTs:BF727344, BG394217, AW297327.
SGPr005, SEQ ID N0:3, SEQ ID N0:38 is 1173 nucleotides long. The open
reading frame starts at position 1 and ends at position 1173, giving an ORF
length of
1173 nucleotides. The predicted protein is 390 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Aspartyl, PepsinAl. This gene maps to chromosomal position 1p33.This
sequence is represented in the database of public ESTs (dbEST) by the
following
ESTs: none.
SGPr078, SEQ ID N0:4, SEQ ID N0:39 is 1239 nucleotides long. The open
reading frame starts at position 1 and ends at position 1239, giving an ORF
length of
1239 nucleotides. The predicted protein is 412 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Aspartyl, PepsinAl. This gene maps to chromosomal position 11p15.
This nucleotide sequence contains the following single nucleotide
polymorphisms
(sequence preceding SNP is given, followed by identity of SNP, the accession
number of SNP, and the allele position of SNP in the reference
sequence):aagtactcccaggy, ss20182 allelePos=101 . SNP ss20182 occurs at
nucleotide 173 (aa 58) of the ORF (C or T = Ala or Val).. This sequence is
represented in the database of public ESTs (dbEST) by the following
ESTs:BG260401, BF025894, BF793219.
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SGPr084, SEQ ID NO:S, SEQ ID N0:40 is 1191 nucleotides long. The open
reading frame starts at position 1 and ends at position 1191, giving an ORF
length of
1191 nucleotides. The predicted protein is 396 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Cysteine, HH. This gene maps to chromosomal position 12q11.
SGPr009, SEQ ID N0:6, SEQ ID N0:41 is 1137 nucleotides long. The open
reading frame starts at position 1 and ends at position 1137, giving an ORF
length of
1137 nucleotides. The predicted protein is 378 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfarnily/group/family):
Protease, Cysteine, ICEplO. This gene maps to chromosomal position l 1q22 This
nucleotide sequence contains the following single nucleotide polymorphisms
(sequence preceding SNP is given, followed by identity of SNP, the accession
number of SNP, and the allele position of SNP in the reference
sequence)agatggaaaataatgtr, ss726380 allelePos=201; gagacagctcaaay,
ss866796 allelePos=187. ss726380 occurs at nucleotide 102 (aa 34) of the ORF
(G
or A = Val or Val) (silent). SNP ss866796 occurs at nucleotide 200 (aa 67) of
the
ORF (C or T = Tyr or Ile).. The nucleic acid contains short repetitive
sequence (the
position and sequence of the repeat): 900 cttcattgctttcaaatcttcc 921; 77
ttgatgatttgatggaaaat 96.
SGPr286, SEQ ID N0:7, SEQ ID N0:42 is 705 nucleotides long. The open reading
frame starts at position 1 and ends at position 705, giving an ORF length of
705
nucleotides. The predicted protein is 234 amino acids long. This sequence
codes
for a full length protein. It is classified as (superfamily/group/family):
Protease,
Cysteine, ICEp20. This gene maps to chromosomal position na. This nucleotide
sequence contains the following single nucleotide polymorphisms (sequence
preceding SNP is given, followed by identity of SNP, the accession number of
SNP,
and the allele position of SNP in the reference
sequence):ytatgtggcctatcgcgatg;
rs551848-allelePos=3135. SNP rs551848 occurs at nucleotide 489 (aa 163) of the
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ORF (C or T = Gly or Gly) silent.. The nucleic acid contains short repetitive
sequence (the position and sequence of the repeat): 574 ctggagctgctgactgagg
592;
388 gtggggcccacagctctcc 406.
SGPr008, SEQ ID NO:B, SEQ ID N0:43 is 2010 nucleotides long. The open
reading frame starts at position l and ends at position 2010, giving an ORF
length of
2010 nucleotides. The predicted protein is 669 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfarnily/group/family):
Protease, Cysteine, PepC2. This gene maps to chromosomal position 2p23 This
nucleotide sequence contains the following single nucleotide polymorphisms
(sequence preceding SNP is given, followed by identity of SNP, the accession
number of SNP, and the allele position of SNP in the reference
sequence):rccgaatggagagggcg, ss678494 allelePos=201 . SNP ss678494 occurs at
nucleotide 838 (aa 280) of the ORF (G or A = Ala or Thr).. This sequence is
represented in the database of public ESTs (dbEST) by the following
ESTs:BE075751.
SGPr198, SEQ ID NO:9, SEQ ID NO:44 is 2112 nucleotides Iong. The open
reading frame starts at position 1 and ends at position 2112, giving an ORF
length of
2112 nucleotides. The predicted protein is 703 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Cysteine, PepC2. This gene maps to chromosomal position 1q42.11.This
sequence is represented in the database of public ESTs (dbEST) by the
following
ESTs:BE047777, AW339160.
SGPr210, SEQ ID NO:10, SEQ ID N0:45 is 2127 nucleotides long. The open
reading frame starts at position l and ends at position 2127, giving an ORF
length of
2127 nucleotides. The predicted protein is 708 amino acids long. Tlus sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Cysteine, PepC2. This gene maps to chromosomal position 19q13.2.
This
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nucleotide sequence contains the following single nucleotide polymorphisms
(sequence preceding SNP is given, followed by identity of SNP, the accession
number of SNP, and the allele position of SNP in the reference
sequence):ggttccttgcagcy, ss1376193 allelePos=473 . SNP ss1376193 occurs at
nucleotide 330 (aa 110) of the ORF (C or T = Ala or Ala) silent.. This
sequence is
represented in the database of public ESTs (dbEST) by the following
ESTs:BE872274. The nucleic acid contains short repetitive sequence (the
position
and sequence of the repeat): 1180 gaggaggatgacgaggatgagg 1201.
SGPr290, SEQ ID NO:11, SEQ ID N0:46 is 2136 nucleotides long. The open
reading frame starts at position 1 and ends at position 2136, giving an ORF
length of
2136 nucleotides. The predicted protein is 711 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Cysteine, PepC2. This gene maps to chromosomal position 2p23. The
nucleic acid contains short repetitive sequence (the position and sequence of
the
repeat): 1835 agcagctgcacgctgccatg 1854.
SGPr116, SEQ ID N0:12, SEQ ID N0:47 is 2109 nucleotides long. The open
reading frame starts at position 1 and ends at position 2109, giving an ORF
length of
2109 nucleotides. The predicted protein is 702 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Cysteine, PepC2. This gene maps to chromosomal position 6p12. The
nucleic acid contains short repetitive sequence (the position and sequence of
the
repeat): 1003 ctggagatctgcaacctcac 1022.
SGPr003, SEQ ID N0:13, SEQ ID N0:48 is 1542 nucleotides long. The open
reading frame starts at position 1 and ends at position 1542, giving an ORF
length of
1542 nucleotides. The predicted protein is 513 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Cysteine, PepC2. This gene maps to chromosomal position 2q37.This
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sequence is represented in the database of public ESTs (dbEST) by the
following
ESTs:AL526645, BG475966, AL529373. The nucleic acid contains short repetitive
sequence (the position and sequence of the repeat): 1520
gctgctgcaggagccgctgctg
1541.
SGPr016, SEQ ID N0:14, SEQ ID N0:49 is 846 nucleotides long. The open
reading frame starts at position 1 and ends at position 846, giving an ORF
length of
846 nucleotides. The predicted protein is 281 amino acids long. This sequence
codes for a partial protein.It is classified as (superfamily/group/family):
Protease,
Metalloprotease, ADAM. This sequence is represented in the database of public
ESTs (dbEST) by the following ESTs:AW589885, AI024863. The nucleic acid
contains short repetitive sequence (the position and sequence of the repeat):
710
ttaaatatatttcttctcataa 731.
SGPr352, SEQ ID NO:15, SEQ ID NO:50 is 3312 nucleotides long. The open
reading frame starts at position 1 and ends at position 3312, giving an ORF
length of
3312 nucleotides. The predicted protein is 1103 amino acids long. This
sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
19p13.3.This sequence is represented in the database ofpublic ESTs (dbEST) by
the
following ESTs:AW027573, AI131032, AI193804. The nucleic acid contains short
repetitive sequence (the position and sequence of the repeat): 1335
agactcgggcctggggctct 1354.
SGPr050, SEQ m N0:16, SEQ ID NO:51 is 3675 nucleotides long. The open
reading frame starts at position 1 and ends at position 3675, giving an ORF
length of
3675 nucleotides. The predicted protein is 1224 amino acids long. This
sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
Sq15.3. This nucleotide sequence contains the following single nucleotide
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polymorphisms (sequence preceding SNP is given, followed by identity of SNP,
the
accession number of SNP, and the allele position of SNP in the reference
sequence)acggctgaaaggcy, ss1483925 allelePos=216 . SNP ss1483925 occurs at
nucleotide 310 (aa 104) of the ORF (C or T = Pro or Ser). This sequence is
represented in the database of public ESTs (dbEST) by the following
ESTs:BF933693. The nucleic acid contains short repetitive sequence (the
position
and sequence of the repeat): 2067 tttcttcttttctttgtcaa 2086; 2061
atttgatttcttcttttctt
2080.
SGPr282, SEQ ID N0:17, SEQ ID N0:52 is 2196 nucleotides long. The open
reading frame starts at position 1 and ends at position 2196, giving an ORF
length of
2196 nucleotides. The predicted protein is 731 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
16p 12.3. This nucleotide sequence contains the following single nucleotide
polymorphisms (sequence preceding SNP is given, followed by identity of SNP,
the
accession number of SNP, and the allele position of SNP in the reference
sequence):ggcaatataaaaggcy, ss679422 allelePos=201; acttcactgggctay,
ss647742 allelePos=201; ggccgagcccaacgcaay, ss1226992 allelePos=101 . SNP
ss679422 occurs at nucleotide 625 (aa 209) of the ORF (C or T = His or Tyr).
SNP
ss647742 occurs at nucleotide 1893 (aa 631) of the ORF (C or T = Tyr or Tyr)
silent. SNP ss1226992 occurs at nucleotide 500 (aa 166) of the ORF (C or T =
Thr
or Met)..
SGPr046, SEQ ID N0:18, SEQ ID N0:53 is 2805 nucleotides long. The open
reading frame starts at position 1 and ends at position 2805, giving an ORF
length of
2805 nucleotides. The predicted protein is 934 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
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16q23The nucleic acid contains short repetitive sequence (the position and
sequence
of the repeat): 2353 gtgaggaagagggagatgaagt 2374.
SGPr060, SEQ ID N0:19, SEQ ID N0:54 is 4287 nucleotides long. The open
reading frame starts at position 1 and ends at position 4287, giving an ORF
length of
4287 nucleotides. The predicted protein is 1428 amino acids long. This
sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
15q26.This sequence is represented in the database of public ESTs (dbEST) by
the
following ESTs:AW575922, AW341169.
SGPr068, SEQ ID N0:20, SEQ ID NO:55 is 3561 nucleotides long. The open
reading frame starts at position 1 and ends at position 3561, giving an ORF
length of
3561 nucleotides. The predicted protein is 1186 amino acids long. This
sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
l Oq22.This sequence is represented in the database of public ESTs (dbEST) by
the
following ESTs:AJ403134, .
SGPr096, SEQ ID N0:21, SEQ ID N0:56 is 5808 nucleotides long. The open
reading frame starts at position 1 and ends at position 5808, giving an ORF
length of
5808 nucleotides. The predicted protein is 1935 amino acids long. This
sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
3p14.This sequence is represented in the database of public ESTs (dbEST) by
the
following ESTs:BE164543, AW995949, BF842288.
SGPr119, SEQ ID N0:22, SEQ ID N0:57 is 4518 nucleotides long. The open
reading frame starts at position 1 and ends at position 4518, giving an ORF
length of
4518 nucleotides. The predicted protein is 1505 amino acids long. This
sequence
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codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
12q11-ql2.This sequence is represented in the database of public ESTs (dbEST)
by
the following ESTs:AU132053, . The nucleic acid contains short repetitive
sequence (the position and sequence of the repeat): 1257 taaagaaatgaaagttacaaa
1277.
SGPr143, SEQ ID N0:23, SEQ ID N0:58 is 2649 nucleotides long. The open
reading frame starts at position 1 and ends at position 2649, giving an ORF
length of
2649 nucleotides. The predicted protein is 882 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, ADAM. This gene maps to chromosomal position
20p13. This nucleotide sequence contains the following single nucleotide
polymorphisms (sequence preceding SNP is given, followed by identity of SNP,
the
accession number of SNP, and the allele position of SNP in the reference
sequence):ggcagtggctactgcy, ss787708 allelePos=201 . SNP ss787708 occurs at
nucleotide 1750 (aa 584) of the ORF (C or T = Arg or Trp). . This sequence is
represented in the database of public ESTs (dbEST) by the following
ESTs:AA442551. The nucleic acid contains short repetitive sequence (the
position
and sequence of the repeat): 2212 tgccactgtgctccaggctg 2231. This protein is
predicted to have a transmembrane helix between amino acids 78 and 100.
(TMHMM, a Hidden Markov Model based transmenbrane prediction program,
Sonnhammer, et al Proc. of Sixth Int. Conf. on Intelligent Systems for
Molecular
Biology, p 175-182 AAAI Press, 1998.)
SGPr164, SEQ ID N0:24, SEQ ID N0:59 is 2937 nucleotides long. The open
reading frame starts at position 1 and ends at position 2937, giving an ORF
length of
2937 nucleotides. The predicted protein is 978 amino acids long. This sequence
codes for a nearly full length protein with only the N terminus missing. It is
classified as (superfamily/group/family): Protease, Metalloprotease, ADAM.
This
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gene maps to chromosomal position 11q25. This nucleotide sequence contains the
following single nucleotide polymorphisms (sequence preceding SNP is given,
followed by identity of SNP, the accession number of SNP, and the allele
position of
SNP in the reference sequence):ataccgatcctgcaay, ss76755 allelePos=87 . SNP
ss76755 occurs at nucleotide 1773 (aa 591) of the ORF (C or T = Asn or Asn)
silent..
SGPr281, SEQ ID N0:25, SEQ ID N0:60 is 3285 nucleotides long. The open
reading frame starts at position 1 and ends at position 3285, giving an ORF
length of
3285 nucleotides. The predicted protein is 1094 amino acids long. This
sequence
codes for a nearly full length protein, with just the amino terminus
missing.It is
classified as (superfamily/group/family): Protease, Metalloprotease, ADAM.
This
gene maps to chromosomal position Sq3l.
SGPr075, SEQ ID N0:26, SEQ ID N0:61 is 375 nucleotides long. The open
reading frame starts at position 1 and ends at position 375, giving an ORF
length of
375 nucleotides. The predicted protein is 125 amino acids long. This sequence
codes for a partial protein. It is classified as (superfamily/group/family):
Protease,
Metalloprotease, ADAM. This gene maps to chromosomal position na.
SGPr292, SEQ ID N0:27, SEQ ID N0:62 is 1710 nucleotides long. The open
reading frame starts at position 1 and ends at position 1710, giving an ORF
length of
1710 nucleotides. The predicted protein is 569 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepMlO. This gene maps to chromosomal position
l Oq26.This sequence is represented in the database of public ESTs (dbEST) by
the
following ESTs:AW665196. The nucleic acid contains short repetitive sequence
(the position and sequence of the repeat): 52 gctccctggcccacccagcc 71; 959
aagcaattcaaaagctgtatg 979.
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SGPr069, SEQ ID N0:28, SEQ ID N0:63 is 2232 nucleotides long. The open
reading frame starts at position 1 and ends at position 2232, giving an ORF
length of
2232 nucleotides. The predicted protein is 743 amino acids long. Tlus sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepMl3. This gene maps to chromosomal position Chr.
SGPr212, SEQ ID N0:29, SEQ ID N0:64 is 2730 nucleotides long. The open
reading frame starts at position 1 and ends at position 2730, giving an ORF
length of
2730 nucleotides. The predicted protein is 909 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepMl. This sequence is represented in the database
of
public ESTs (dbEST) by the following ESTs:AL523882, T11456.
SGPr049, SEQ ID N0:30, SEQ ID N0:65 is 2973 nucleotides long. The open
reading frame starts at position 1 and ends at position 2973, giving an ORF
length of
2973 nucleotides. The predicted protein is 990 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepMl. This sequence is represented in the database
of
public ESTs (dbEST) by the following ESTs:AI222989. The nucleic acid contains
short repetitive sequence (the position and sequence of the repeat): 2269
aatttaatatggaatatttat 2289.
SGPr026, SEQ ID N0:31, SEQ ID N0:66 is 1953 nucleotides long. The open
reading frame starts at position 1 and ends at position 1953, giving an ORF
length of
1953 nucleotides. The predicted protein is 650 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepMl..
SGPr203, SEQ ID N0:32, SEQ ID N0:67 is 2175 nucleotides long. The open
reading frame starts at position l and ends at position 2175, giving an ORF
length of
2175 nucleotides. The predicted protein is 724 amino acids long. This sequence
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codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepMl. This gene maps to chromosomal position 2q37.
This sequence is represented in the database of public ESTs (dbEST) by the
following ESTs:AU132908, BE735172, BE563549 (many). The nucleic acid
contains short repetitive sequence (the position and sequence of the repeat):
83
tggacgtggcctcggcctcca 103.
SGPr157, SEQ ID N0:33, SEQ ID N0:68 is 1524 nucleotides long. The open
reading frame starts at position 1 and ends at position 1524, giving an ORF
length of
1524 nucleotides. The predicted protein is 507 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepM20. This gene maps to chromosomal position
18q22.3.This sequence is represented in the database of public ESTs (dbEST) by
the
following ESTs:BE386438, BE386547, BF920454 (many). The nucleic acid
contains short repetitive sequence (the position and sequence of the repeat):
614
ccctggaggaacttgtggaa 633; 561 tcctgtgaatatcaaattca 580.
SGPr154, SEQ ID N0:34, SEQ ID N0:69 is 1422 nucleotides long. The open
reading frame starts at position 1 and ends at position 1422, giving am ORF
length of
1422 nucleotides. The predicted protein is 473 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepM20. This nucleotide sequence contains the
following
single nucleotide polymorphisms (sequence preceding SNP is given, followed by
identity of SNP, the accession number of SNP, and the allele position of SNP
in the
reference sequence):gtcatctatggty, ss1289877 allelePos=223. SNP ss1289877
occurs at nucleotide 457 (aa 153) of the ORF (C or T = Arg or Trp). The
nucleic
acid contains short repetitive sequence (the position and sequence of the
repeat):
806 tccttgcagctgctgtcagc 825.
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SGPr088, SEQ ID N0:35, SEQ ID N0:70 is 1428 nucleotides long. The open
reading frame starts at position 1 and ends at position 1428, giving an ORF
length of
1428 nucleotides. The predicted protein is 475 amino acids long. This sequence
codes for a full length protein. It is classified as
(superfamily/group/family):
Protease, Metalloprotease, PepM20. This gene maps to chromosomal position
18q23.This sequence is represented in the database of public ESTs (dbEST) by
the
following ESTs:AL541127, AL542184, AL529661 (many).
EXAMPLE 2: Exuression Analvsis of Mammalian Proteases
Materials and Methods
Quantitative PCR Analysis
RNA is isolated from a variety of normal human tissues and cell lines.
Single stranded cDNA is synthesized from 10 p,g of each RNA as described above
using the Superscript Preamplification System (GibcoBRL). These single strand
templates are then linearly amplified with a pair of specific primers in a
real time
PCR reaction on a Light Cycler (Roche Molecular Biochemical). Graphical
readout
can provide quantitative analysis of the relative abundance of the targeted
gene in
the total RNA preparation.
DNA Arrav Based Expression Anal~is
DNA-free RNA is isolated from a variety of normal human tissues, cryostat
sections, and cell lines. Single stranded cDNA is synthesized from l0ug RNA or
lug mRNA using a modification of the SMART PCR cDNA synthesis technique
(Clontech). The procedure can be modified to allow asymmetric labeling of the
5'
and 3' ends of each transcript with a unique oligonucleotide sequence. The
resulting
sscDNAs are then linearly amplified using Advantage long-range PCR (Clontech)
on a Light Cycler PCR machine. Reactions are halted when the graphical real-
time
display demonstrates the products have begun to plateau. The double stranded
cDNA products are purified using Millipore DNA purification matrix, dried,
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resuspended, quantified, and analyzed on an agarose gel. The resulting
elements are
referred to as "tissue cDNAs".
Tissue cDNAs are spotted onto GAPS coated glass slides (Corning) using a
Genetic Microsystems (GMS) arrayer at 500 ng/ul.
Fluorescent labeled oligonucleotides are synthesized to each novel exon,
ensuring they contained internal mismatches with the closest known homologue.
Typically oligos are 45 nucleotides long, labeled on the 5' end with CyS.
Exon-specific Cy5-labeled oligos are hybridized to the tissue cDNAs arrayed
onto glass slides, and washed using standard buffers and conditions.
Hybridizing
signals are then quantified using a GMS Scanner.
Alternatively, tissue cDNAs are manually spotted onto Nylon membranes
using a 384 pin replicator, and hybridized to 32P-end labeled oligo probes.
Tissue cDNAs are generated from multiple RNA templates selected to
provide information of relevance to the disease areas of interest and to
reflect the
biological mechanism of action for each protease. These templates include:
human
tumor cell lines, cryostat sections of primary human tumors and 32 normal
human
tissues to identify cancer-related genes; sections of normal, Alzheimer's,
Parkinson's, and Schizophrenia brain regions for CNS-related genes; normal and
diabetic or obese skeletal muscle, adipose, or liver for metabolic-related
genes; and
purified hematopoeitic cells, and lymphoid tissues for immune-related genes.
To
characterize gene mechanism of action, tissue cDNAs are generated to reflect
angiogenesis (cultured endothelial cells treated with VEGF ligand, anti-
angiogenic
drugs, or hypoxia), motility (A549 cells stimulated with HGF ligand,
orthotopic
metastases, primary tumors with matched metastatic tumors), cell cycle (Hela,
H1299, and other cell lines synchronized by drug block and harvested at
various
times in the cell cycle), checkpoint integrity and DNA repair (p53 normal or
defective cells treated with y-radiation, UV, cis-platinum, or oxidative
stress), and
cell survival (cells induced to differentiate or at various stages of
apoptosis).
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DESCRIPTION OF NOVEL PROTEASE POLYPEPTIDES
SGPr140, SEQ ID NO:l, SEQ ID N0:36 encodes a protein that is 379 amino acids
long. It is classified as an Aspartylprotease, of the PepsinAl family. The
protease
domain in this protein matches the hidden Markov profile for a Eukaryotic
aspartyl
protease, from amino acid 65 to amino acid 378. The positions within the
H1VIMIt
profile that match the protein sequence are from profile position 1 to profile
position
356. Other domains identified within this protein are: none. The results of a
Smith
Waterman search (PAM100, gap open and extend penalties of 12 and 2) of the
public database of amino acid sequences (NR.AA) with this protein sequence
yielded
the following results: Pscore =1.40E-160; number of identical amino acids =
263;
percent identity = 66%; percent similarity = 76%; the accession number of the
most
similar entry in NRAA is CAC19554.1; the name or description, and species, of
the
most similar protein in NRAA is: Chymosin [Camelus dromedarius].
SGPr197, SEQ ID N0:2, SEQ ID N0:37 encodes a protein that is 499 amino acids
long. It is classified as an Aspartylprotease, of the PepsinAl family. The
protease
domain in this protein matches the hidden Markov profile for a LTbiquitin
carboxyl-
terminal hydrolases family 2, from amino acid 199 to amino acid 230. The
positions
within the HMMR profile that match the protein sequence are from profile
position
1 to profile position 32. Other domains identified within this protein are: Zn-
finger
in ubiquitin-hydrolases (amino acid 26 to amino acid 96) P Score = 5.6e-025.
The
results of a Smith Waterman search (PAM100, gap open and extend penalties of
12
and 2) of the public database of amino acid sequences (NR.A.A) with this
protein
sequence yielded the following results: Pscore = 6.90E-137; number of
identical
amino acids = 296; percent identity = 46%; percent similarity = 56%; the
accession
number of the most similar entry in NR.A.A is CAB66759.1; the name or
description, and species, of the most similar protein in NRAA is: Hypothetical
histone deacetylase [Homo sapiens].
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SGPr005, SEQ ID NO:3, SEQ ID N0:38 encodes a protein that is 390 amino acids
long. It is classified as an Aspartylprotease, of the PepsinAl family. The
protease
domain in this protein matches the hidden Markov profile for a Eukaryotic
aspartyl
protease, from amino acid 65 to amino acid 389. The positions within the
H1VIMR
profile that match the protein sequence are from profile position 1 to profile
position
356. Other domains identified within this protein are: none. The results of a
Smith
Waterman search (PAM100, gap open and extend penalties of 12 and 2) of the
public database of amino acid sequences (NRAA) with this protein sequence
yielded
the following results: Pscore =1.40E-130; number of identical amino acids =
230;
percent identity = 62%; percent similarity = 76%; the accession number of the
most
similar entry in NRAA is BAB11755.1; the name or description, and species, of
the
most similar protein in NR.AA is: Pepsinogen C [Rhinolophus ferrumequinum].
SGPr078, SEQ ID N0:4, SEQ ID NO:39 encodes a protein that is 412 amino acids
long. It is classified as an Aspartylprotease, of the PepsinAl family. The
protease
domain in this protein matches the hidden Markov profile for a Eukaryotic
aspartyl
protease, from amino acid 70 to amino acid 409. The positions within the HMMR
profile that match the protein sequence are from profile position 1 to profile
position
356. Other domains identified within this protein are: none. The results of a
Smith
Waterman search (PAM100, gap open and extend penalties of 12 acid 2) of the
public database of amino acid sequences (NR.AA) with this protein sequence
yielded
the following results: Pscore = 3.20E-285; number of identical amino acids =
412;
percent identity = 100%; percent similarity = 100%; the accession number of
the
most similar entry in NRAA is NP_001900.1; the name or description, and
species,
of the most similar protein in NR.A.A is: Cathepsin D (lysosomal aspartyl
protease)
[Homo sapiens].
SGPr084, SEQ ID NO:S, SEQ ID N0:40 encodes a protein that is 396 amino acids
long. It is classified as a Cysteineprotease, of the HH family. The protease
domain
in this protein matches the hidden Markov profile for a Hedgehog amino-
terminal
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signaling domain, from amino acid 23 to amino acid 185. The positions within
the
IiMMR profile that match the protein sequence are from profile position 1 to
profile
position 163. Other domains identified within this protein are: Hint module
amino
acids 188-396; P Score = 5.9e-120. The results of a Smith Waterman search
(PAM100, gap open and extend penalties of 12 and 2) of the public database of
amino acid sequences (NR.AA) with this protein sequence yielded the following
results: Pscore = 3.00E-259; number of identical amino acids = 396; percent
identity
=100%; percent similarity =100%; the accession number of the most similar
entry
in NRAA is 043323; the name or description, and species, of the most similar
protein in NR.AA is: DESERT HEDGEHOG PROTEIN PRECURSOR (DHH)
(HHG-3) [Homo sapiens].
SGPr009, SEQ ID N0:6, SEQ ID N0:41 encodes a protein that is 378 amino acids
long. It is classified as a Cysteineprotease, of the ICEplO family. The
protease
domain in this protein matches the hidden Markov profile for a ICE-like
protease
(caspase) p20 domain, from amino acid 131 to amino acid 264. The positions
within
the HMMR profile that match the protein sequence are from profile position 1
to
profile position 141. Other domains identified within this protein are: ICE-
like
protease (caspase) p10 domain, amino acids 291-376; profile from 1-95: Caspase
recruitment domain from amino acids 2-91. The results of a Smith Waterman
search
(PAM100, gap open and extend penalties of 12 and 2) of the public database of
amino acid sequences (NRAA) with this protein sequence yielded the following
results: Pscore = 3.50E-129; number of identical amino acids = 223; percent
identity
= 55%; percent similarity = 67%; the accession number of the most similar
entry in
NR.AA is NP_033938.1; the name or description, and species, of the most
similar
protein in NR.AA is: Caspase 12 [Mus musculus].
SGPr286, SEQ ID N0:7, SEQ ID N0:42 encodes a protein that is 234 amino acids
long. It is classified as a Cysteineprotease, of the ICEp20 family. The
protease
domain in this protein matches the hidden Markov profile for a ICE-like
protease
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(caspase) p20 domain, from amino acid 19 to amino acid 58. The positions
within
the HIVIMR profile that match the protein sequence are from profile position
22 to
profile position 61. Other domains identified within this protein are: ICE-
like
protease (caspase) p10 domain, amino acids 144-202; profile from 1-61. . The
results of a Smith Waterman search (PAM100, gap open and extend penalties of
12
and 2) of the public database of amino acid sequences (NRAA) with this protein
sequence yielded the following results: Pscore = 4.60E-42; number of identical
amino acids = 108; percent identity = 46%; percent similarity = 65%; the
accession
number of the most similar entry in NR.AA is NP 036246.1; the name or
description, and species, of the most similar protein in NRAA is: Caspase 14,
apoptosis-related cysteine protease [Homo sapiens].
SGPr008, SEQ ID N0:8, SEQ ID NO:43 encodes a protein that is 669 amino acids
long. It is classified as a Cysteineprotease, of the PepC2 family. The
protease
domain in this protein matches the hidden Markowprofile for a Calpain family
cysteine protease; Peptidase C2, from amino acid 35 to amino acid 333. The
positions within the HIVIMR profile that match the protein sequence are from
profile
position 2 to profile position 344. The results of a Smith Waterman search
(PAM100, gap open and extend penalties of 12 and 2) of the public database of
amino acid sequences (NRAA) with this protein sequence yielded the following
results: Pscore = 9.10E-86; number of identical amino acids = 229; percent
identity
= 33%; percent similarity = 53%; the accession number of the most similar
entry in
NRAA is A.AD34601.1; the name or description, and species, of the most similar
protein in NR.A.A is: Lens-specific calpain Lp82 [Oryctolagus cuniculus].
SGPr198, SEQ ID N0:9, SEQ ID N0:44 encodes a protein that is 703 amino acids
long. It is classified as a Cysteineprotease, of the PepC2 family. The
protease
domain in this protein matches the hidden Markov profile for a Calpain family
cysteine protease; Peptidase C2, from amino acid 45 to amino acid 344. The
positions within the HIVIIVIR profile that match the protein sequence are from
profile
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position 1 to profile position 344. Other domains identified within this
protein are:
Calpain large subunit, domain III, amino acids 355-512, profile from 1-163.
Also
three EF hand motifs at amino acids 579-607, 609-637 and 674-701; all EF hands
match from 1-26 of profile. . The results of a Smith Waterman search (PAM100,
gap open and extend penalties of 12 and 2) of the public database of amino
acid
sequences (NRAA) with this protein sequence yielded the following results:
Pscore
= 0; number of identical amino acids = 593; percent identity = 84%; percent
similarity = 92%; the accession number of the most similar entry in NRAA is
BAA03369.1; the name or description, and species, of the most similar protein
in
NRAA is: Calpain [Rattus norvegicus].
SGPr210, SEQ ID NO:10, SEQ ID N0:45 encodes a protein that is 708 amino acids
long. It is classified as a Cysteineprotease, of the PepC2 family. The
protease
domain in this protein matches the hidden Markov profile for a Calpain family
cysteine protease; Peptidase C2, from amino acid 45 to amino acid 341. The
positions within the I~VEVIR profile that match the protein sequence are from
profile
position 1 to profile position 344. Other domains identified within this
protein are:
Calpain large subunit, domain III, amino acids 353-499, profile from 1-163.
Also
one EF hand motif at amino acids 613-641; EF hand matches from 1-26 of
profile. .
The results of a Smith Waterman search (PAM100, gap open and extend penalties
of
12 and 2) of the public database of amino acid sequences (NRAA) with this
protein
sequence yielded the following results: Pscore = 0; number of identical amino
acids
= 569; percent identity = 79%; percent similarity = 86%; the accession number
of
the most similar entry in NRA.A is CAC10066.1; the name or description, and
species, of the most similar protein in NRAA is: Calpain 12 [Mus musculus].
SGPr290, SEQ ID NO:11, SEQ ID N0:46 encodes a protein that is 711 amino acids
long. It is classified as a Cysteineprotease, of the PepC2 family. The
protease
domain in this protein matches the hidden Markov profile for a Calpain family
cysteine protease; Peptidase C2, from amino acid 43 to amino acid 346. The
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positions within the HIVIMR profile that match the protein sequence are from
profile
position 1 to profile position 344. Other domains identified within this
protein are:
Calpain large subunit, domain III, amino acids 347-490, profile from 1-163.
Also
two EF hand motifs at amino acids 561-593 and 595-622; EF hands match from 1-
26 of profile. . The results of a Smith Waterman search (PAM100, gap open and
extend penalties of 12 and 2) of the public database of amino acid sequences
(NR.AA) with this protein sequence yielded the following results: Pscore =
6.20E-
103; number of identical amino acids = 256; percent identity = 39%; percent
similarity = 56%; the accession number of the most similar entry in NR.AA is
AAD34601.1; the name or description, and species, of the most similar protein
in
NR.A.A is: Lens-specific calpain Lp82 [Oryctolagus cuniculus].
SGPr116, SEQ ID N0:12, SEQ ID N0:47 encodes a protein that is 702 amino acids
long. It is classified as a Cysteineprotease, of the PepC2 family. The
protease
domain in this protein matches the hidden Maxkov profile for a Calpain family
cysteine protease; Peptidase C2, from amino acid 42 to amino acid 341. The
positions within the HMMR profile that match the protein sequence axe from
profile
position 1 to profile position 344. Other domains identified within this
protein are:
Calpain large subunit, domain III, amino acids 352-510, profile from 1-163.
Also
two EF hand motifs at amino acids 577-605 and 607-635; EF hands match from 1-
26 of profile. . The results of a Smith Waterman search (PAM100, gap open and
extend penalties of 12 and 2) of the public database of amino acid sequences
(NR.AA) with this protein sequence yielded the following results: Pscore = 0;
number of identical amino acids = 702; percent identity =100%; percent
similarity =
100%; the accession number of the most similar entry in NRAA is NP_008989.1;
the name or description, and species, of the most similar protein in NRAA is:
Calpain 11 [Homo Sapiens].
SGPr003, SEQ ID N0:13, SEQ ID N0:48 encodes a protein that is 513 amino acids
long. It is classified as a Cysteineprotease, of the PepC2 family. The
protease
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domain in this protein matches the hidden Markov profile for a Calpain family
cysteine protease; Peptidase C2, from amino acid 13 to amino acid 322. The
positions within the HM1VB~ profile that match the protein sequence are from
profile
position 1 to profile position 344. Other domains identified within this
protein are:
Calpain large subunit, domain III, amino acids 338-494, profile from 3-163..
The
results of a Smith Waterman search (PAM100, gap open and extend penalties of
12
and 2) of the public database of amino acid sequences (NRAA) with this protein
sequence yielded the following results: Pscore = 0; number of identical amino
acids
= 513; percent identity = 100%; percent similarity = 100%; the accession
number of
the most similar entry in NR.AA is NP_075574.1; the name or description, and
species, of the most similar protein in NRA.A is: Calpain 10 [Homo sapiens].
SGPr016, SEQ ID NO:14, SEQ ID N0:49 encodes a protein that is 281 amino acids
long. It is classified as a Metalloprotease, of the ADAM family. The protease
domain in this protein matches the hidden Markov profile for a Reprolysin
family
propeptide, Pep M12B_propep, from amino acid 58 to amino acid 175. The
positions within the HMMR profile that match the protein sequence are from
profile
position 1 to profile position 119. Other domains identified within this
protein are:
none. The results of a Smith Waterman search (PAM100, gap open and extend
penalties of 12 and 2) of the public database of amino acid sequences (NRAA)
with
this protein sequence yielded the following results: Pscore =1.30E-89; number
of
identical amino acids = 215; percent identity = 52%; percent similarity = 58%;
the
accession number of the most similar entry in NR.AA is 547656; the~name or
description, and species, of the most similar protein in NRAA is: tMDC II
(ADAM
5-like) protein - crab-eating macaque.
SGPr352, SEQ ID NO:15, SEQ ID NO:50 encodes a protein that is 1103 amino
acids long. It is classified as a Metalloprotease, of the ADAM family. The
protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 239 to amino acid 457. The
positions
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within the HIVP.VIR profile that match the protein sequence are from profile
position
1 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide, from amino acids 90-201, matclung profile from 1-
119. Also five Thrombospondin type 1 domains from 551-601, 829-884, 888-944,
946-1002, 1007-1057. All thrombospondin type 1 domains match profile from 1-
54.. The results of a Smith Waterman search (PAM100, gap open and extend
penalties of 12 and 2) of the public database of amino acid sequences (NRAA)
with
this protein sequence yielded the following results: Pscore = 0; number of
identical
amino acids = 1072; percent identity = 100%; percent similarity = 100%; the
accession number of the most similar entry in NRAA is AAG35563.1; the name or
description, and species, of the most similar protein in NR.AA is: Zinc
metalloendopeptidase [Homo Sapiens].
SGPr050, SEQ ID N0:16, SEQ ID NO:51 encodes a protein that is 1224 amino
acids long. It is classified as a Metalloprotease, of the ADAM family. The
protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 292 to amino acid 495. The
positions
within the HMMR profile that match the protein sequence are from profile
position
3 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide from 111-235, matching profile from 1-119. Also
has
five Thrombospondin type 1 domains from 590-640, 930-986, 990-1047, 1055-
1101, 1128-1180.. The results of a Smith Waterman search (PAM100, gap open and
extend penalties of 12 and 2) of the public database of amino acid sequences
(NR.AA) with this protein sequence yielded the following results: Pscore =
6.80E-
149; number of identical amino acids = 385; percent identity = 37%; percent
similarity = 53%; the accession number of the most similar entry in NRAA is
AAG35563.1; the name or description, and species, of the most similar protein
in
NR.AA is: Zinc metalloendopeptidase [Homo Sapiens].
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SGPr282, SEQ ID N0:17, SEQ ID N0:52 encodes a protein that is 731 amino acids
long. It is classified as a Metalloprotease, of the ADAM family. The protease
domain in this protein matches the hidden Markov profile for a Reprolysin
family
propeptide, Pep Ml2B~ropep, from amino acid 75 to amino acid 190. The
positions within the I~VIMR profile that match the protein sequence are from
profile
position 1 to profile position 119. Other domains identified within this
protein are:
Disintegrin domain at amino acids 415-487; matches profile from 4-86. Also EGF-
like domain at amino acids 633-661.. The results of a Smith Watennan search
(PAM100, gap open and extend penalties of 12 and 2) of the public database of
amino acid sequences (NRAA) with this protein sequence yielded the following
results: Pscore = 0; number of identical amino acids = 619; percent identity =
85%;
percent similarity = 91 %; the accession number of the most similar entry in
NRA.A
is I52361; the name or description, and species, of the most similar protein
in
NRAA is: Metalloproteinase-like, disintegrin-like, cysteine-rich protein IVa
[crab-
eating macaque].
SGPr046, SEQ ID N0:18, SEQ ID N0:53 encodes a protein that is 934 amino acids
long. It is classified as a Metalloprotease, of the ADAM family. The protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 1 to amino acid 194. The
positions
within the HMMR profile that match the protein sequence are from profile
position
13 to profile position 203. Other domains identified within this protein are:
Six
Thrombospondin type 1 domains at 289-339, 569-627, 634-687, 689-736, 769-828,
844-890.. The results of a Smith Waterman search (PAM100, gap open and extend
penalties of 12 and 2) of the public database of amino acid sequences (NRAA)
with
this protein sequence yielded the following results: Pscore = 1.10E-162;
number of
identical amino acids = 320; percent identity = 39%; percent similarity = 56%;
the
accession number of the most similar entry in NRAA is AAG35563.1; the name or
description, and species, of the most similar protein in NR_AA is: Zinc
metalloendopeptidase [Homo sapiens].
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SGPr060, SEQ ID N0:19,. SEQ ID N0:54 encodes a protein that is 1428 amino
acids long. It is classified as a Metalloprotease, of the ADAM family. The
protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 639 to amino acid 860. The
positions
within the HMMR profile that match the protein sequence are from profile
position
1 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide, Pep Ml2B~ropep from amino acids 502-615.
Matches profile from 1-119. Also has one thrombospondin type 1 domain from
954-1004, matching profile from 1-54.. The results of a Smith Waterman search
(PAM100, gap open and extend penalties of 12 and 2) of the public database of
amino acid sequences (NRAA) with this protein sequence yielded the following
results: Pscore = 5.20E-87; number of identical amino acids = 250; percent
identity
= 39%; percent similarity = 55%; the accession number of the most similar
entry in
NR.AA is NP_055087.1; the name or description, and species, of the most
similar
protein in NR.A.A is: Disintegrin-like and metalloprotease with thrombospondin
type
1 motif, 7 [Homo sapiens].
SGPr068, SEQ ID N0:20, SEQ ID NO:55 encodes a protein that is 1186 amino
acids long. It is classified as a Metalloprotease, of the ADAM family. The
protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 261 to amino acid 460. The
positions
within the HMMR profile that match the protein sequence are from profile
position
3 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide, Pep Ml2B~ropep from amino acids 120-240,
matching profile from 1-119. Also has four thrombospondin type 1 domains
between 556 - 1021.. The results of a Smith Waterman search (PAM100, gap open
and extend penalties of 12 and 2) of the public database of amino acid
sequences
(NRAA) with this protein sequence yielded the following results: Pscore = 0;
number of identical amino acids = 624; percent identity = 64%; percent
similarity =
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77%; the accession number of the most similar entry in NRAA is 015072; the
name
or description, and species, of the most similar protein in NR.AA is: ADAM-TS
3
PRECURSOR [Homo sapiens].
SGPr096, SEQ ID N0:21, SEQ ID N0:56 encodes a protein that is 1935 amino
acids long. It is classified as a Metalloprotease, of the ADAM family. The
protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 293 to amino acid 499. The
positions
within the HMMR profile that match the protein sequence are from profile
position
1 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide, Pep Ml2B~ropep from amino acids 112-242,
matching profile from 1-119. Also has 13 thrombospondin type 1 domains between
589 - 1733.. The results of a Smith Waterman search (PAM100, gap open and
extend penalties of 12 and 2) of the public database of amino acid sequences
(NRAA) with this protein sequence yielded the following results: Pscore = 0;
number of identical amino acids = 1465; percent identity = 100%; percent
similarity
= 100%; the accession number of the most similar entry in NRAA is BAA92550.1;
the name or description, and species, of the most similar protein in NR.A.A
is:
KIAA1312 protein [Homo Sapiens].
SGPr119, SEQ ID N0:22, SEQ ID N0:57 encodes a protein that is 1505 amino
acids long. It is classified as a Metalloprotease, of the ADAM family. The
protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 259 to amino acid 467. The
positions
within the HMMR profile that match the protein sequence are from profile
position
1 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide, Pep Ml2B~ropep from amino acids 92-215,
matching profile from 1-119. Also has eight thrombospondin type 1 domains
between 561 - 1416.. The results of a Smith Waterman search (PAM100, gap open
and extend penalties of 12 and 2) of the public database of amino acid
sequences
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(NRAA) with this protein sequence yielded the following results: Pscore = 0;
number of identical amino acids = 699; percent identity = 53%; percent
similarity =
70%; the accession number of the most similar entry in NRAA is BAA92550.1; the
name or description, and species, of the most similar protein in NR.AA is:
KIAA1312 (ADAMS 9-like) protein [Homo sapiens].
SGPr143, SEQ ID N0:23, SEQ ID N0:58 encodes a protein that is 882 amino acids
long. It is classified as a Metalloprotease, of the ADAM family. The protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 275 to amino acid 478. The
positions
within the HMMR profile that match the protein sequence are from profile
position
1 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide, Pep Ml2B~ropep from amino acids 145-263,
matching profile from 1-119. Also has Disintegrin motif 495-570.. The results
of a
Smith Waterman search (PAM100, gap open and extend penalties of 12 and 2) of
the public database of amino acid sequences (hTRAA) with this protein sequence
yielded the following results: Pscore = 0; number of identical amino acids =
726;
percent identity = 99%; percent similarity = 99%; the accession number of the
most
similar entry in NRAA is CAC16509.2; the name or description, and species, of
the
most similar protein in NR.AA is: Novel disintegrin and reprolysin
metalloproteinase
[Homo Sapiens].
SGPr164, SEQ ID N0:24, SEQ ID N0:59 encodes a protein that is 978 amino acids
long. It is classified as a Metalloprotease, of the ADAM family. The protease
domain in this protein matches the ludden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 243 to amino acid 452. The
positions
within the HIVIZVIR profile that match the protein sequence are from profile
position
1 to profile position 203. Other domains identified within this protein are:
Reprolysin family propeptide, Pep Ml2B~ropep from amino acids 92-206,
matching profile from 1-119. Also has three Thrombospondin type 1' domains
from
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amino acids 545 to 978. Also has Glucose-6-phosphate dehydrogenase motif at
855-
878.. The results of a Smith Waterman search (PAM100, gap open and extend
penalties of 12 and 2) of the public database of amino acid sequences (NRAA)
with
this protein sequence yielded the following results: Pscore =1.80E-264; number
of
identical amino acids = 465; percent identity = 50%; percent similarity = 67%;
the
accession number of the most similar entry in NRAA is XP_012978.1; the name or
description, and species, of the most similar protein in NRAA is: ADAMS-1
preproprotein [Homo Sapiens].
SGPr281, SEQ ID N0:25, SEQ ID N0:60 encodes a protein that is 1094 amino
acids long. It is classified as a Metalloprotease, of the ADAM family. The
protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 317 to amino acid 432. The
positions
within the H1VIMR profile that match the protein sequence are from profile
position
89 to profile position 203. Other domains identified within this protein are:
Six
Thrombospondin type 1 domains from amino acid 346 to 1030.. The results of a
Smith Waterman search (PAM100, gap open and extend penalties of 12 and 2) of
the public database of amino acid sequences (NR.A.A) with this protein
sequence
yielded the following results: Pscore = 4.4e-075; number of identical amino
acids =
287; percent identity = 39%; percent similarity = 55%; the accession number of
the
most similar entry in NRAA is NP_112217.1; the name or description, and
species,
of the most similar protein in NRAA is: ADAMTS 12 [Homo Sapiens].
SGPr075, SEQ ID N0:26, SEQ ID N0:61 encodes a protein that is 125 amino acids
long. It is classified as a Metalloprotease, of the ADAM family. The protease
domain in this protein matches the hidden Markov profile for a Reprolysin
(M12B)
family zinc metalloprotease, from amino acid 1 to amino acid 123. The
positions
within the I~VM2 profile that match the protein sequence are from profile
position
14 to profile position 203. Other domains identified within this protein are:
none.
The results of a Smith Waterman search (PAM100, gap open and extend penalties
of
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12 and 2) of the public database of amino acid sequences (NRAA) with this
protein
sequence yielded the following results: Pscore =1.10E-54; number of identical
amino acids = 98; percent identity = 65%; percent similarity = 73%; the
accession
number of the most similar entry in NR.AA is CAC18729; the name or
description,
and species, of the most similar protein in NRAA is:
Metalloprotease/disintegrin
[Rattus norvegicus].
SGPr292, SEQ ID N0:27, SEQ ID N0:62 encodes a protein that is 569 amino acids
long. It is classified as a Metalloprotease, of the PepMlO family. The
protease
domain in this protein matches the hidden Markov profile for a Peptidase M10,
Matrixin, from amino acid 56 to amino acid 267. The positions within the
HIVIMR
profile that match the protein sequence are from profile position 1 to profile
position
171. Other domains identified within this protein are: Also has four Hemopexin
domains at amino acids 333-391, 394-449, 451-499, 506-549.. The results of a
Smith Waterman search (PAM100, gap open and extend penalties of 12 and 2) of
the public database of amino acid sequences (NRAA) with this protein sequence
yielded the following results: Pscore = 6.00E-137; number of identical amino
acids
= 333; percent identity = 57%; percent similarity = 74%; the accession number
of
the most similar entry in NR.A.A is AAC21447.1; the name or description, and
species, of the most similar protein in NR.AA is: Matrix metalloproteinase
[Xenopus
laevis].
SGPr069, SEQ ID N0:28, SEQ ID N0:63 encodes a protein that is 743 amino acids
long. It is classified as a Metalloprotease, of the PepMl3 family. The
protease
domain in this protein matches the hidden Markov profile for a Peptidase
family
M13, from amino acid 535 to amino acid 742. The positions witlun the HMlVm
profile that match the protein sequence are from profile position 1 to profile
position
225. Other domains identified within this protein are: None. The results of a
Smith
Waterman search (PAM100, gap open and extend penalties of 12 and 2) of the
public database of amino acid sequences (NRAA) with this protein sequence
yielded
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the following results: Pscore = 0; number of identical amino acids = 581;
percent
identity = 78%; percent similarity = 90%; the accession number of the most
similar
entry in NR.AA is AAG18446.1; the name or description, and species, of the
most
similar protein in NRAA is: Neprilysin-like peptidase alpha [Mus musculus].
This
protein is predicted to have a transmembrane helix between amino acids 13 and
35.
This transmembrane region could function as a signal peptide. (TMF3MM, a
Hidden
Markov Model based transmenbrane prediction program, Sonnhammer, et al Proc.
of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182
AAAI
Press, 1998.)
SGPr212, SEQ ID N0:29, SEQ ID N0:64 encodes a protein that is 909 amino acids
long. It is classified as a Metalloprotease, of the PepMl family. The protease
domain in this protein matches the hidden Markov profile for a Peptidase
family
M1, from amino acid 275 to amino acid 306. The positions within the HMMR
profile that match the protein sequence are from profile position 343 to
profile
position 374. Other domains identified within this protein are: None. The
results of
a Smith Waterman search (PAM100, gap open and extend penalties of 12 and 2) of
the public database of amino acid sequences (NRAA) with this protein sequence
yielded the following results: Pscore =1.40E-31; number of identical amino
acids =
55; percent identity = 77%; percent similarity = 87%; the accession number of
the
most similar entry in NRAA is BAB25647.1; the name or description, and
species,
of the most similar protein in NR.A.A is: Probable zinc metal proteinase [Mus
musculus] .
SGPr049, SEQ ID N0:30, SEQ ID N0:65 encodes a protein that is 990 amino acids
long. It is classified as a Metalloprotease, of the PepMl family. The protease
domain in this protein matches the hidden Markov profile for a Peptidase
family
M1, from amino acid 98 to amino acid 506. The positions within the I~VM2
profile
that match the protein sequence are from profile position 1 to profile
position 441.
Other domains identified within this protein are: None. The results of a Smith
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Waterman search (PAM100, gap open and extend penalties of 12 and 2) of the
public database of amino acid sequences (NRAA) with this protein sequence
yielded
the following results: Pscore = 4.10E-220; number of identical amino acids =
375;
percent identity = 68%; percent similarity = 79%; the accession number of the
most
similar entry in NR.A.A is BAB29490.1; the name or description, and species,
of the
most similar protein in NR.AA is: Putative aminopeptidase [Mus musculus]. This
protein is predicted to have a transmembrane helix between amino acids 13 and
35.
This transmembrane region could function as a signal peptide. (TMHMM, a Hidden
Markov Model based transmenbrane prediction program, Sonnhammer, et al Proc.
of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182
AAAI
Press, 1998)
SGPr026, SEQ ID N0:31, SEQ ID N0:66 encodes a protein that is 650 amino acids
long. It is classified as a Metalloprotease, of the PepMl family. The protease
domain in this protein matches the hidden Markov profile for a Peptidase
family
Ml, from amino acid 32 to amino acid 417. The positions within the HMMR
profile
that match the protein sequence are from profile position 1 to profile
position 441.
Other domains identified within this protein are: None. The results of a Smith
Waterman search (PAM100, gap open and extend penalties of 12 and 2) of the
public database of amino acid sequences (hIRA.A) with this protein sequence
yielded
the following results: Pscore = 0; number of identical amino acids = 650;
percent
identity = 100%; percent similarity =100%; the accession number of the most
similar entry in NRAA is AAH01064; the name or description, and species, of
the
most similar protein in NRAA is: Hypothetical protein DKFZp547H084 [Homo
Sapiens].
SGPr203, SEQ ID N0:32, SEQ ID N0:67 encodes a protein that is 724 amino acids
long. It is classified as a Metalloprotease, of the PepMl family. The protease
domain in this protein matches the hidden Markov profile for a Peptidase
family
Ml, from amino acid I94 to amino acid 444. The positions within the I~VIMR
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profile that match the protein sequence are from profile position 161 to
profile
position 441. Other domains identified within this protein are: None. The
results of
a Smith Waterman search (PAM100, gap open and extend penalties of 12 and 2) of
the public database of amino acid sequences (NRAA) with this protein sequence
yielded the following results: Pscore =1.90E-276; number of identical amino
acids
= 493; percent identity =100%; percent similarity =100%; the accession number
of
the most similar entry in NRAA is AAG22080.1; the name or description, and
species, of the most similar protein in NRAA is: RNPEP-like protein [Homo
sapiens].
SGPr157, SEQ ID N0:33, SEQ ID N0:68 encodes a protein that is 507 amino acids
long. It is classified as a Metalloprotease, of the PepM20 family. The
protease
domain in this protein matches the hidden Markov profile for a Peptidase M20 ,
from amino acid 106 to amino acid 450. The positions within the HMMR profile
that match the protein sequence are from profile position 42 to profile
position 368.
Other domains identified within this protein are: None. The results of a Smith
Waterman search (PAM100, gap open and extend penalties of 12 and 2) of the
public database of amino acid sequences (NRAA) with this protein sequence
yielded
the following results: Pscore = 7.50E-202; number of identical amino acids =
310;
percent identity = 100%; percent similarity = 100%; the accession number of
the
most similar entry in NR.A.A is AAH04271.1; the name or description, and
species,
of the most similar protein in NR.A.A is: Hypothetical protein [Homo Sapiens].
SGPr154, SEQ ID N0:34, SEQ ID N0:69 encodes a protein that is 473 amino acids
long. It is classified as a Metalloprotease of the PepM20 family. The protease
domain in this protein matches the hidden Markov profile for a Peptidase M20,
from amino acid 55 to amino acid 286. The positions within the profile that
match the protein sequence are from profile position 1 to profile position
247. Other
domains identified within this protein are: None. The results of a Smith
Waterman
search (PAM100, gap open and extend penalties of 12 and 2) of the public
database
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of amino acid sequences (I~TRAA) with this protein sequence yielded the
following
results: Pscore = 1.90E-28; number of identical amino acids = 122; percent
identity
= 31 %; percent similarity = 48%; the accession number of the most similar
entry in
NR.A.A is AAK22721.1; the name or description, and species, of the most
similar
protein in NRAA is: M20/M25/M40 family peptidase [Caulobacter crescentus].
SGPr088, SEQ ID N0:35, SEQ ID N0:70 encodes a protein that is 475 amino acids
long. It is classified as a Metalloprotease of the PepM20 family. The protease
domain in this protein matches the hidden Markov profile for a Peptidase M20,
from amino acid 22 to amino acid 417. The positions within the HMMR profile
that
match the protein sequence are from profile position 1 to profile position
368. Other
domains identified within this protein are: None. The results of a Smith
Waterman
search (PAM100, gap open and extend penalties of 12 and 2) of the public
database
of amino acid sequences (NR.AA) with this protein sequence yielded the
following
results: Pscore = 9.8e-315; number of identical amino acids = 475; percent
identity =
100%; percent similarity =100%; the accession number of the most similar entry
in
NRA.A is XP-008819.1; the name or description, and species, of the most
similar
protein in NR.A.A is: Hypothetical protein FLJ10830 [Homo sapiens].
EXAMPLE 3: Isolation of cDNAs Encoding Mammalian Proteases
Materials and Methods
Identification of novel clones
Total RNAs are isolated using the Guanidine Salts/Phenol extraction
protocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal.
Biochem. 162:156 (1987)) from primary human tumors, normal and tumor cell
lines,
normal human tissues, and sorted human hematopoietic cells. These RNAs are
used
to generate single-stranded cDNA using the Superscript Preamplification System
(GIBCO BRL, Gaithersburg, MD; Geraxd, GF et al. (1989), FOCUS 11, 66) under
conditions recommended by the manufacturer. A typical reaction uses 10 ~,g
total
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RNA with 1.5 ~,g oligo(dT)la-la in a reaction volume of 60 ~,L. The product is
treated with RNaseH and diluted to 100 pL with H20. For subsequent PCR
amplification, 1-4 ~,L of this sscDNA is used in each reaction.
Degenerate oligonucleotides are synthesized on an Applied Biosystems 3948
DNA synthesizer using established phosphoramidite chemistry, precipitated with
ethanol and used unpurified for PCR. These primers are derived from the sense
and
antisense strands of conserved motifs within the catalytic domain of several
proteases. Degenerate nucleotide residue designations are: N = A, C, G, or T;
R =
Aorta;Y=CorT;H=A,CorTnotG;D=A,GorTnotC;S=CorG;andW=
AorT.
PCR reactions are performed using degenerate primers applied to multiple
single-stranded cDNAs. The primers are added at a final concentration of 5 ~,M
each to a mixture containing 10 mM TrisHCl, pH 8.3, 50 mM KCI, 1.5 mM MgCl2,
200 p.M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA
Polymerase (Perkin-Elmer/Cetus), and 1-4 ~,L cDNA. Following 3 min
denaturation
at 95 °C, the cycling conditions are 94 °C for 30 s, 50
°C for 1 min, and 72 °C for 1
min 45 s for 35 cycles. PCR fragments migrating between 300-350 by are
isolated
from 2% agarose gels using the GeneClean Kit (Bio101), and T-A cloned into the
pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's
protocol.
Colonies are selected for mini plasmid DNA-preparations using Qiagen
columns and the plasmid DNA is sequenced using a cycle sequencing dye-
terminator kit with AmpliTaq DNA Polyrnerase, FS (ABI, Foster City, CA).
Sequencing reaction products are run on an ABI Prism 377 DNA Sequencer, and
analyzed using the BLAST alignment algorithm (Altschul, S.F. et al.,
J.Mol.Biol.
215:403-10).
Additional PCR strategies are employed to connect various PCR fragments
or ESTs using exact or near exact oligonucleotide primers. PCR conditions are
as
described above except the annealing temperatures are calculated for each
oligo pair
using the formula: Tm = 4(G+C)+2(A+T).
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Isolation of cDNA clones:
Human cDNA libraries are probed with PCR or EST fragments
corresponding to protease-related genes. Probes are 32P-labeled by random
priming
and used at 2x106 cpm/mL following standard techniques for library screening.
Pre-
hybridization (3 h) and hybridization (overnight) are conducted at 42
°C in 5X SSC,
5X Denhart's solution, 2.5% dextran sulfate, 50 mM NaaP04/NaHP04, pH 7.0, 50%
formamide with 100 mg/mL denatured salmon sperm DNA. Stringent washes are
performed at 65 °C in O.1X SSC and 0.1% SDS. DNA sequencing is carried
out on
both strands using a cycle sequencing dye-terminator kit with AmpliTaq DNA
Polymerase, FS (ABI, Foster City, CA). Sequencing reaction products are run on
an
ABI Prism 377 DNA Sequencer.
EXAMPLE 4: Expression Analysis of Mammalian Proteases
Materials and Methods
Northern blot analysis
Northern blots are prepared by running 10 ~.g total RNA isolated from 60
human tumor cell lines (such as HOP-92, EKVX, NCI-H23, NCI-H226, NCI-
H322M, NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-4, OVCAR-5,
OVCAR-8, IGROV1, SK-OV-3, SNB-19, SNB-75, U251, SF-268, SF-295, SF-539,
CCRF-CEM, K-562, MOLT-4, HL-60, RPMI 8226, SR, DU-145, PC-3, HT-29,
HCC-2998, HCT-116, SW620, Colo 205, HTC15, KM-12, UO-31, SN12C, A498,
CaKil, RXF-393, ACHN, 786-0, TK-10, LOX IMVI, Maline-3M, SK-MEL-2, SK-
MEL-5, SIB-MEL-28, UACC-62, UACC-257, M14, MCF-7, MCF-7/ADR RES,
Hs578T, MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D), from human
adult tissues (such as thymus, lung, duodenum, colon, testis, brain,
cerebellum,
cortex, salivary gland, liver, pancreas, kidney, spleen, stomach, uterus,
prostate,
skeletal muscle, placenta, mammary gland, bladder, lymph node, adipose
tissue),
and 2 human fetal normal tissues (fetal liver, fetal brain ), on a denaturing
formaldehyde 1.2% agarose gel and transferring to nylon membranes.
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Filters are hybridized with random primed [oc32P]dCTP-labeled probes
synthesized from the inserts of several of the protease genes. Hybridization
is
performed at 42 °C overnight in 6X SSC, 0.1% SDS, 1X
Denhardt's'solution, 100
~,g/mL denatured herring sperm DNA with 1-2 x 106 cpm/mL of 32P-labeled DNA
probes. The filters are washed in O.1X SSC/0.1% SDS, 65 °C, and exposed
on a
Molecular Dynamics phosphorimager.
Quantitative PCR analysis
RNA is isolated from a variety of normal human tissues and cell lines.
Single stranded cDNA is synthesized from 10 ~,g of each RNA as described above
using the Superscript Preamplification System (GibcoBRL). These single strand
templates are then used in a 25 cycle PCR reaction with primers specific to
each
clone. Reaction products are electrophoresed on 2% agarose gels, stained with
ethidium bromide and photographed on a UV light box. The relative intensity of
the
STK-specific bands were estimated for each sample.
DNA Array Based Expression Analysis
Plasmid DNA array blots are prepared by loading 0.5 ~,g denatured plasmid
for each protease on a nylon membrane. The [y3aP]dCTP labeled single stranded
DNA probes are synthesized from the total RNA isolated from several human
immune tissue sources or tumor cells (such as thymus, dendrocytes, mast cells,
monocytes, B cells (primary, Jurkat, RPMI8226, SR), T cells (CD8/CD4+, TH1,
TH2, CEM, MOLT4), K562 (megakaryocytes). Hybridization is performed at 42
°C
for 16 hours in 6X SSC, 0.1% SDS, 1X Denhardt's solution, 100 ~,g/mL denatured
herring sperm DNA with 106 cpm/mL of [y3zP]dCTP labeled single stranded probe.
The filters are washed in O.1X SSC/0.1% SDS, 65 °C, and exposed for
quantitative
analysis on a Molecular Dynamics phosphorimager.
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EXAMPLE 5: Protease Gene Ex reb scion
Vector Construction
Materials and Methods
Expression Vector Construction
Expression constructs are generated for some of the human cDNAs
including: a) full-length clones in a pCDNA expression vector; and b) a GST-
fusion
construct containing the catalytic domain of the novel protease fused to the C-
terminal end of a GST expression cassette; and c) a full-length clone
containing a
mutation within the predicted polypeptide cleaving site within the protease
domain,
inserted in the pCDNA vector.
These mutants of the protease might function as dominant negative
constructs, and will be used to elucidate the function of these novel
proteases.
EXAMPLE 6: Generation of Specific Immunorea~ents to Proteases
Materials and Methods
Specific immunoreagents are raised in rabbits against KLH- or MAP-
conjugated synthetic peptides corresponding to isolated protease polypeptides.
C-
terminal peptides were conjugated to KLH with glutaraldehyde, leaving a free C-
terminus. Internal peptides were MAP-conjugated with a blocked N-terminus.
Additional immunoreagents can also be generated by immunizing rabbits with the
bacterially expressed GST-fusion proteins containing the cytoplasmic domains
of
each novel PTK or STK.
The various immune sera are first tested for reactivity and selectivity to
recombinant protein, prior to testing for endogenous sources.
Western blots
Proteins in SDS PAGE are transferred to immobilon membrane. The
waslung buffer is PBST (standard phosphate-buffered saline pH 7.4 + 0.1%
Triton
X-100). Blocking and antibody incubation buffer is PBST +5% milk. Antibody
dilutions are varied from 1:1000 to 1:2000.
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EXAMPLE 7: Recombinant Expression and Biological Assays for Proteases
Materials and Methods
Transient Expression of Proteases in Mammalian Cells
The pcDNA expression plasmids (10 ~,g DNA/100 mm plate) containing the
protease constructs are introduced into 293 cells with lipofectamine (Gibco
BRL).
After 72 hours, the cells are harvested in 0.5 mL solubilization buffer (20 mM
HEPES, pH 7.35, 150 mM NaCI, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1
mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ~,g/mL aprotinin). Sample
aliquots are resolved by SDS polyacrylamide gel electrophoresis (PAGE) on 6%
acrylamide/0.5% bis-acrylamide gels and electrophoretically transferred to
nitrocellulose. Non-specific binding is blocked by preincubating blots in
Blotto
(phosphate buffered saline containing 5% w/v non-fat dried milk and 0.2% v/v
nonidet P-40 (Sigma)), and recombinant protein is detected using the various
anti-
peptide or anti-GST-fusion specific antisera.
Ih Vitro Protease Assays
In vitro Protease Assay Using Fluoro~enic Peptides
Assays are carried out using a spectrofluorometer, such as Perkin-Eliner
2045. The standard reaction mixtures (100 ~wl) contains 200 mM Tris-HCI,
pH8.5,
and 200 ~M fluorogenic peptide substrate. After enzyme addition, reaction
mixtures
are incubated at 37 °C for 30 min and terminated by addition of 1.9 ml
of 125 rnM
ZnS04 (Brenner, C., and Fuller, R. S., 1992, P~oc. Natl. Acad. Sci. U. S. A.
89:922-
926). The precipitate is removed by centrifugation for 1 min in a
microcentrifuge
(15,000 X g), and the rate of product (7-amino-4-methyl-coumarin) released
into the
supernatant solution is determined fluorometrically [ (excitation) = 385 nm,
(emission) = 465 nm]. Examples of substrates used in the literature include:
Boc-
Gly-Arg-Arg-4-methylcoumaryl-7-amide (MCA), Boc-Gln-Arg-Arg-MCA, Z-Arg-
Arg-MCA, and pGlu-Arg-Thr-Lys-Arg-MCA. Stock solutions (100 mM) are
prepared by dissolving peptides in dimethyl sulfoxide that are then diluted in
water
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to 1 mM working stock before use. (Details of this assay can be found in: R.
Yosuf,
et al. J. Biol. Chem., Vol. 275, Tssue 14, 9963-9969, April 7, 2000 which is
incorporated herein by reference in its entirety including any figures,
tables, or
drawings.)
Protease assa~in intact cells using fluoro epic peptides-
Calpain activity is measured by the rate of generation of the fluorescent
product, AMC, from intracellular thiol-conjugated Boc-Leu-Met-CMAC (Rosser, B.
G., Powers, S. P., and Gores, G. J. (1993) J. Biol. Chem. 268, 23593-23600).
Cells
are dispersed, grown on glass coverslips, continuously superfused with
physiologic
saline solution at 37 °C, and sequentially imaged with a quantitative
fluorescence
imaging system. At t = 0, Boc-Leu-Met-CMAC (10 ~,M, Molecular Probes) is
introduced into the superfusion solution, and mean fluorescence intensity
(excitation
350 nm, emission 470 nm) of individual cells is measured at 60-s intervals. At
10
min, TNF- (30 ng/ml) is added to the superfusion solution with 10 ~M Boc-Leu-
Met-CMAC. The slope of the fluorescence change with respect to time represents
the intracellular calpain activity (Rosser, et al., 1993, J. Biol. Chen2.
268:23593-
23600). For calpain assays in whole cell populations, suspension cultures of
cells
are loaded with 10 ~M Boc-Leu-Met-CMAC, and changes in intracellular
fluorescence are measured prior to and after TNFalpha addition at 37 °C
using a
FAGS Vantage system. Cellular fluorescence of AMC is measured using a 360-nm
excitation filter and a 405-nm long-pass emission filter. (Details of this
assay can be
found in: Han, et al., 1999, JBiol Chem, 274:787-794 which is incorporated
herein
by reference in its entirety including any figures, tables, or drawings)
Protease assay using chromo.~enic substrates
The proteolytic activity of enzymes is measured using a commercially
available assay system (Athena Environmental Sciences, Inc.). The assay
employs a
universal substrate of a dye-protein conjugate cross linked to a matrix.
Protease
activity is determined spectrophotometrically by measuring the absorbance of
the
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dye released from the matrix to the supernatant. Reaction vials containing the
enzyme and substrate are incubated for 3 h at 37 °C. The activity is
measured at
different incubation times, and reactions are terminated by adding 500 ~,1 of
0.2 N
NaOH to each vial. The absorbance of the supernatant in each reaction vial is
measured at 450 nm. The proteolytic activity is monitored using 10 ~1
(approximately 10 fig) of purified protein incubated with 5 ~,g of -casein
(Sigma) in
50 mM Tris-HCl (pH 7.5) for 30 min, 1 h or 2 h at 37 °C. The reaction
products are
resolved by SDS-polyacrylamide gel electrophoresis and proteins visualized by
staining with Coomassie Blue (Details of this assay can be found in: Faccio,
et al.,
2000, JBiol Chem, 275:2581-2588 which is incorporated herein by reference in
its
entirety including any figures, tables, or drawings).
Protease assay using radiolabeled substrate bound to membranes-
Unlabeled protease is mixed with radiolabeled substrate-containing
membranes in buffer (100 mM HEPES, 100 mM NaCI, 125 ~M magnesium acetate,
125 wM zinc acetate, pH 7.5) and incubated at 30 °C. Typically, each
reaction had a
final volume of 80-100 ~,1. Each reaction is normalized to the same final
concentration of lysis buffer components (25 mM Tris, 0.1 M sorbitol, 0.5 mM
EDTA, 0.01 % NaN3, pH 7.5) because the amount of membranes added to each
reaction is varied. To examine metal ion specificity, reactions are assembled
without substrate and pretreated with 1.125 mM 1,10-orthophenanthroline for 20
min on ice. Subsequently, metal ions and substrate-containing membranes are
added, and reactions are initiated by incubation at 30 °C; the
additions result in
dilution of the 1,10-orthophenanthroline to a final concentration of 1 mM. The
metal
ions are added in the form of acetate salts from 25-100 mM stock solutions
(Zn2+,
Mga+, Cu2+, Coa+, or Ca2+) that are first acidified with 2 mM concentrated HCl
and
then neutralized with 1 mM HEPES, pH 7.5; this step is necessary to achieve
full
solubilization of zinc acetate. For analysis by immunoprecipitation, samples
are
diluted 10-20~ with immunoprecipitation buffer (Berkower, C., and Michaelis,
S.
(1991) EMBO J. 10:3777-3785) containing 0.1% SDS, cleared of insoluble
material
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(13,000 ~ g for 5-10 min at 4 °C), and immunoprecipitated with
substrate-specific
antibody. Alternatively, samples are solubilized by SDS (final concentration,
0.5%),
boiled for 3 min, and directly immunoprecipitated after dilution with
immunoprecipitation buffer. Immunoprecipitates are subjected to SDS-
polyacrylamide gel electrophoresis as described, fixed for 7 min with 20%
trichloroacetic acid, dried, and exposed to a PhosphorImager screen for
detection
and quantitation (Molecular Dynamics, Sunnyvale, CA). All of the above
reagents
can be purchased from Sigma. (Details of this assay can be found in: Schmidt,
et
al., 2000, JBiol Chem, 275:6227-6233 which is incorporated herein by reference
in
its entirety including any figures, tables, or drawings). Variation of this
assay to
apply to substrate not bound to membrane is straightforward.
A comprehensive discussion of various protease assays can be found in: The
Handbook of Proteolytic Enzymes by Alan J. Barrett (Editor), Neil D. Rawlings
(Editor), J. Fred Woessner (Editor) (February 1998) Academic Press, San Diego;
ISBN: 0-12-079370-9 (which is incorporated herein by reference in its entirety
including any figures, tables, or drawings).
Similar assays are performed on bacterially expressed GST-fusion constructs
of the proteases.
EXAMPLE 8a: Chromosomal Localization of Proteases
Materials And Methods
Several sources were used to find information about the chromosomal
localization of
each of the genes described in this patent application. First, -cytogenetic
map
locations of these contigs were found in the title or text of their Genbank
record, or
by inspection through the NCBI human genome map viewer
(http://www.ncbi.nhn.nih.gov/cgi-bin/Entrez/hum srch?). Alternatively, the
accession number of a genomic contig (identified by BLAST against NRNA) was
used to query the Entrez Genome Browser
(http://www.ncbi.nhn.nih.gov/PMGifs/Genomes/MapViewerHelp.html ), and the
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cytogenetic localization was read from the NCBI data. A thorough search of
available literature for the cytogenetic region is also made using Medline
(http://www.ncbi.nhn.nih.gov/PubMedlmedline.html). References for association
of
the mapped sites with chromosomal amplifications found in human cancer can be
found in: Knuutila, et al., Am J Pathol, 1998, 152:1107-1123.
Results
The chromosomal regions for mapped genes are listed Table 2. The chromosomal
positions were cross-checked with the Online Mendelian Inheritance in Man
database (OMIM, http://www.ncbi.nhn.nih.~ov/htbin-post/Omim)., which tracks
genetic information for many human diseases, including cancer. References for
association of the mapped sites with chromosomal abnormalities found in human
cancer can be found in: Knuutila, et al., Am J Pathol, 1998, 152:1107-1123. A
third
source of information on mapped positions was searching published literature
(at
NCBI, http://www.ncbi.nlm.nih.~ov/entrez/dueuy.fc~i) for documented
association
of the mapped position with human disease.
The following section describes various diseases that map to chromosomal
locations established for proteases included in this patent application. The
protease
polynucleotides of the present invention can be used to identify individuals
who
have, or are at risk for developing, relevant diseases. As discussed elsewhere
in this
application, the polypeptides and polynucleotides of the present invention are
useful
in identifying compounds that modulate protease activity, and in turn
ameliorate
various diseases.
SGPr140 SEQ ID NO:1 1p33/1p13.3
Novel recurrent genetic imbalances in human hepatocellular carcinoma cell
lines
identified by comparative genomic hybridization. (Hepatology. 1999
Apr;29(4):1208-14.) Chromosome 1 alterations in breast cancer: allelic loss on
1p
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and 1q is related to lymphogenic metastases and poor prognosis. (Genes
Chromosomes Cancer. 1992 Nov;S(4):311-20.).
SGPr197 SEQ ID N0:2 6p21.1
Genetic imbalances with impact on survival in head and neck cancer patients.
(Am J
Pathol. 2000 Aug;157(2):369-75.). Systematic screening of the LDL-PLA2 gene
for
polymorphic variants and case-control analysis in schizophrenia. (Biochem
Biophys
Res Commun. 1997 Dec 29;241(3):630-5.)
SGPr005 SEQ ID N0:3 1p33
Novel recurrent genetic imbalances in human hepatocellular carcinoma cell
lines
identified by comparative genomic hybridization. (Hepatology. 1999
Apr;29(4):1208-14.) Chromosome 1 alterations in breast cancer: allelic loss on
1p
and 1q is related to lymphogenic metastases and poor prognosis. (Genes
Chromosomes Cancer. 1992 Nov;S(4):311-20.).
SGPr078 SEQ ID N0:4 11p15
Use of horizontal ultrathin gel electrophoresis to analyze allelic deletions
in
chromosome band 11p15.5 in gliomas. (Neuro-oncol. 2000 Jan;2(1):1-5.).
Loss of heterozygosity and heterogeneity of its appearance and persisting in
the
course of acute myeloid leukemia and myelodysplastic syndromes. (Leak Res.
2001
Jan;25(1):45-53.). Chromosomal localization of two genes underlying late-
infantile
neuronal ceroid lipofuscinosis. (Neurogenetics. 1998 Mar;l(3):217-22.).
The usher syndromes also map to this location. (Am J Med Genet. 1999 Sep
24;89(3):158-66.)
SGPr084.2 SEQ ID NO:S 12q11
Fine genetic mapping of diffuse non-epidermolytic palinoplantar keratoderma to
chromosome 12q11-q13: exclusion of the mapped type II keratins. (Exp Dermatol.
1999 Oct;B(5):388-91.).
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SGPr009 SEQ ID N0:6 l 1q22
Restricted chromosome breakpoint sites on 11 q22-q23.1 and 11 q25 in various
hematological malignancies without MLL/ALL-1 gene rearrangement. (Cancer
Genet Cytogenet. 2001 Jan 1;124(1):27-35.). Molecular characterization of
deletion
at 11q22.I-23.3 in mantle cell lymphoma. (Br J Haematol. 1999 Mar;104(4):665-
71.). Structure and chromosome localization of the human CASP8 gene
(implicated
in tumorigenesis, with loss of heterogeneity (LOH)). (Gene. 1999 Jan
21;226(2):225-32. Reduced expression of adhesion molecules and cell signaling
receptors by chronic lymphocytic leukemia cells with l 1q deletion. (Blood.
1999 Jan
15;93(2):624-31.).
SGPr286 SEQ ID N0:7 16p13.3
Monosomy for the most telomeric, gene-rich region of the short arm of human
chromosome 16 causes minimal phenotypic effects (Eur J Hum Genet. 2001
Mar;9(3):217-225.). Identification of a subtle t(16;19)(p13.3;p13.3) in an
infant
with multiple congenital abnormalities using a 12-colour multiplex FISH
telomere
assay, M-TEL. (Eur J Hum Genet. 2000 Dec;B(12):903-10). Familial Mediterranean
fever in the 'Chuetas' of Mallorca: a question of Jewish origin or genetic
heterogeneity (Eur J Hum Genet. 2000 Apr;B(4):242-6.). Familial mental
retardation syndrome ATR-16 due to an inherited cryptic subtelomeric
translocation,
t(3;16)(q29;p13.3) (Am J Hum Genet. 2000 Jan;66(1):16-25). Autosomal dominant
polycystic kidney disease: clues to pathogenesis. (Hum Mol Genet.
1999;8(10):1861-6. Review).
SGPr008 SEQ ID N0:8 2p23
Familial syndromic esophageal atresia (Am J Hum Genet. 2000 Feb;66(2):436-
44.).
Chromosomal rearrangements in acute myelogenous leukemia involving loci on
chromosome (Leukemia. 1999 Oct;l3(10):1534-8.). Association and linkage
analysis of candidate chromosomal regions in multiple sclerosis: indication of
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disease genes in disease genes in 12q23 and 7ptr-15 (Eur J Hum Genet. 1999 Feb-
Mar;7(2):110-6.).
SGPr198 SEQ ID N0:9 1q42
Familial effort polymorphic ventricular arrhythmias in arrhythmogenic right
ventricular cardiomyopathy map to chromosome 1q42-43 (Am J Cardiol. 2000 Mar
1;85(5):573-9.). Replication linkage study for prostate cancer susceptibility
genes
(Prostate. 2000 Oct 1;45(2):106-14.). Linkage analyses at the chromosome 1
loci
1q24-25 (HPC1), 1q42.2-43 (PCAP), and 1p36 (CAPB) in families with hereditary
prostate cancer (Am J Hum Genet. 2000 Feb;66(2):539-46.). Clinical profile and
long-term follow-up of 37 families with arrhythmogenic right ventricular
cardiomyopathy
(J Am Coll Cardiol. 2000 Dec;36(7):2226-33.) Arrhythmic disorder mapped to
chromosome 1q42-q43 causes malignant polymorphic ventricular tachycardia in
structurally normal hearts (J Am Coll Cardiol. 1999 Dec;34(7):2035-42.).
Analysis
of chromosome 1q42.2-43 in 152 families with high risk of prostate cancer. (Am
J
Hum Genet. 1999 Apr;64(4):1087-95.). A genome-wide search for susceptibility
genes in human systemic lupus erythematosus sib-pair families (Proc Natl Acad
Sci
U S A. 1998 Dec 8;95(25):14875-9.).
SGPr210 SEQ ID NO:10 19q13.2
A microdeletion in 19q13.2 associated with mental retardation, skeletal
malformations, and Diamond-Blackfan anaemia suggests a novel contiguous gene
syndrome (J Med Genet. 2000 Feb;37(2):128-31.). A microdeletion syndrome due
to a 3-Mb deletion on 19q13.2--Diamond-Blackfan anemia associated with
macrocephaly, hypotonia, and psychomotor retardation. (Clin Genet. 1999
Jun;55(6):487-92.). Diamond-Blackfan Anaemia: an overview. (Paediatr Drugs.
2000 Sep-Oct;2(5):345-55. Review.) A microdeletion in 19q13.2 associated with
mental retardation, skeletal malformations, and Diamond-Blackfan anaemia
suggests
a novel contiguous gene syndrome. (J Med Genet. 2000 Feb;37(2):128-31.).
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SGPr290.2 SEQ ID NO:11 2p23
Familial syndromic esophageal atresia (Am J Hum Genet. 2000 Feb;66(2):436-
44.).
Chromosomal rearrangements in acute myelogenous leukemia involving loci on
chromosome (Leukemia. 1999 Oct;13(10):1534-8.). Association and linkage
analysis of candidate chromosomal regions in multiple sclerosis: indication of
disease genes in disease genes in 12q23 and 7ptr-15 (Eur J Hum Genet. 1999 Feb-
Mar;7(2):110-6.).
SGPr116 SEQ ID N0:12 6p12
Familial patent ductus arteriosus and bicuspid aortic valve with hand
anomalies: a
novel heart-hand syndrome. (Am J Med Genet. 1999 Nov 19;87(2):175-9.) Char
syndrome, an inherited disorder with patent ductus arteriosus, maps to
chromosome
6p12-p21. (Circulation. 1999 Jun 15;99(23):3036-42.). Clinical features of
autosomal dominant congenital nystagmus linked to chromosome 6p 12. (Am J
Ophthalinol. 1998 Jan;125(1):64-70.). Linkage analysis of candidate regions
for
coeliac disease genes. (Hum Mol Genet. 1997 Aug;6(8):1335-9.). Fine mapping of
MEP1A, the gene encoding the alpha subunit of the metalloendopeptidase meprin,
to
human chromosome 6P21. (Biochem Biophys Res Commun. 1995 Nov
13;216(2):630-5.). Genetic linkage studies in familial frontal epilepsy:
exclusion of
the human chromosome regions homologous to the El-1 mouse locus. (Epilepsy
Res. 1995 Nov;22(3):227-33.)
SGPr003 SEQ ID N0:13 2q37
The expression of fragile sites in lymphocytes of patients with rectum cancer
and
their first-degree relatives. (Cancer Lett. 2000 May 1;152(2):201-9.).
Anterior
chamber eye anomalies, redundant skin and syndactyly--a new syndrome
associated
with breakpoints at 2q37.2 and 7q36.3. (Clin Dysmorphol. 1999 Jul;8(3):157-
63.).
Wilins' tumor and gonadal dysgenesis in a child with the 2q37.1 deletion
syndrome.
(Clin Genet. 1998 Apr;53(4):278-80.). A case of Albright's hereditary
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osteodystrophy-like syndrome complicated by several endocrinopathies: normal
Gs
alpha gene and chromosome 2q37. (J Clin Endocrinol Metab. 1998 May;83(5):1563-
5.). Albright hereditary osteodystrophy and del(2) (q37.3) in four unrelated
individuals. (Am J Med Genet. 1995 Jul 31;58(1):1-7.).
Oguchi disease: suggestion of linkage to markers on chromosome 2q.
(J Med Genet. 1995 May;32(5):396-8.). Malformation syndrome with t(2;22) in a
cancer family with chromosome instability. (Cancer Genet Cytogenet. 1989
Apr;38(2):223-7.).
SGPr016 SEQ ID N0:14 8p11.1
FGFRl and MOZ, two key genes involved in malignant hemopathies linked to
rearrangements within the chromosomal region 8p1 1-12. (Bull Cancer. 2000
Dec;87(12):887-94. Review). Sql l, 8p11, and 10q22 are recurrent chromosomal
breakpoints in prostate cancer cell lines. (Genes Chromosomes Cancer. 2001
Feb;30(2):187-95.). Unusual breakpoint distribution of 8p abnormalities in T-
prolymphocytic leukemia: a study with YACS mapping to 8p11-p12.
(Cancer Genet Cytogenet. 2000 Sep;l21(2):128-32). Loss ofheterozygosity at
chromosome segments 8p22 and 8p11.2-21.1 in transitional-cell carcinoma of the
urinary bladder. (Int J Cancer. 2000 May 15;86(4):501-5).
SGPr352 SEQ ID NO:15 19p13.3
Clinical characteristics of hereditary cerebrovascular disease in a large
family from
Colombia (Rev Neurol. 2000 Nov 16-30;31(10):901-7.). Molecular genetic
alterations in hamartomatous polyps and carcinomas of patients with Peutz-
Jeghers
syndrome. (J Clin Pathol. 2001 Feb;54(2):126-31.). Identification of a subtle
t(16;19)(p13.3;p13.3) in an infant with multiple congenital abnormalities
using a 12-
colour multiplex FISH telomere assay, M-TEL. (Eur J Hum Genet. 2000
Dec;B(12):903-10.). Identification of a locus for autosomal dominant
polycystic
liver disease, on chromosome 19p13.2-13.1. (Am J Hum Genet. 2000
Dec;67(6):1598-604.). Fine mapping of a distinctive autosomal dominant
vacuolar
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neuromyopathy using 11 novel microsatellite markers from chromosome band
19p13.3. (Eur J Hum Genet. 2000 Oct;B(10):809-12.).
Genomewide scan in german families reveals evidence for a novel psoriasis-
susceptibility locus on chromosome 19p13. (Am J Hum Genet. 2000
Oct;67(4):1020-4.). Genomewide Search in Canadian Families with Inflammatory
Bowel Disease Reveals Two Novel Susceptibility Loci. (Am J Hum Genet. 2000
Jun;66(6):1863-1870.)
SGPr050 SEQ ID N0:16 Sq15.3
Mucolipidosis type IV: Novel MCOLNl mutations in Jewish and non-Jewish
patients and the frequency of the disease in the Ashkenazi Jewish population.
(Hum
Mutat. 2001 May;l7(5):397-402.) Myocarditis, a Rare but Severe Manifestation
of
Q Fever: Report of 8 Cases and Review of the Literature. (Clin Infect Dis.
2001 May
15;32(10):1440-1447.)
SGPr282 SEQ ID N0:17 16p12.3
Linkage of benign familial infantile convulsions to chromosome 16p12-ql2
suggests
allelism to the infantile convulsions and choreoathetosis syndrome. (Am J Hum
Genet. 2001 Mar;68(3):788-94.). A second-generation genomewide screen for
asthma-susceptibility alleles in a founder population. (Am J Hum Genet. 2000
Nov;67(5):1154-62.). Evidence of further genetic heterogeneity in autosomal
dominant medullary cystic kidney disease. (Nephrol Dial Transplant. 2000
Jun;15(6):818-21.) Localization of a gene for familial juvenile hyperuricemic
nephropathy causing underexcretion-type gout to 16p12 by genome-wide linkage
analysis of a large family (Arthritis Rheum. 2000 Apr;43(4):925-9.).
Localization of
a hereditary neuroblastoma predisposition gene to 16p12-p13 (Med Pediatr
Oncol.
2000 Dec;35(6):526-30.). Identifying genes predisposing to atopic eczema (J
Allergy Clin Tmmunol. 1999 ov;104(5):1066-70.). Molecular genetics of the
neuronal ceroid lipofuscinoses. (Epilepsia. 1999;40 Suppl 3:29-32.). Thirty
years of
Batten disease research: present status and future goals.
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(Mol Genet Metab. 1999 Apr;66(4):231-3.).
SGPr046 SEQ ID N0:18 16q23
A genome-wide family-based linkage study of coeliac disease.
(Ann Hum Genet. 2000 Nov;64(Pt 6):479-90.). Pleiotropic syndrome of dehydrated
hereditary stomatocytosis, pseudohyperkalemia, and perinatal edema maps to
16q23-q24. (Blood. 2000 Oct 1;96(7):2599-605.). Identification and fine
mapping
of a region showing a high frequency of allelic imbalance on chromosome
16q23.2
that corresponds to a prostate cancer susceptibility locus. (Cancer Res. 2000
Jul
1;60(13):3645-9.). Concurrent and independent genetic alterations in the
stromal
and epithelial cells of mammary carcinoma: implications for tumorigenesis.
(Cancer
Res. 2000 May 1;60(9):2562-6.). A 700-kb physical map of a region of 16q23.2
homozygously deleted in multiple cancers and spanning the common fragile site
FRA16D. (Cancer Res. 2000 Mar 15;60(6):1690-7.). Prognostic significance of
allelic imbalance of chromosome arms 7q, 8p, 16q, and 18q in stage T3NOM0
prostate cancer. (Genes Chromosomes Cancer. 1998 Feb;21(2):131-43.) Loss of
heterozygosity at 16q24.1-q24.2 is significantly associated with metastatic
and
aggressive behavior of prostate cancer. (Cancer Res. 1997 Aug 15;57(16):3356-
9.).
SGPr060 SEQ ID N0:19 15q26
A genome-wide search for susceptibility genes in human systemic lupus
erythematosus sib-pair families. (Pros Natl Acad Sci U S A. 1998 Dec
8;95(25):14875-9.). Linkage analysis of candidate regions for coeliac disease
genes.
(Hum Mol Genet. 1997 Aug;6(8):1335-9.).
SGPr068 SEQ ID NO:20 10q22
Autosomal dominant myofibrillar myopathy with arrhythmogenic right ventricular
cardiomyopathy linked to chromosome 10q. (Ann Neurol. 1999 Nov;46(5):684-92.)
Construction of a high-resolution physical map of the chromosome 1Oq22-q23
dilated cardiomyopathy locus and analysis of candidate genes. (Genomics. 2000
Jul
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15;67(2):109-27.). Chromosomal basis of adenocaxcinoma of the prostate.
(Cancer
Invest. 1999;17(6):441-7.) Allele loss in colorectal cancer at the Cowden
disease/juvenile polyposis locus on lOq (Cancer Genet Cytogenet. 1997
Aug;97(1):64-9.) Identification of a genetic locus for familial atrial
fibrillation. (N
Engl J Med. 1997 Mar 27;336(13):905-1 l.).
SGPr096 SEQ ID N0:21 3p14
The relationship between genetic susceptibility to head and neck cancer with
the
expression of common fragile sites. (Head Neck. 2000 Sep;22(6):591-8.).
Concurrent and independent genetic alterations in the stromal and epithelial
cells of
mammary carcinoma: implications for tumorigenesis. (Cancer Res. 2000 May
1;60(9):2562-6.) Prognostic implication of microsatellite alteration profiles
in early-
stage non-small cell lung cancer. (Clin Cancer Res. 2000 Feb;6(2):559-65).
Loss of heterozygosity at chromosomes 3, 6, 8, 11, 16, and 17 in ovarian
cancer:
correlation to clinicopathological variables. (Cancer Genet Cytogenet. 2000
Oct
1;122(1):49-54.).
SGPr119 SEQ ID N0:22 12q1 I
Fine genetic mapping of diffuse non-epidermolytic palmoplantax keratoderma to
chromosome 12q11-q13: exclusion of the mapped type II keratins. (Exp Dermatol.
1999 Oct;8(5):388-91.).
SGPr143 SEQ ID N0:23 20p13
Hallervorden-Ppatz disease (OMIM 234200).
SGPr164 SEQ ID N0:24 11q25
Deletion mapping of chromosome segment l 1q24-q25, exhibiting extensive
allelic
loss in early onset breast cancer. (Int J Cancer. 2001 Apr 15;92(2):208-13.).
Restricted chromosome breakpoint sites on 11q22-q23.1 and 11q25 in various
hematological malignancies without MLL/ALL-1 gene rearrangement. (Cancer
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Genet Cytogenet. 2001 Jan 1;124(1):27-35.). Autozygosity mapping, to
chromosome 11q25, of a rare autosomal recessive syndrome causing
histiocytosis,
joint contractures, and sensorineural deafness. (Am J Hum Genet. 1998
May;62(5):1123-8.). Tertiary trisomy (22q1 lq),47,+der(22),t(11;22). (Hum
Genet.
1980 Feb;53(2):173-7.).
SGPr281 SEQ ID N0:25 Sq31
Interleukin-5 is at Sq31 and is deleted in the Sq- syndrome. (Blood. 1988
Apr;71(4):1150-2.). Lack of association between the interferon regulatory
factor-1
(IRF1) locus at Sq31.1 and multiple sclerosis in Germany, northern Italy,
Sardinia
and Sweden. (Genes Immun. 2000;1(4):290-2.). Childhood asthma: aspects of
global environment, genetics and management. (Changgeng Yi Xue Za Zhi. 2000
Nov;23(11):641-61. Review.). Association and linkage of atopic dermatitis with
chromosome 13q12-14 and Sq31-33 markers. J Invest Dermatol. 2000
Nov;115(5):906-8. Deletion of Sq31 is observed in megakaryocytic cells in
patients
with myelodysplastic syndromes and a del(Sq), including the Sq- syndrome.
(Genes
Chromosomes Cancer. 2000 Dec;29(4):350-2.). Ethnic differences in genetic
susceptibility to atopy and asthma in Singapore. (Ann Acad Med Singapore. 2000
May;29(3):346-50. Review.). Genomewide scan for prostate cancer-aggressiveness
loci. (Am J Hum Genet. 2000 Ju1;67(1):92-9.). Molecular genetic analysis of
malignant ovarian germ cell tumors. (Gynecol Oncol. 2000 May;77(2):283-8.).
SGPr075 SEQ ID N0:26 Unmapped
SGPr292.2 SEQ ID N0:27 10q26
Sequence homology between 4qter and lOqter loci facilitates the instability of
subtelomeric I~pnI repeat units implicated in facioscapulohumeral muscular
dystrophy. (Am J Hum Genet. 1998 Ju1;63(1):181-90.) Frequent loss of
heterozygosity on chromosome lOq in muscle-invasive transitional cell
carcinomas
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of the bladder. (Oncogene. 1997 Jun 26;14(25):3059-66.). Allelic loss on
chromosome 10 in prostate adenocarcinoma. (Cancer Res. 1996 May 1;56(9):2143-
7.) Severe midline fusion defects in a newborn with l Oq26----qter deletion.
(Ann
Genet. 1989;32(2):124-5.)
SGPr069 SEQ ID N0:28 1p36.3
Neurodevelopmental profile of a new dysmorphic syndrome associated with
submicroscopic partial deletion of 1p36.3. (Dev Med Child Neurol. 2000
Mar;42(3):201-6.). Molecular Cytogenetics in Ewing Tumors: Diagnostic and
Prognostic Information. (Onkologie. 2000 Oct;23(5):416-422.). Significance of
the
small subtelomeric area of chromosome 1 (1p36.3) in the progression of
malignant
melanoma: FISH deletion screening with YAC DNA probes. (Virchows Arch. 1999
Aug;435(2):105-11). Allelic loss on chromosome 1 is associated with tumor
progression of cervical carcinoma. (Cancer. 1999 Oct 1;86(7):1294-8). Terminal
deletion, del(1)(p36.3), detected through screening for terminal deletions in
patients
with unclassified malformation syndromes. (Am J Med Genet. 1999 Jan
29;82(3):249-53). Partial monosomy of chromosome 1p36.3: characterization of
the
critical region and delineation of a syndrome. ( Am J Med Genet. 1995 Dec
4;59(4):467-75). Consistent association of 1p loss of heterozygosity with
pheochromocytomas from patients with multiple endocrine neoplasia type 2
syndromes. (Cancer Res. 1992 Feb 15;52(4):770-4.).
SGPr212 SEQ ID N0:29 9q22
Chromosome 9 deletions and recurrence of superficial bladder cancer:
identification
of four regions of prognostic interest. (Oncogene. 2000 Dec 14;19(54):6317-
23).
Exclusion of NFIL3 as the gene causing hereditary sensory neuropathy type I by
mutation analysis. (Hum Genet. 2000 Jun;106(6):594-6). Chromosomal imbalances
are associated with a high risk of progression in early invasive (pT1) urinary
bladder
cancer (Cancer Res. 1999 Nov 15;59(22):5687-91). Brachydactyly type B: linkage
to chromosome 9q22 and evidence for genetic heterogeneity. (Am J Hum Genet.
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1999 Feb;64(2):578-85). A YAC-based transcript map of human chromosome
9q22.1-q22.3 encompassing the loci for hereditary sensory neuropathy type I
and
multiple self healing squamous epithelioma. (Genomics. 1998 Jul 15;51(2):277-
81).
Molecular analysis of childhood primitive neuroectodermal tumors defines
markers
associated with poor outcome. (J Clin Oncol. 1998 Jul;16(7):2478-85).
Mutilating
neuropathic ulcerations in a chromosome 3q13-q22 linked Charcot-Marie-Tooth
disease type 2B family. (J Neurol Neurosurg Psychiatry. 1997 Jun;62(6):570-3).
SGPr049 SEQ ID N0:30 Sq23.3 / Sq31
Interleukin-5 is at Sq31 and is deleted in the Sq- syndrome. (Blood. 1988
Apr;71(4):1150-2.). Lack of association between the interferon regulatory
factor-1
(IRF1) locus at Sq31.1 and multiple sclerosis in Germany, northern Italy,
Sardinia
and Sweden. (Genes Immun. 2000;1(4):290-2.). Cluldhood asthma: aspects of
global environment, genetics and management. (Changgeng Yi Xue Za Zhi. 2000
Nov;23(11):641-61. Review.). Association and linkage of atopic dermatitis with
chromosome 13q12-14 and Sq31-33 markers. J Invest Dermatol. 2000
Nov;115(5):906-8. Deletion of Sq31 is observed in megakaryocytic cells in
patients
with myelodysplastic syndromes and a del(Sq), including the Sq- syndrome.
(Genes Chromosomes Cancer. 2000 Dec;29(4):350-2.). Ethnic differences in
genetic susceptibility to atopy and asthma in Singapore. (Ann Acad Med
Singapore.
2000 May;29(3):346-50. Review.). Genomewide scan for prostate cancer-
aggressiveness loci. (Am J Hum Genet. 2000 Ju1;67(1):92-9.). Molecular genetic
analysis of malignant ovarian germ cell tumors. (Gynecol Oncol. 2000
May;77(2):283-8.).
SGPr026 SEQ ID N0:31 1q31
Genomewide search and genetic localization of a second gene associated with
autosomal dominant branchio-oto-renal syndrome: clinical and genetic
implications.
(Am J Hum Genet. 2000 May;66(5):1715-20.). Jumping translocations involving
chromosome 1 q in a patient with Crohn disease and acute monocytic leukemia: a
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review of the literature on jumping translocations in hematological
malignancies and
Crohn disease (Cancer Genet Cytogenet. 1999 Mar;109(2):144-9. Review).
Molecular analysis of childhood primitive neuroectodennal tumors defines
markers
associated with poor outcome. (J Clin Oncol. 1998 Jul;16(7):2478-85).
Mapping a gene (SRNl) to chromosome 1q25-q31 in idiopathic nephrotic syndrome
confirms a distinct entity of autosomal recessive nephrosis. (Hum Mol Genet.
1995
Nov;4(11):2155-8).
SGPr203 SEQ ID N0:32 2q37
The expression of fragile sites in lymphocytes of patients with rectum cancer
and
their first-degree relatives. (Cancer Lett. 2000 May 1;152(2):201-9.).
Anterior
chamber eye anomalies, redundant skin and syndactyly--a new syndrome
associated
with breakpoints at 2q37.2 and 7q36.3. (Clin Dysmorphol. 1999 Jul;B(3):157-
63.).
Wilms' tumor and gonadal dysgenesis in a child with the 2q37.1 deletion
syndrome.
(Clin Genet. 1998 Apr;53(4):278-80). Albright hereditary osteodystrophy and
del(2)
(q37.3) in four unrelated individuals. (Am J Med Genet. 1995 Jul 31;58(1):1-
7).
Oguchi disease: suggestion of linkage to markers on chromosome 2q. (J Med
Genet.
1995 May;32(5):396-8). Malformation syndrome with t(2;22) in a cancer family
with chromosome instability. (Cancer Genet Cytogenet. 1989 Apr;38(2):223-7).
SGPr157 SEQ ID N0:33 18q22.3
Psychiatric disorder in a familial 15;18 translocation and sublocalization of
myelin
basic protein of 18q22.3. (Am J Med Genet. 1996 Apr 9;67(2):154-61.).
SGPr154 SEQ ID N0:34 1q32.1
Oncogene amplification in human gliomas: a molecular cytogenetic analysis.
(Oncogene. 1994 Sep;9(9):2717-22).
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SGPr088 SEQ ID N0:35 18q23
Molecular characterization of patients with 18q23 deletions. (Am J Hum Genet.
1997 Apr;60(4):860-8.) Unbalanced translocation, t(18;21), detected by
fluorescence in situ hybridization (FISH) in a child with 18q- syndrome and a
ring
chromosome 21. (Am J Med Genet. 1993 Jul 1;46(6):647-51).
EXAMPLE 8b: Candidate Single Nucleotide Polymorphisms (SNPsI
Materials And Methods
The most common variations in human DNA are single nucleotide polymorphisms
(SNPs), which occur approximately once every 100 to 300 bases. Because SNPs
are
expected to facilitate large-scale association genetics studies, there has
recently been
great interest in SNP discovery and detection. Candidate SNPs for the genes in
this
patent were identified by blastn searching the nucleic acid sequences against
the
public database of sequences containing documented SNPs (dbSNP, at NCBI,
http://www.ncbi.nhn.nih.gov/SNP/snpblastpretty.html). dbSNP accession numbers
for the SNP-containing sequences are given. SNPs were also identified by
comparing several databases of expressed genes (dbEST, NRNA) and genomic
sequence (i.e., NRNA) for single basepair mismatches. The results are shown in
Table 1, in the column labeled "SNPs". These are candidate SNPs - their actual
frequency in the human population was not determined. The code below is
standard
for representing DNA sequence:
G = Guanosine
A =Adenosine
T = Thymidine
C = Cytidine
R = G or A, puRine
Y = C or T, pYrimidine
K = G or T, Keto
W =A or T, Weak (2 H-bonds)
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S = C or G, Strong (3 H-bonds)
M = A or C, aMino
B =C, G or T (i.e.,
not A)
D =A, G or T (i.e.,
not C)
H =A, C or T (i.e.,
not G)
V = A, C or G (i.
e., not T)
N = A, C, G or T,
aNy
X - A, C, G or T
complementary G A T C R Y W S K M B V D H N X
DNA +-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+
strands C T A G Y R S W M K V B H D N X
For example, if two versions of a gene exist, one with a "C" at a given
position,
and a second one with a "T: at the same position, then that position is
represented as
a Y, which means C or T. SNPs may be important in identifying heritable traits
associated with a gene.
Results
The results of SNP identification are contained in Table 2 above, and
in Example 1, under the section entitled DESCRIPTION OF NOVEL PROTEASE
POLYNUCLEOTIDES. As discussed above, a varietyof SNPs were identified in
the protease polynucleotides of the present invention.
EXAMPLE 9: Demonstration Of Gene Amplification By Southern Blotting
Materials and Methods
Nylon membranes are purchased from Boehringer Mannheim. Denaturing
solution contains 0.4 M NaOH and 0.6 M NaCI. Neutralization solution contains
0.5 M Tris-HCL, pH 7.5 and 1.5 M NaCI. Hybridization solution contains 50%
formamide, 6X SSPE, 2.5X Denhardt's solution, 0.2 mg/mL denatured salmon
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DNA, 0.1 mg/mL yeast tRNA, and 0.2 % sodium dodecyl sulfate. Restriction
enzymes are purchased from Boehringer Mannheim. Radiolabeled probes are
prepared using the Prime-it II kit by Stratagene. The (3-actin DNA fragment
used for
a probe template is purchased from Clontech.
Genomic DNA is isolated from a variety of tumor cell lines (such as MCF-7,
MDA-MB-231, Calu-6, A549, HCT-15, HT-29, Colo 205, LS-180, DLD-l, HCT-
116, PC3, CAPAN-2, MIA-PaCa-2, PANG-1, AsPc-1, BxPC-3, OVCAR-3,
SKOV3, SW 626 and PA-1, and from two normal cell lines.
A 10 ~,g aliquot of each genomic DNA sample is digested with EcoR I
restriction enzyme and a separate 10 ~,g sample is digested with Hind III
restriction
enzyme. The restriction-digested DNA samples are loaded onto a 0.7% agarose
gel
and, following electrophoretic separation, the DNA is capillary-transferred to
a
nylon membrane by standard methods (Sambrook, J. et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory).
EXAMPLE 10: Detection Of Protein-Protein Interaction Throush Phase Disnla
Materials And Methods
Phage display provides a method for isolating molecular interactions based
on affinity for a desired bait. cDNA fragments cloned as fusions to phage coat
proteins are displayed on the surface of the phage. Phage(s) interacting with
a bait
are enriched by affinity purification and the insert DNA from individual
clones is
analyzed.
T7 Phage Display Libraries
All libraries were constructed in the T7Selectl-lb vector (Novagen)
according to the manufacturer's directions.
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Bait Presentation
Protein domains to be used as baits are generated as C-terminal fusions to
GST and expressed in E. coli. Peptides are chemically synthesized and
biotinylated
at the N-terminus using a long chain spacer biotin reagent.
Selection
Aliquots of refreshed libraries (101°-1012 pfu) supplemented with
PanMix
and a cocktail of E. coli inhibitors (Sigma P-8465) are incubated for 1-2 hrs
at room
temperature with the immobilized baits. Unbound phage is extensively washed
(at
least 4 times) with wash buffer.
After 3-4 rounds of selection, bound phage is eluted in 100 ~,L of 1% SDS
and plated on agarose plates to obtain single plaques.
Identification of insert DNAs
Individual plaques are picked into 25 ~,L of 10 mM EDTA and the phage is
disrupted by heating at 70 °C for 10 min. 2 ~,L of the disrupted phage
are added to
50 ~.L PCR reaction mix. The insert DNA is amplified by 35 rounds of thermal
cycling (94 °C, 50 sec; 50 °C, lmin; 72 °C, lmin).
Composition of Buffer
lOx PanMix
5% Triton X-100
10% non-fat dry milk (Carnation)
10 mM EGTA
250 mM NaF
250 ~,g/mL Heparin (sigma)
250 ~.g/mL sheared, boiled salmon sperm DNA (sigma)
0.05% Na azide
Prepared in PBS
Wash Buffer
PBS supplemented with:
0.5 % NP-40
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25 ~,l g/mL heparin
PCR reaction mix
1.0 mL l Ox PCR buffer (Perkin-Eliner, with 15 mM Mg)
0.2 mL each dNTPs (10 mM stock)
0.1 mL T7UP primer (15 pmol/~,L) GGAGCTGTCGTATTCCAGTC
0.1 mL T7DN primer (15 pmol/p,L)
AACCCCTCAAGACCCGTTTAG
0.2 mL25 mM MgCl2 or MgS04 to compensate for EDTA
Q.S. to 10 mL with distilled water
Add 1 unit of Taq polymerise per 50 ~.L reaction
LIBRARY: T7 Selectl-H441
CONCLUSION
One skilled in the art would readily appreciate that the present invention is
well adapted to carry out the objects and obtain the ends and advantages
mentioned,
as well as those inherent therein. The molecular complexes and the methods,
procedures, treatments, molecules, specific compounds described herein are
presently representative of preferred embodiments, are exemplary, and are not
intended as limitations on the scope of the invention. It will be readily
apparent to
one skilled in the art that varying substitutions and modifications may be
made to
the invention disclosed herein without departing from the scope and spirit of
the
invention.
All patents and publications mentioned in the specification are indicative of
the levels of those skilled in the art to which the invention pertains. All
patents and
publications are herein incorporated by reference to the same extent as if
each
individual publication was specifically and individually indicated to be
incorporated
by reference.
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The invention illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which is not
specifically disclosed herein. Thus, for example, in each instance herein any
of the
terms "comprising," "consisting essentially of and "consisting of ' may be
replaced
with either of the other two-terms. The terms and expressions which have been
employed are used as terms of description and not of limitation, and there is
no
intention that in the use of such terms and expressions of excluding any
equivalents
of the features shown and described or portions thereof, but it is recognized
that
various modifications are possible within the scope of the invention claimed.
Thus,
it should be understood that although the present invention has been
specifically
disclosed by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by those skilled
in the
art, and that such modifications and variations are considered to be within
the scope
of this invention as defined by the appended claims.
In addition, where features or aspects of the invention are described in terms
of Markush groups, those skilled in the art will recognize that the invention
is also
thereby described in terms of any individual member or subgroup of members of
the
Markush group. For example, if X is described as selected from the group
consisting of bromine, chlorine, and iodine, claims for X being bromine and
claims
for X being bromine and chlorine are fully described.
In view of the degeneracy of the genetic code, other combinations of nucleic
acids also encode the claimed peptides and proteins of the invention. For
example,
all four nucleic acid sequences GCT, GCC, GCA, and GCG encode the amino acid
alanine. Therefore, if for an amino acid there exists an average of three
codons, a
polypeptide of 100 amino acids in length will, on average, be encoded by 3100,
or 5
x 1047, nucleic acid sequences. Thus, a nucleic acid sequence can be modified
to
form a second nucleic acid sequence, encoding the same polypeptide as encoded
by
the first nucleic acid sequences, using routine procedures and without undue
experimentation. Thus, all possible nucleic acids that encode the claimed
peptides
and proteins are also fully described herein, as if all were written out in
full taking
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into account the codon usage, especially that preferred in humans.
Furthermore,
changes in the amino acid sequences of polypeptides, or in the corresponding
nucleic acid sequence encoding such polypeptide, may be designed or selected
to
take place in an area of the sequence where the significant activity of the
polypeptide remains unchanged. For example, an amino acid change may take
place
within a [3-turn, away from the active site of the polypeptide. Also changes
such as
deletions (e.g. removal of a segment of the polypeptide, or in the
corresponding
nucleic acid sequence encoding such polypeptide, which does not affect the
active
site) and additions (e.g. addition of more amino acids to the polypeptide
sequence
without affecting the function of the active site, such as the formation of
GST-fusion
proteins, or additions in the corresponding nucleic acid sequence encoding
such
polypeptide without affecting the function of the active site) are also within
the
scope of the present invention. Such changes to the polypeptides can be
performed
by those with ordinary skill in the art using routine procedures and without
undue
experimentation. Thus, all possible nucleic and/or amino acid sequences that
can
readily be determined not to affect a significant activity of the peptide or
protein of
the invention are also fully described herein.
The invention has been described broadly and generically herein. Each of
the narrower species and subgeneric groupings falling within the generic
disclosure
also form part of the invention. This includes the generic description of the
invention with a proviso or negative limitation removing any subject matter
from the
genus, regardless of whether or not the excised material is specifically
recited
herein.
Other embodiments are within the following claims.
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