Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
MOTILIN HOMOLOGS
BACKGROUND OF THE INVENTION
Many of the regulatory peptides that are
important in maintaining nutritional homeostasis are found
in the gastrointestinal environment. These peptides may
be synthesized in the digestive system and act locally,
but can also be identified in the brain as well. In
addition, the reverse is also found, i.e., peptides are
synthesized in the brain, but found to regulate cells in
the gastrointestinal tract. This phenomena has been
called the "brain-gut axis" and is important for signaling
satiety, regulating body temperature and other
physiological processes that require feedback between the
brain and gut.
The gut peptide hormones include gastrin,
cholecystokinin (CCK), secretin, gastric inhibitory
peptide (GIP), vasoactive intestinal polypeptide (VIP),
motilin, somatostatin, pancreatic peptide (PP), substance
P and neuropeptide Y (NPY), and use several different
mechanisms of action. For example, gastrin, rnotilin and
CCK function as endocrine- and neurocrine-type hormones.
Others, such as gastrin and GIP, are thought to act
exclusively in an endocrine fashion. Other modes of
action include a combination of endocrine, neurocrine and
paracrine action (somatostatin); exclusively neurocrine
action (NPY); and a combination of neurocrine and
paracrine actions (VIP and Substance P). Most of the gut
hormone actions are mediated by membrane-bound receptors
and activate second messenger systems. For a review of
gut peptides see, Mulvihill et al., in Basic and Clinical
Endocrinology, pp.551-570, 4th edition Greenspan F. S. and
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Baxter, J. D. editors., Appleton & Lange: Norwalk,
Connecticut, 1994.
Many of these gut peptides are synthesized as
inactive precursor molecules that require multiple peptide
cleavages to be activated. The family known as the
"glucagon-secretin" family which includes VIP, gastrin,
secretin, motilin, glucagon and galanin exemplifies
peptides regulated by multiple cleavages and post-
translational modifications.
Motilin is a 22 amino acid peptide found in gut
tissue of mammalian species (Domschke, W., Digestive
Diseases 22 5 :454-461, 1977). The DNA and amino acid
sequences for porcine prepromotilin have been identified
(U.S. Patent 5,006,469). Motilin has been identified as a
factor capable of increasing gastric motility, affecting
the secretory function of the stomach by stimulating
pepsin secretion (Brown et al., Canadian J. of Physiol.
Pharmacol. 49:399-405, 1971), and recent evidence suggests
a role in myoelectric regulation of stomach and small
intestine. Cyclic increases of motilin have been
correlated with phase III of the interdigestive
myoelectric complex and the hunger contraction of the
duodenum (Chey et al., in Gut Hormones, (eds.) Bloom,
S.R., pp. 355-358, Edinburgh, Churchill Livingstone, 1978;
Lee et al, Am. J. Digestive Diseases, 23:789-795, 1978;
and Itoh et al., Am. J. Digestive Diseases, 23:929-935,
1978). Motilin and analogues of motilin have been
demonstrated to produce contraction of gastrointestinal
smooth muscle, but not other types of smooth muscle cells
(Strunz et al., Gastroenterology 68:1485-1491, 1975).
The present invention is directed to a novel
secreted protein with homology to motilin, found to be
transcribed in the gastrointestinal system. The discovery
of this novel peptide is important for further elucidation
of the how the body maintains its nutritional homeostasis
and development of therapeutics to intervene in those
CA 02284733 2006-02-10
3
processes, as well as other uses that will be apparent
from the teachings therein.
SUMMARY OF THE INVENTION
Within one aspect, the present invention
provides an isolated polynucleotide molecule encoding a
polypeptide selected from the group consisting of: (a)
polynucleotide molecules comprising a nucleotide sequence
as shown in SEQ ID NO: 1 from nucleotide 70 to nucleotide
111; (b) polynucleotide molecules comprising a nucleotide
sequence that is at least 50% identical to the nucleotide
sequence as shown in SEQ ID NO: 1 from nucleotide 70 to
nucleotide 111, wherein the polynucleotide encodes a
polypeptide capable of stimulating gastric motility ; (c)
allelic variants of (a); (d) orthologs of (a) and (c); and
(e) degenerate nucleotide sequences of (a), (b), (c) or
(d). In one embodiment, the isolated polynucleotide is
DNA.
The present invention further provides an
isolated polynucleotide molecule selected from the group
consisting of: (a) polynucleotide molecules comprising a
nucleotide sequence as shown in SEQ ID NO:l from
nucleotide 70 to nucleotide 120; (b) polynucleotide
molecules comprising a nucleotide sequence that is at
least 50% identical to the nucleotide sequence as shown in
SEQ ID NO:1 from nucleotide 70 to nucleotide 120, wherein
the polynucleotide encodes a polypeptide capable of
stimulating gastric motility; and (c)degenerate nucleotide
sequences of (a) or (b).
The present invention further provides an
isolated polynucleotide molecule selected from the group
consisting of: (a)polynucleotide molecules comprising a
nucleotide sequence as shown in SEQ ID NO:1 from
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4a
nucleotide 70 to nucleotide 351; (b)a polynucleotide
molecule comprising a nucleotide sequence that is at least
50% identical to the nucleotide sequence as shown in SEQ
ID NO:1 from nucleotide 70 to nucleotide 351; wherein the
polynucleotide encodes a polypeptide capable of
stimulating gastric motility; and (c)degenerate nucleotide
sequences of (a) or (b).
The present invention further provides an
isolated polynucleotide molecule selected from the group
consisting of: (a) polynucleotide molecules comprising a
nucleotide sequence as shown in SEQ ID NO:1 from
nucleotide 1 to nucleotide 111; (b)polynucleotide
molecules comprising a nucleotide sequence that is at
least 50% identical to the nucleotide sequence as shown in
SEQ ID NO:1 from nucleotide 1 to nucleotide 111, wherein
the polynucleotide encodes a polypeptide capable of
stimulating gastric motility; and (c)degenerate nucleotide
sequences of (a) or (b).
The present invention further provides an
isolated polynucleotide molecule comprising the nucleotide
sequence as shown in SEQ ID NO: 1 from nucleotide 1 to
nucleotide 351.
Within another aspect, the present invention
provides an isolated polypeptide selected from the group
consisting of: (a)polypeptide molecules comprising an
amino acid sequence as shown in SEQ ID NO: 2 from residue
24 to residue 37; (b) allelic variants of (a); and (c)
orthologs of (a) or (b); polypeptide molecules comprising
an amino acid sequence as shown in SEQ ID NO: 2 from
residue 24 to residue 37 that is at least 80% identical to
the amino acid sequence as shown in SEQ ID NO:2 from
residue 24 to residue 37, wherein the polypeptides are
capable of stimulating gastric motility.
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4b
The invention further provides an isolated
polypeptide selected from the group consisting of: (a)
polypeptide molecules comprising an amino acid sequence as
shown in SEQ ID NO:2 from residue 24 to 41; (b)polypeptide
molecules comprising an amino acid sequence that is at
least 80% identical to the amino acid sequence as shown in
SEQ ID NO: 2 from residue 24 to residue 41, wherein the
polypeptide is capable of stimulating gastric motility.
The invention further provides an isolated
polypeptide selected from the group consisting of:
(a) polypeptide molecules comprising an amino acid
sequence as shown in SEQ ID NO:2 from residue 24 to 117;
(b) polypeptide molecules comprising an amino acid
sequence that is at least 80% identical to the amino acid
sequence as shown in SEQ ID NO:2 from residue 24 to
residue 117, wherein the polypeptides are capable of
stimulating gastric motility.
The invention further provides an isolated
polypeptide selected from the group consisting of:
(a)polypeptide molecules comprising an amino acid sequence
as shown in SEQ ID NO:2 from residue 1 to residue 37;
(b)polypeptide molecules comprising an amino acid sequence
that is at least 80% identical to the amino acid sequence
as shown in SEQ ID NO:2 from residue 1 to residue 37,
wherein the polypeptides are capable of stimulating
gastric motility.
The invention further provides an isolated
polypeptide molecule comprising an amino acid sequence as
shown in SEQ ID NO: 2 from residue 1 to residue 117.
In another aspect, the present invention
provides an expression vector comprising the following
operably linked elements: a transcription promoter; a DNA
segment selected from the group consisting of: (a)
polynucleotide molecules comprising a nucleotide sequence
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as shown in SEQ ID NO: 1 from nucleotide 70 to nucleotide
111; (b) allelic variants of (a); (c) orthologs of (a) or
(b); (d) polynucleotide molecules comprising a nucleotide
sequence that is at least 50% identical to the nucleotide
sequence as shown in SEQ ID NO:1 from nucleotide 70 to
nucleotide 111, wherein the polynucleotide encodes a
polypeptide capable of stimulating gastric motility ; and
(e)degenerate nucleotide sequences of (a), (b) or (c); a
transcription terminator.
In another aspect, the present invention
provides a cultured cell into which has been introduced an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment selected
from the group consisting of: (a) polynucleotide molecules
comprising a nucleotide sequence as shown in SEQ ID NO: 1
from nucleotide 70 to nucleotide 111; (b) allelic variants
of (a); (c) orthologs of (a) or (b);(d) polynucleotide
molecules comprising a nucleotide sequence that is at
least 50% identical to the nucleotide sequence as shown
in SEQ ID NO:l from nucleotide 70 to nucleotide 111,
wherein the polynucleotide encodes a polypeptide capable
of stimulating gastric motility ; and(e)degenerate
nucleotide sequences of (a), (b),(c) or (d); a
transcription terminator, wherein said cell expresses the
polypeptide encoded by the DNA segment.
In another aspect, the present invention
provides a pharmaceutical composition comprising purified
polypeptide selected from the group consisting of: (a)
polypeptide molecules comprising an amino acid sequence as
shown in SEQ ID NO: 2 from residue 24 to residue 37; (b)
allelic variants of (a);(c) orthologs of (a) or (b),and
(d)polypeptide molecules that are at least 80% identical
to the amino acid sequence as shown in SEQ ID NO: 2 from
residue 24 to residue 37, wherein the polypeptide is
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4d
capable of stimulating gastric motility, in combination
with a pharmaceutically acceptable vehicle.
In another aspect, the present invention
provides an antibody that binds to an epitope of a
polypeptide selected from the group consisting of: (a)
polypeptide molecules comprising an amino acid sequence as
shown in SEQ ID NO: 2 from residue 24 to residue 117; (b)
allelic variants of (a); and (c) orthologs of (a) or (b).
In another aspect, the present invention
provides for an antibody which specifically binds to a
polypeptide consisting of amino acid residues 24 to 117 of
SEQ ID NO:2.
In another aspect, the present invention
provides an antibody that binds to an epitope of a
polypeptide selected from the group consisting of: (a) a
polypeptide consisting of the amino acid sequence of SEQ
ID NO: 2; (b) the polypeptide consisting of residue 24 to
residue 117 of SEQ ID NO: 2; (c) the polypeptide
consisting of residue 1 to residue 23 of SEQ ID NO:2; (d)
the polypeptide consisting of residue 24 to residue 37 of
SEQ ID NO:2; (e) the polypeptide consisting of residue 24
to residue 41 of SEQ ID NO:2; and (f) the polypeptide
consisting of residue 42 to residue 117 of SEQ ID NO:2.
In one embodiment, the antibody of the present invention
is a monoclonal antibody.
In another aspect, the present invention provides a
method of producing an antibody comprising the following
steps in order: inoculating an animal with a polypeptide
selected from the group consisting of: (a)a polypeptide
consisting of the amino acid sequence of SEQ ID NO:2 from
residue 24 to residue 117; (b) a polypeptide consisting
of the amino acid sequence of SEQ ID NO:2 from residue 1
to residue 23; (c) a polypeptide consisting of the amino
acid sequence of SEQ ID NO:2 from residue 24 to residue
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4e
37; (d)a polypeptide consisting of the amino acid sequence
of SEQ ID NO:2 from residue 24 to residue 41; (e) a
polypeptide consisting of the amino acid sequence of SEQ
ID NO:2 from residue 42 to residue 117; wherein the
polypeptide elicits an immune response in the animal to
produce the antibody; and isolating the antibody from the
animal,wherein the antibody specifically binds to an
epitope on the polypeptide having the amino acid sequence
of SEQ ID NO:2 from amino acid number 1 to amino acid
number 117.
In another aspect, the present invention provides a
method of producing a zsig33 polypeptide comprising:
culturing a cell into which has been introduced an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment selected
from the group consisting of: (a) polynucleotide molecules
comprising a nucleotide sequence as shown in SEQ ID NO: 1
from nucleotide 70 to nucleotide 111; (b) allelic variants
of (a); (c) orthologs of (a) or (b);(d) polypeptide
molecules comprising a nucleotide sequence that is at
least 50% identical to the nucleotide sequence set forth
in SEQ ID NO: 1 from nucleotide 1 to nucleotide 111,
wherein the polynucleotide encodes a polypeptide capable
of stimulating gastric motility; (e) degenerate sequences
of (a),(b),(c)or (d); and a transcription terminator,
whereby said cell expresses a polypeptide encoded by the
DNA segment; and recovering the polypeptide.
In another aspect, the present invention
provides a use of an isolated polypeptide selected from
the group consisting of: (a) polypeptide molecules
comprising an amino acid sequence as shown in SEQ ID NO:2
from residue 24 to residue 37;(b) polypeptide molecules
comprising an amino acid sequence that is at least 80%
identical to the amino acid sequence as shown in SEQ ID
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4f
NO:2 from residue 24 to residue 37, wherein the
polypeptides are capable of stimulating gastric motility;
for stimulating gastric motility by increasing transit
time or gastric emptying of an ingested substance. In one
embodiment, the transit time or gastric emptying is
measured using a radiolabeled substance.
In another aspect, the present invention
provides a method of stimulating gastric motility
comprising administering to a mammal in need thereof, an
amount of a composition comprising an isolated polypeptide
selected from the group consisting of: (a) polypeptide
molecules comprising an amino acid sequence as shown in
SEQ ID NO: 2 from residue 24 to residue 37; (b)allelic
variants of (a); and (c) orthologs of (a) or (b); in a
pharmaceutically acceptable vehicle, sufficient to
increase transit time or gastric emptying of an ingested
substance.
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DETAILED DESCRIPTION OF THE INVENTION
Prior to describing the present invention in
detail, it may be helpful to define certain terms used
herein:
The term "ortholog" denotes a polypeptide or
protein obtained from one species that is the functional
counterpart of a polypeptide or protein from a different
species. Sequence differences among orthologs are the
result of speciation.
"Paralogs" are distinct but structurally related
proteins made by an organism. Paralogs are believed to
arise through gene duplication. For example, a-globin, R-
globin, and myoglobin are paralogs of each other.
The term "allelic variant" denotes any of two or
more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally
through mutation, and may result in phenotypic
polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may
encode polypeptides having altered amino acid sequence.
The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
The term "expression vector" denotes a DNA
molecule, linear or circular, that comprises a segment
encoding a polypeptide of interest operably linked to
additional segments that provide for its transcription.
Such additional segments may include promoter and
terminator sequences, and may optionally include one or
more origins of replication, one or more selectable
markers, an enhancer, a polyadenylation signal, and the
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like. Expression vectors are generally derived from
plasmid or viral DNA, or may contain elements of both.
The term "isolated", when applied to a
polynucleotide molecule, denotes that the polynucleotide
has been removed from its natural genetic milieu and is
thus free of other extraneous or unwanted coding
sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such
isolated molecules are those that are separated from their
natural environment and include cDNA and genomic clones.
Isolated DNA molecules of the present invention are free
of other genes with which they are ordinarily associated,
but may include naturally occurring 5' and 3' untranslated
regions such as promoters and terminators. The
identification of associated regions will be evident to
one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985). When applied to a
protein, the term "isolated" indicates that the protein is
found in a condition other than its native environment,
such as apart from blood and animal tissue. In a
preferred form, the isolated protein is substantially free
of other proteins, particularly other proteins of animal
origin. It is preferred to provide the protein in a
highly purified form, i.e., greater than 95% pure, more
preferably greater than 99% pure.
The term "operably linked", when referring to
DNA segments, denotes that the segments are arranged so
that they function in concert for their intended purposes,
e.g. transcription initiates in the promoter and proceeds
through the coding segment to the terminator
The term "polynucleotide" denotes a single- or
double-stranded polymer of deoxyribonucleotide or
ribonucleotide bases read from the 5' to the 3' end.
Polynucleotides include RNA and DNA, and may be isolated
from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules.
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The term "complements of polynucleotide
molecules" denotes polynucleotide molecules having a
complementary base sequence and reverse orientation as
compared to a reference sequence. For example, the
sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT
3'.
The term "degenerate nucleotide sequence"
denotes a sequence of nucleotides that includes one or
more degenerate codons (as compared to a reference
polynucleotide molecule that encodes a polypeptide).
Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e.,
GAU and GAC triplets each encode Asp).
The term "promoter" denotes a portion of a gene
containing DNA sequences that provide for the binding of
RNA polymerase and initiation of transcription. Promoter
sequences are commonly, but not always, found in the 5'
non-coding regions of genes.
The term "secretory signal sequence" denotes a
DNA sequence that encodes a polypeptide (a "secretory
peptide") that, as a component of a larger polypeptide,
directs the larger polypeptide through a secretory pathway
of a cell in which it is synthesized. The larger peptide
is commonly cleaved to remove the secretory peptide during
transit through the secretory pathway.
The term "receptor" denotes a cell-associated
protein that binds to a bioactive molecule (i.e., a
ligand) and mediates the effect of the ligand on the cell.
Membrane-bound receptors are characterized by a multi-
domain structure comprising an extracellular ligand-
binding domain and an intracellular effector domain that
is typically involved in signal transduction. Binding of
ligand to receptor results in a conformational change in
the receptor that causes an interaction between the
effector domain and other molecule(s) in the cell. This
interaction in turn leads to an alteration in the
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metabolism of the cell. Metabolic events that are linked
to receptor-ligand interactions include gene
transcription, phosphorylation, dephcsphorylation,
increases in cyclic AMP production, mobilization of
cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids. Most nuclear receptors also exhibit a
multi-domain structure, including an amino-terminal,
transactivating domain, a DNA binding domain and a ligand
binding domain. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid
stimulating hormone receptor, beta-ad,eneraic receptor) or
multimeric (e.g., PDGF receptor, growth hormone receptor,
IL-3 receptor, GM-CSF receptor, G-CSF receptor,
erythropoietin receptor and IL-6 receptor).
The term "complement/anti-complement pair"
denotes non-identical moieties that form a non-covalently
associated, stable pair under appropriate conditions. For
instance, biotin and avidin (or streptavidin) are
prototypical members of a complement/anti-complement pair.
Other exemplary complement/anti-complement pairs include
receptor/ligand pairs, antibody/antigen (or hapten or
epitope) pairs, sense/antisense polynucleotide pairs, and
the like. Where subsequent dissociation of the
complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding
affinity of <109 M-1.
The present invention is based in part upon the
discovery of a novel human DNA sequence that encodes a
novel secreted polypeptide having homology to motilin, of
which the closest homolog is porcine motilin (shown in SEQ
ID NOs : 3 and 4) . Motilin is member of a family of
polypeptides that regulate the gastrointestinal
physiology. The family of polypeptides important in
RCV. VON: E!'9-NILF_NCHLN u4 3- 4.-CA 02284733 1999-09-21 cc I'CC EcM-. +49
8:3 -13994465: 7
PCT/VS96/05620 9
gastrointestinal regulation to which motilin belongs
includes glucagoz, gastrin, galanin, and vasoactive
intestinal peptide (VIP). These polypeptides are
synthesized in a precursor form that requires multiple
S -steps of processing to the active form, Particularly
relevant to the polypeptide of the present invention are
motilin, VIP and galanin, where processing involves
removal of signal sequence, followed by cleavage of one or
more accessory peptides to release the active peptide.
The resulting active peptide is generally small (10-30
amino acids) and may require further post-translational
modifications, such as amidation, sulfation or pyrroiidan
carbonylic acid modification of glutamic residues.
Analysis of the tissue distribution of the mRNA
corresponding to this novel DNA showed that expression was
highest in stomach, followed by apparent but decreased
expression levels in small intestine and pancreas. The
EST is also present in lung cDNA libraries. The
polypeptide has been designated zsig33.
The novel zsig33 polynucleotides and
polypeptides of the present invention were initially
identified by querying an EST database for sequences
possessing a putative secretion signal. An EST sequence
was discovered and predicted to be related to the motilin
family. The EST sequence was derived from a fetal
pancreatic library.
The novel polypeptide encoded by the full length
cDNA is 117 amino acids. The predicted signal sequence is
23 amino acid. residues (amino acid residues 1 to 23 of SEQ
ID NO; 2). The active peptide was predicted to be 18
amino acid residues (amino acid residues 24 to 41 of SEQ
ID NO: 2), with a C-terminal cleavage after amino acid
residue 41 of SEQ ID NO. 2 (Ser). However, many of the
gut-brain peptides require multiple cleavages. For
example, progastrin peptide is 101 amino acids, and ie
cleaved at the N-terminus resulting in sequentially
AMENDED 1SW 1iEEr
FCV.'Y)\:EPA-%IL'E\CHEN i)4b 3- 4.-CA 02284733 1999-09-21 (:CITY ECM- +4=;)
89 2t39944E35:# 8
PCT/U896/05630 10
smaller peptides (G34, G17 and G14) (Sugano et al.,
Biol. Chem._ Z.Q,:11724-11729, 1985). Other peptides that
require multiple processing steps include glucagon, for
which C-terminal cleavages result in glucagon-like peptide
1 and glucagon-like peptide 2 and galanin, in which
processing involves cleavage of a C-terminal peptide known
as GMAP. Therefore, an additional peptide based on
cleavage after amino acid 37 of SEQ ID NO: 2 (Gln) was
synthesized and resulted in a 14 amino acid peptide with
biological activity (from amino acid residue 24 (Gly) to
amino acid residue 37 (G1n) of SEQ ID NO; 2).
The C-terminal peptide (amino acid 42 to 117 of
SEQ ID NO: 2) may have some specialized activity as well.
Processing of the active peptide for mocilin (shown in SEQ
ID NO: 4) results in a release of a C-terminal peptide of
70 amino acids, amino acid residue 5o (Ser) to amino acid
residue 119 (Lys), known as motilin-associated peptide
(MAP). Adelman et al., (U.S. Patent 5,006,469) have
postulated that HAD plays a role in regulation of
digestion, appetite and nutrient absorption.
The highly conserved amino acids in the
polypeptide zaig33 can be used as a tool to identify new
family members. For instance, reverse transcription-
polymerase chain reaction (RT-PCR) can be used to amplify
sequences encoding the conserved motif from RNA obtained
from a variety of tissue sources. Two such conserved
domains have been identified using sequences from the
present invention. The first domain is found at amino
acid residues 31 to 36 of SEQ ID NO; 2, wherein the motif
identified is GLu X Gin Arg X Gin, wherein X is any amino
acid residue, and the second domain is found at amino acid
residues 78 to 84 of SEQ ID NO: 2, wherein the motif
identified is Ala Pro X Asp X Gly Ile, wherein X is any
amino acid residue. in particular, highly degenerate
primers designed from these sequences are useful for this
purpose.
AMENDED SHEET
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Those skilled in the art will readily recognize
that, in view of the degeneracy of the genetic code,
considerable sequence variation is possible among these
polynucleotide molecules encoding SEQ ID NO:2, including
all RNA sequences by substituting U for T. Thus, zsig33
polypeptide-encoding polynucleotides and their RNA
equivalents are contemplated by the present invention.
Table 1 sets forth the one-letter codes used to denote
degenerate nucleotide positions. "Resolutions" are the
nucleotides denoted by a code letter. "Complement"
indicates the code for the complementary nucleotide(s).
For example, the code Y denotes either C or T, and its
complement R denotes A or G, A being complementary to T,
and G being complementary to C.
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TABLE 1
Nucleotide Resolution Nucleotide Complement
A A T T
C C G G
G G C C
T T A A
R AIG Y CST
Y CIT R AEG
M AIC K GIT
K GAT M AIC
S CMG S CMG
W AST W AIT
H AICIT D AIGIT
B CIGIT V AICIG
V AICIG B CIGIT
D AIGIT H AICIT
N AICIGIT N AICIGIT
The degenerate codons encompassing all possible
codons for a given amino acid are set forth in Table 2.
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TABLE 2
One
Amino Letter Codons Degenerate
Acid Code Codon
Cys C TGC TGT TGY
Ser S AGC AGT TCA TCC TCG TCT WSN
Thr T ACA ACC ACG ACT ACN
Pro P CCA CCC CCG CCT CCN
Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN
Asn N AAC AAT AAY
Asp D GAC GAT GAY
Glu E GAA GAG GAR
Gln Q CAA CAG CAR
His H CAC CAT CAY
Arg R AGA AGG CGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
Ile I ATA ATC ATT ATH
Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN
Phe F TTC TTT TTY
Tyr Y TAC TAT TAY
Trp W TGG TGG
Ter TAA TAG TGA TRR
AsnlAsp B RAY
GluIGln Z SAR
Any X NNN
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One of ordinary skill in the art will appreciate
that some ambiguity is introduced in determining a
degenerate codon, representative of all possible codons
encoding each amino acid. For example, the degenerate
codon for serine (WSN) can, in some circumstances, encode
arginine (AGR), and the degenerate codon for arginine
(MGN) can, in some circumstances, encode serine (AGY) . A
similar relationship exists between codons encoding
phenylalanine and leucine. Thus, some polynucleotides
encompassed by the degenerate sequence may encode variant
amino acid sequences, but one of ordinary skill in the art
can easily identify such variant sequences by reference to
the amino acid sequence of SEQ ID NO:2. Variant sequences
can be readily tested for functionality as described
herein.
Within preferred embodiments of the invention
the isolated polynucleotides will hybridize to similar
sized regions of SEQ ID NO: 1, or a sequence complementary
thereto, under stringent conditions. In general,
stringent conditions are selected to be about 5 C lower
than the thermal melting point (Tm) for the specific
sequence at a defined ionic strength and pH. The Tm is
the temperature (under defined ionic strength and pH) at
which 500 of the target sequence hybridizes to a perfectly
matched probe. Typical stringent conditions are those in
which the salt concentration is at least about 0.02 M at
pH 7 and the temperature is at least about 60 C.
As previously noted, the isolated
polynucleotides of the present invention include DNA and
RNA. Methods for isolating DNA and RNA are well known in
the art. It is generally preferred to isolate RNA from
stomach, although DNA can also be prepared using RNA from
other tissues or isolated as genomic DNA. Total RNA can
be prepared using guanidine HC1 extraction followed by
isolation by centrifugation in a CsCl gradient (Chirgwin
et al., Biochemistry 18:52-94, 1979). Poly (A) + RNA is
CA 02284733 1999-09-21
WO 98/42840 PCT/US98/05620
prepared from total RNA using the method of Aviv and Leder
(Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972).
Complementary DNA (cDNA) is prepared from poly (A)+ RNA
using known methods. Polynucleotides encoding zsig33
5 polypeptides are then identified and isolated by, for
example, hybridization or PCR.
The present invention further provides
counterpart polypeptides and polynucleotides from other
species (orthologs). Of particular interest are zsig33
10 polypeptides from other mammalian species, including
murine, rat, porcine, ovine, bovine, canine, feline,
equine and other primate proteins. Orthologs of the human
proteins can be cloned using information and compositions
provided by the present invention in combination with
15 conventional cloning techniques. For example, a cDNA can
be cloned using mRNA obtained from a tissue or cell type
that expresses the protein. Suitable sources of mRNA can
be identified by probing Northern blots with probes
designed from the sequences disclosed herein. A library
is then prepared from mRNA of a positive tissue of cell
line. A zsig33 ortholog-encoding cDNA can then be
isolated by a variety of methods, such as by probing with
a complete or partial human cDNA or with one or more sets
of degenerate probes based on the disclosed sequences. A
cDNA can also be cloned using the polymerase chain
reaction, or PCR (Mullis, U.S. Patent 4,683,202), using
primers designed from the sequences disclosed herein.
Within an additional method, the cDNA library can be used
to transform or transfect host cells, and expression of
the cDNA of interest can be detected with an antibody to
zsig33 Similar techniques can also be applied to the
isolation of genomic clones.
Those skilled in the art will recognize that the
sequences disclosed in SEQ ID NO: 1, and polypeptide
encoded thereby, represent a single allele of the human
zsig33 gene and polypeptide, and that allelic variation
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16
and alternative splicing are expected to occur. Allelic
variants can be cloned by probing cDNA or genomic
libraries from different individuals according to standard
procedures. Allelic variants of the DNA sequence shown in
SEQ ID NO: 1, including those containing silent mutations
and those in which mutations result in amino acid sequence
changes, are within the scope of the present invention, as
are proteins which are the product of allelic variation of
SEQ ID NO: 2.
The present invention also provides isolated
zsig33 polypeptides that are substantially homologous to
the polypeptides of SEQ ID NO: 2 and their orthologs. The
term "substantially homologous" is used herein to denote
polypeptides having 50%, preferably 60%, more preferably
at least 80%, sequence identity to the sequences shown in
SEQ ID NO: 2 or their orthologs. Such polypeptides will
more preferably be at least 90% identical, and most
preferably 95% or more identical to SEQ ID NO: 2 or its
orthologs. Percent sequence identity is determined by
conventional methods. See, for example, Altschul et al.,
Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.
Briefly, two amino acid sequences are aligned to optimize
the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "blosum 62" scoring
matrix of Henikoff and Henikoff (ibid.) as shown in Table
3 (amino acids are indicated by the standard one-letter
codes).
CA 02284733 1999-09-21
WO 98/42840 PCT/US98/05620
17
H N M
H I
H L11 N N O
d1 H M N N
I I 1
a N H H 4 M N
I I t
4+ l0 d1 N N H M r--I
1 I I
LJ O N H r-i H H I-1
I I I I I
~4 LC) H M H O -1 M N N
I I I I I
M I~ I'll N N O M N r-i N H H
u) I I I I I
r-I d1 N M H O M N H M rl M
I I I 1 I I
'~". tb M M r-1 N H N H N N N M
I I I I I I 1 I
C l0 N cr d1 N M M N O N N M M
I 1 I I t I t I I
I!) N O M M H N M H O H M N N
I I I I I I I
O1 Ln N N O M N H O M H O H N H N
I I I U IT M dr M M r1 H M rl N M r i rt N N ri
I 1 I t I I I I I I t I
A l0 M O N ri r1 M dr H M M H O H Vr M M
I I I I I I I I I
z l~ r-1 M O O O H
i M M O N M N ri O cjr N M
I i I t t 1 I
R tll O N M H O N O M N N H M N H H M N M
I I I I 1 t I I 1 I I I
FC ~N r~ N N O H H O N H ri H H N r-1 rl O M N O
I I I I I I I I I I I I I
4 C4 Zi A U 01 W C~ x' - 1 ~4 .' 4 X r4 a4 al E-1 v~ >+
L(1 O LO 0
r-1 H N
CA 02284733 2003-12-02
18
The percent identity is then calculated as:
Total number of identical matches
x 100
[length of the longer sequence plus the number
of caps introduced into the longer sequence in
order to align the two sequences]
Sequence identity of polynucleotide molecules is
determined by similar methods using a ratio as disclosed
above.
Substantially homologous proteins and
polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes
are preferably of a minor nature, that is conservative
amino acid substitutions (see Table 4) and other
substitutions that do not significantly affect the folding
or activity of the protein or polypeptide; small
deletions, typically of one to about 30 amino acids; and
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide
of up to about 20-25 residues, or a small extension that
facilitates purification (an affinity tag) , such as a poly-
histidine tract, protein A (Nilsson et al., EMBO J.
4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3,
1991) , glutathione S transferase (Smith and Johnson, Gene
67:31, 1988), maltose binding protein (Kellerman and
Ferenci, Methods Enzymol. 90:459-463, 1982; Guan et al.,
Gene 67:21-30, 1987), thioredoxin, ubiquitin, cellulose
binding protein, T7 polymerase, or other antigenic epitope
or binding domain. See, in general Ford et al., Protein
Expression and Purification 2: 95-107, 1991.
DNAs encoding affinity
tags are available from commercial suppliers (e.g.,
Pharmacia Biotech, Piscataway, NJ; New England Biolabs,
Beverly, MA).
CA 02284733 1999-09-21
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19
Table 4
Conservative amino acid substitutions
Basic: arginine
lysine
histidine
Acidic: glutamic acid
aspartic acid
Polar: glutamine
asparagine
Hydrophobic: leucine
isoleucine
valine
Aromatic: phenylalanine
tryptophan
tyrosine
Small: glycine
alanine
serine
threonine
methionine
In addition to the 20 standard amino acids, non-
standard amino acids (such as 4-hydroxyproline, 6-N-methyl
lysine, 2-aminoisobutyric acid, isovaline and a-methyl
serine) may be substituted for amino acid residues of
zsig33. A limited number of non-conservative amino acids,
amino acids that are not encoded by the genetic code, and
unnatural amino acids may be substituted for zsig33 amino
acid residues. "Unnatural amino acids" have been modified
after protein synthesis, and/or have a chemical structure
in their side chain(s) different from that of the standard
amino acids. Unnatural amino acids can be chemically
synthesized, or preferably, are commercially available,
and include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and 3,3-
dimethylproline.
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Essential amino acids in the zsig33 polypeptides
of the present invention can be identified according to
procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham
5 and Wells, Science 244: 1081-1085, 1989) . In the latter
technique, single alanine mutations are introduced at
every residue in the molecule, and the resultant mutant
molecules are tested for biological activity (e.g.,
stimulation of gastrointestinal cell contractility,
10 modulation of nutrient uptake and/or secretion of
digestive enzymes) to identify amino acid residues that
are critical to the activity of the molecule. See also,
Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites
of ligand-receptor interaction can also be determined by
15 physical analysis of structure, as determined by such
techniques as nuclear magnetic resonance, crystallography,
electron diffraction or photoaffinity labeling, in
conjunction with mutation of putative contact site amino
acids. See, for example, de Vos et al., Science 255:306-
20 312, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992;
Wlodaver et al., FEBS Lett. 309:59-64, 1992. The
identities of essential amino acids can also be inferred
from analysis of homologies with related members of the
glucagon-secretin family of gut-brain peptide hormones.
Multiple amino acid substitutions can be made
and tested using known methods of mutagenesis and
screening, such as those disclosed by Reidhaar-Olson and
Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc.
Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these
authors disclose methods for simultaneously randomizing
two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the
mutagenized polypeptides to determine the spectrum of
allowable substitutions at each position. Other methods
that can be used include phage display (e.g., Lowman et
al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S.
CA 02284733 1999-09-21
WO 98/42840 PCT/US98/05620
21
Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204)
and region-directed mutagenesis (Derbyshire et al., Gene
46:145, 1986; Ner et al., DNA 7:127, 1988).
Mutagenesis methods as disclosed above can be
combined with high-throughput, automated screening methods
to detect activity of cloned, mutagenized polypeptides in
host cells. Mutagenized DNA molecules that encode active
polypeptides (e.g., stimulation of gastrointestinal cell
contractility, modulation of nutrient uptake and/or
secretion of digestive enzymes) can be recovered from the
host cells and rapidly sequenced using modern equipment.
These methods allow the rapid determination of the
importance of individual amino acid residues in a
polypeptide of interest, and can be applied to
polypeptides of unknown structure.
Using the methods discussed above, one of
ordinary skill in the art can identify and/or prepare a
variety of polypeptides that are substantially homologous
to residues 24 to 37 of SEQ ID NO: 2 or allelic variants
thereof and retain properties of the wild-type protein.
Such polypeptides may also include additional polypeptide
segments as generally disclosed above.
The polypeptides of the present invention,
including full-length proteins and fragments thereof, can
be produced in genetically engineered host cells according
to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with
exogenous DNA and grown in culture, and include bacteria,
fungal cells, and cultured higher eukaryotic cells.
Eukaryotic cells, particularly cultured cells of
multicellular organisms, are preferred. Techniques for
manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed
by Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, 1989, and Ausubel et al. (eds.),
CA 02284733 2003-12-02
22
Current Protocols in Molecular Biology, John Wiley and
Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a zsig33
polypeptide of the present invention is operably linked to
other genetic elements required for its expression,
generally including a transcription promoter and
terminator within an expression vector. The vector will
also commonly contain one or more selectable markers and
one or more origins of replication, although those skilled
in the art will recognize that within certain systems
selectable markers may be provided on separate vectors,
and replication of the exogenous DNA may be provided by
integration into the host cell genome. Selection of
promoters, terminators, selectable markers, vectors and
other elements is a matter of routine design within the
level of ordinary skill in the art. Many such elements
are described in the literature and are available through
commercial suppliers.
To direct a zsig33 polypeptide into the
secretory pathway of a host cell, a secretory signal
sequence (also known as a leader sequence, prepro sequence
or pre sequence) is provided in the expression vector.
The secretory signal sequence may be that of the zsig33
polypeptide, or may be derived from another secreted
protein (e.g., t-PA) or synthesized de novo. The
secretory signal sequence is joined to the zsig33 DNA
sequence in the correct reading frame. Secretory signal
sequences are commonly positioned 5' to the DNA sequence
encoding the propeptide of interest, although certain
signal sequences may be positioned elsewhere in the DNA
sequence of interest (see, e.g., Welch et al., U.S. Patent
No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).
Cultured mammalian cells are also preferred
hosts within the present invention. Methods for
introducing exogenous DNA into mammalian host cells
CA 02284733 2003-12-02
23
include calcium phosphate-mediated transfection (Wialer et
al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell
Genetics 7:603, 1981: Graham and Van der Eb, Virology
52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection
(Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987), liposome-
mediated transfection (Hawley-Nelson et al., Focus 15:73,
1993; Ciccarone et al., Focus 15:80, 1993), and viral
vectors (A. Miller and G. Rosman, BioTechnigues 7:980-90,
1989; Q. Wang and M. Finer, Nature Med. 2:714-16, 1996).
The
production of recombinant polypeptides in cultured
mammalian cells is disclosed, for example, by Levinson et
al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent
No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821;
and Ringold, U.S. Patent No. 4,656,134.
Preferred cultured
mammalian cells include the COS-1 (ATCC No. CRL 1650),
COS-7 (ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314),
293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol.
36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1;
ATCC No. CCL 61) cell lines. Additional suitable cell
lines are known in the art and available from public
depositories such as the American Type Culture Collection,
Rockville, Maryland. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
Other suitable promoters include those from
metallothionein genes (U.S. Patent Nos. 4,579,821 and
4,601,978 ) and
the aderovirus major late promoter.
Drug selection is generally used to select for
cultured mammalian cells into which foreign DNA has been
inserted. Such cells are commonly referred to as
"transfectants". Cells that have been cultured in the
CA 02284733 1999-09-21
WO 98/42840 PCT/US98/05620
24
presence of the selective agent and are able to pass the
gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is
a gene encoding resistance to the antibiotic neomycin.
Selection is carried out in the presence of a neomycin-
type drug, such as G-418 or the like. Selection systems
may also be used to increase the expression level of the
gene of interest, a process referred to as
"amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of
the selective agent and then increasing the amount of
selective agent to select for cells that produce high
levels of the products of the introduced genes. A
preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate.
Other drug resistance genes (e.g., hygromycin resistance,
multi-drug resistance, puromycin acetyltransferase) can
also be used. Alternative markers that introduce an
altered phenotype, such as green fluorescent protein, or
cell surface proteins such as CD4, CD8, Class I MHC,
placental alkaline phosphatase may be used to sort
transfected cells from untransfected cells by such means
as FACS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used
as hosts, including plant cells, insect cells and avian
cells. The use of Agrobacterium rhizogenes as a vector
for expressing genes in plant cells has been reviewed by
Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.
Transformation of insect cells and production of foreign
polypeptides therein is disclosed by Guarino et al., U.S.
Patent No. 5,162,222 and WIPO publication WO 94/06463.
Insect cells can be infected with recombinant baculovirus,
commonly derived from Autographa californica nuclear
polyhedrosis virus (AcNPV). DNA encoding the zsig33
polypeptide is inserted into the baculoviral genome in
place of the AcNPV polyhedrin gene coding sequence by one
CA 02284733 1999-09-21
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of two methods. The first is the traditional method of
homologous DNA recombination between wild-type AcNPV and a
transfer vector containing the zsig33 flanked by AcNPV
sequences. Suitable insect cells, e.g. SF9 cells, are
5 infected with wild-type AcNPV and transfected with a
transfer vector comprising a zsig33 polynucleotide
operably linked to an AcNPV polyhedrin gene promoter,
terminator, and flanking sequences. See, King, L.A. and
Possee, R.D., The Baculovirus Expression System: A
10 Laboratory Guide, London, Chapman & Hall; O'Reilly, D.R.
et al., Baculovirus Expression Vectors: A Laboratory
Manual, New York, Oxford University Press., 1994; and,
Richardson, C. D., Ed., Baculovirus Expression Protocols.
Methods in Molecular Biology, Totowa, NJ, Humana Press,
15 1995. Natural recombination within an insect cell will
result in a recombinant baculovirus which contains zsig33
driven by the polyhedrin promoter. Recombinant viral
stocks are made by methods commonly used in the art.
The second method of making recombinant
20 baculovirus utilizes a transposon-based system described
by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993).
This system is sold in the Bac-to-Bac kit (Life
Technologies, Rockville, MD). This system utilizes a
transfer vector, pFastBaclTM (Life Technologies) containing
25 a Tn7 transposon to move the DNA encoding the zsig33
polypeptide into a baculovirus genome maintained in E.
coli as a large plasmid called a "bacmid." The pFastBaclTM
transfer vector utilizes the AcNPV polyhedrin promoter to
drive the expression of the gene of interest, in this case
zsig33. However, pFastBaclTM can be modified to a
considerable degree. The polyhedrin promoter can be
removed and substituted with the baculovirus basic protein
promoter (also known as Pcor, p6.9 or MP promoter) which
is expressed earlier in the baculovirus infection, and has
been shown to be advantageous for expressing secreted
proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen
CA 02284733 1999-09-21
WO 98/42840 PCT/US98/05620
26
Virol 71:971-6, 1990; Bonning, B.C. et al., J Gen Virol
75:1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B.,
J Biol Chem 270:1543-9, 1995. In such transfer vector
constructs, a short or long version of the basic protein
promoter can be used. Moreover, transfer vectors can be
constructed which replace the native zsig33 secretory
signal sequences with secretory signal sequences derived
from insect proteins. For example, a secretory signal
sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus
gp67 (PharMingen, San Diego, CA) can be used in constructs
to replace the native zsig33 secretory signal sequence.
In addition, transfer vectors can include an in-frame
fusion with DNA encoding an epitope tag at the C- or N-
terminus of the expressed zsig33 polypeptide, for example,
a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc Natl
Acad Sci. 82:7952-4, 1985). Using a technique known in
the art, a transfer vector containing zsig33 is
transformed into E. Coli, and screened for bacmids which
contain an interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant
baculovirus genome is isolated, using common techniques,
and used to transfect Spodoptera frugiperda cells, e.g.
Sf9 cells. Recombinant virus that expresses zsig33 is
subsequently produced. Recombinant viral stocks are made
by methods commonly used the art.
The recombinant virus is used to infect host
cells, typically a cell line derived from the fall
armyworm, Spodoptera frugiperda. See, in general, Glick
and Pasternak, Molecular Biotechnology: Principles and
Applications of Recombinant DNA, ASM Press, Washington,
D.C., 1994. Another suitable cell line is the High FiveOTM
cell line (Invitrogen) derived from Trichoplusia ni (U.S.
Patent #5,300,435). Commercially available serum-free
media are used to grow and maintain the cells. Suitable
media are Sf900 IITM (Life Technologies) or ESF 921TM
CA 02284733 2003-12-02
27
(Expression Systems) for the Sf9 cells; and Ex-cell0405TM
(JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life
Technologies) for the T. ni cells. The cells are grown up
from an inoculation density of approximately 2-5 x 105
cells to a density of 1-2 x 106 cells at which time a
recombinant viral stock is added at a multiplicity of
infection (MOI) of 0.1 to 10, more typically near 3. The
recombinant virus-infected cells typically produce the
recombinant zsig33 polypeptide at 12-72 hours post-
infection and secrete it with varying efficiency into the
medium. The culture is usually harvested 48 hours post-
infection. Centrifugation is used to separate the cells
from the medium (supernatant). The supernatant containing
the zsig33 polypeptide is filtered through micropore
filters, usually 0.45 m pore size. Procedures used are
generally described in available laboratory manuals (King,
L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al.,
ibid.; Richardson, C. D., ibid.). Subsequent purification
of the zsig33 polypeptide from the supernatant can be
achieved using methods described herein.
Fungal cells, including yeast cells, and
particularly cells of the genera Saccharomyces and Pichia,
can also be used within the present invention, such as for
producing zsig33 fragments or polypeptide fusions.
Methods for transforming yeast cells with exogenous DNA
and producing recombinant polypeptides therefrom are
disclosed by, for example, Kawasaki, U.S. Patent No.
4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373;
Brake, U.S. Patent No. 4,870,008; Welch et al., U.S.
Patent No. 5,037,743; and Murray et al., U.S. Patent No.
4,845,075.
Transformed cells are selected by phenotype determined by
the selectable marker, commonly drug resistance or th=
ability to grow in the absence of a particular nutrient
(e.g., leucine) . A preferred vector system for use in
yeast is the POT1 vector system disclosed by Kawasaki et
CA 02284733 2003-12-02
28
al. (U.S. Patent No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing
media. Suitable promoters and terminators for use in
yeast include those from glycolytic enzyme genes (see,
e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et
al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent
No. 4,977,092.
and alcohol dehydrogenase genes. See also U.S. Patents
Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation
systems for other yeasts, including Hansenula polymorpha,
Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris,
Pichia guillermondii, Pichia methanolica and' Candida
maltosa are known in the art. See, for example, Gleeson
et al., J. Gen. Microbiol. 132:3459-3465, 1986 and Cregg,
U.S. Patent No. 4,882,279. Aspergillus cells may be
utilized according to the methods of McKnight et al., U.S.
Patent No. 4,935,349.
Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Patent
No. 5,162,228.
Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Patent No. 4,486,533.
Transformed or transfected host cells are
cultured according to conventional procedures in a culture
medium containing nutrients and other components required
for the growth of the chosen host cells. A variety of
suitable media, including defined media and complex media,
are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins
and minerals. Media may also contain such components as
growth factors or serum, as required. The growth medium
will generally select for cells containing the exogenously
CA 02284733 2003-12-02
29
added DNA by, for example, drug selection or deficiency in
an essential nutrient which is complemented by the
selectable marker carried on the expression vector or co-
transfected into the host cell. P. methanolica cells are
cultured in a medium comprising adequate sources of
carbon, nitrogen and trace nutrients at a temperature of
about 25 C to 35 C. Liquid cultures are provided with
sufficient aeration by conventional means, such as shaking
of small flasks or sparging of fermentors. A preferred
culture medium for P. methanolica is YEPD (21 D-glucose,
21 BactoTM Peptone (Difco Laboratories, Detroit, MI), 11
BactoTM yeast extract (Difco Laboratories), 0.0041 adenine
and 0.006% L-leucine).
Expressed recombinant zsig33 polypeptides can be
purified using fractionation and/or conventional
purification methods and media. Ammonium sulfate
precipitation and acid or chaotrope extraction may be used
for fractionation of samples. Exemplary purification
steps may include hydroxyapatite, size exclusion, FPLC and
reverse-phase high performance liquid chromatography.
Suitable anion exchange media include derivatized
dextrans, agarose, cellulose, polyacrylamide, specialty
silicas, and the like. PEI, DEAE, QAE and Q derivatives
are preferred, with DEAE Fast-Flow Sepharose* (Pharmaca,
Piscataway, NJ) being particularly preferred. Exemplary
chromatographic media include those media derivatized with
phenyl, butyl, or octyl groups, such as Phenyl-Sepharose
FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, PA), Octyl-Sepharose* (Pharmacia) and the
like; or polyacrylic resins, such as Amberchrom CG 71
(Toso Haas) and the like. Suitable solid supports include
glass beads, silica-based resins, cellulosic resins,
agarose beads, cross-linked agarose beads, polystyrene
beads, cross-linked polyacrylamide resins and the like
that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive
* trade-mark
CA 02284733 1999-09-21
WO 98/42840 PCTIUS98/05620
groups that allow attachment of proteins by amino groups,
carboxyl groups, sulfhydryl groups, hydroxyl groups and/or
carbohydrate moieties. Examples of coupling chemistries
include cyanogen bromide activation, N-hydroxysuccinimide
5 activation, epoxide activation, sulfhydryl activation,
hydrazide activation, and carboxyl and amino derivatives
for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and
are available from commercial suppliers. Methods for
10 binding receptor polypeptides to support media are well
known in the art. Selection of a particular method is a
matter of routine design and is determined in part by the
properties of the chosen support. See, for example,
Affinity Chromatography: Principles & Methods, Pharmacia
15 LKB Biotechnology, Uppsala, Sweden, 1988.
The polypeptides of the present invention can be
isolated by exploitation of small size and low pI. For
example, polypeptides of the present invention can be
bound to anionic exchanges at low pH values. Other
20 methods of purification include purification of
glycosylated proteins by lectin affinity chromatography
and ion exchange chromatography (Methods in Enzymol., Vol.
182, "Guide to Protein Purification", M. Deutscher, (ed.),
Acad. Press, San Diego, 1990, pp.529-39). Alternatively,
25 a fusion of the polypeptide of interest and an affinity
tag (e.g., polyhistidine, maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification.
Protein refolding (and optionally reoxidation)
30 procedures may be advantageously used. It is preferred to
purify the protein to >800i purity, more preferably to >90%
purity, even more preferably >95%, and particularly
preferred is a pharmaceutically pure state, that is
greater than 99.9% pure with respect to contaminating
macromolecules, particularly other proteins and nucleic
acids, and free of infectious and pyrogenic agents.
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31
Preferably, a purified protein is substantially free of
other proteins, particularly other proteins of animal
origin.
zsig33 polypeptides or fragments thereof may
also be prepared through chemical synthesis. zsig33
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; pegylated or non-pegylated; amidated or
non-amidated; sulfated or non-sulfated; and may or may not
include an initial methionine amino acid residue. For
example, zsig33 polypeptides can also be synthesized by
exclusive solid phase synthesis, partial solid phase
methods, fragment condensation or classical solution
synthesis. The polypeptides are preferably prepared by
solid phase peptide synthesis, for example as described by
Merrifield, J. Am. Chem. Soc. 85:2149, 1963. The
synthesis is carried out with amino acids that are
protected at the alpha-amino terminus. Trifunctional
amino acids with labile side-chains are also protected
with suitable groups to prevent undesired chemical
reactions from occurring during the assembly of the
polypeptides. The alpha-amino protecting group is
selectively removed to allow subsequent reaction to take
place at the amino-terminus. The conditions for the
removal of the alpha-amino protecting group do not remove
the side-chain protecting groups.
The alpha-amino protecting groups are those
known to be useful in the art of stepwise polypeptide
synthesis. Included are acyl type protecting groups
(e.g., formyl, trifluoroacetyl, acetyl), aryl type
protecting groups (e.g., biotinyl), aromatic urethane type
protecting groups [e.g., benzyloxycarbonyl (Cbz),
substituted benzyloxycarbonyl and 9-fluorenylmethyloxy-
carbonyl (Fmoc)], aliphatic urethane protecting groups
[e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,
cyclohexloxycarbonyl] and alkyl type protecting groups
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32
(e.g., benzyl, triphenylmethyl). The preferred protecting
groups are tBoc and Fmoc.
The side-chain protecting groups selected must
remain intact during coupling and not be removed during
the deprotection of the amino-terminus protecting group or
during coupling conditions. The side-chain protecting
groups must also be removable upon the completion of
synthesis using reaction conditions that will not alter
the finished polypeptide. In tBoc chemistry, the side-
chain protecting groups for trifunctional amino acids are
mostly benzyl based. In Fmoc chemistry, they are mostly
tert-butyl or trityl based.
In tBoc chemistry, the preferred side-chain
protecting groups are tosyl for arginine, cyclohexyl for
aspartic acid, 4-methylbenzyl (and acetamidomethyl) for
cysteine, benzyl for glutamic acid, serine and threonine,
benzyloxymethyl (and dinitrophenyl) for histidine, 2-Cl-
benzyloxycarbonyl for lysine, formyl for tryptophan and 2-
bromobenzyl for tyrosine. In Fmoc chemistry, the
preferred side-chain protecting groups are 2,2,5,7,8-
pentamethylchroman-6-sulfonyl (Pmc) or 2,2,4,6,7-
pentamethyldihydrobenzofuran- 5-sulfonyl (Pbf) for
arginine, trityl for asparagine, cysteine, glutamine and
histidine, tert-butyl for aspartic acid, glutamic acid,
serine, threonine and tyrosine, tBoc for lysine and
tryptophan.
For the synthesis of phosphopeptides, either
direct or post-assembly incorporation of the phosphate
group is used. In the direct incorporation strategy, the
phosphate group on serine, threonine or tyrosine may be
protected by methyl, benzyl, or tert-butyl in Fmoc
chemistry or by methyl, benzyl or phenyl in tBoc
chemistry. Direct incorporation of phosphotyrosine without
phosphate protection can also be used in Fmoc chemistry.
In the post-assembly incorporation strategy, the
unprotected hydroxyl groups of serine, threonine or
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33
tyrosine are derivatized on solid phase with di-tert-
butyl-, dibenzyl- or dimethyl-N,N'-
diisopropylphosphoramidite and then oxidized by tert-
butylhydroperoxide.
Solid phase synthesis is usually carried out
from the carboxyl-terminus by coupling the alpha-amino
protected (side-chain protected) amino acid to a suitable
solid support. An ester linkage is formed when the
attachment is made to a chloromethyl, chlortrityl or
hydroxymethyl resin, and the resulting polypeptide will
have a free carboxyl group at the C-terminus.
Alternatively, when an amide resin such as benzhydrylamine
or p-methylbenzhydrylamine resin (for tBoc chemistry) and
Rink amide or PAL resin (for Fmoc chemistry) are used, an
amide bond is formed and the resulting polypeptide will
have a carboxamide group at the C-terminus. These resins,
whether polystyrene- or polyamide-based or
polyethyleneglycol-grafted, with or without a handle or
linker, with or without the first amino acid attached, are
commercially available, and their preparations have been
described by Stewart et al., "Solid Phase Peptide
Synthesis" (2nd Edition), (Pierce Chemical Co., Rockford,
IL, 1984) and Bayer & Rapp Chem. Pept. Prot. 3:3 (1986);
and Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach, IRL Press, Oxford, 1989.
The C-terminal amino acid, protected at the side
chain if necessary, and at the alpha-amino group, is
attached to a hydroxylmethyl resin using various
activating agents including dicyclohexylcarbodiimide
(DCC), N,N'-diisopropylcarbodiimide (DIPCDI) and
carbonyldiimidazole (CDI). It can be attached to
chloromethyl or chlorotrityl resin directly in its cesium
tetramethylammonium salt form or in the presence of
triethylamine (TEA) or diisopropylethylamine (DIEA). First
amino acid attachment to an amide resin is the same as
amide bond formation during coupling reactions.
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Following the attachment to the resin support,
the alpha-amino protecting group is removed using various
reagents depending on the protecting chemistry (e.g.,
tBoc, Fmoc). The extent of Fmoc removal can be monitored
at 300-320 nm or by a conductivity cell. After removal of
the alpha-amino protecting group, the remaining protected
amino acids are coupled stepwise in the required order to
obtain the desired sequence.
Various activating agents can be used for the
coupling reactions including DCC, DIPCDI, 2-chloro-l,3-
dimethylimidium hexafluorophosphate (CIP), benzotriazol-l-
yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro-
phosphate (BOP) and its pyrrolidine analog (PyBOP), bromo-
tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),
0-(benzotriazol-l-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (HBTU) and its tetrafluoroborate
analog (TBTU) or its pyrrolidine analog (HBPyU), 0-(7-
azabenzotriazol-l-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (HATU) and its tetrafluoroborate
analog (TATU) or its pyrrolidine analog (HAPyU). The most
common catalytic additives used in coupling reactions
include 4-dimethylaminopyridine (DMAP), 3-hydroxy-3,4-
dihydro-4-oxo-1,2,3-benzotriazine (HODhbt), N-
hydroxybenzotriazole (HOBt) and 1-hydroxy-7-
azabenzotriazole (HOAt). Each protected amino acid is
used in excess (>2.0 equivalents), and the couplings are
usually carried out in N-methylpyrrolidone (NMP) or in
DMF, CH2C12 or mixtures thereof. The extent of completion
of the coupling reaction can be monitored at each stage,
e.g., by the ninhydrin reaction as described by Kaiser et
al., Anal. Biochem. 34:595, 1970.
After the entire assembly of the desired
peptide, the peptide-resin is cleaved with a reagent with
proper scavengers. The Fmoc peptides are usually cleaved
and deprotected by TFA with scavengers (e.g., H2O,
ethanedithiol, phenol and thioanisole). The tBoc peptides
CA 02284733 2006-02-10
are usually cleaved and deprotected with liquid HF for 1-2
hours at -5 to 00 C, which cleaves the polypeptide from
the resin and removes most of the side-chain protecting
groups. Scavengers such as anisole, dimethylsulfide and
5 p-thiocresol are usually used with the liquid HF to
prevent cations formed during the cleavage from alkylating
and acylating the amino acid residues present in the
polypeptide. The formyl group of tryptophan and the
dinitrophenyl group of histidine need to be removed,
10 respectively by piperidine and thiophenyl in DMF prior to
the HF cleavage. The acetamidomethyl group of cysteine
can be removed by mercury(II)acetate and alternatively by
iodine, thallium(III)trifluoroacetate or silver
tetrafluoroborate which simultaneously oxidize cysteine to
15 cystine. Other strong acids used for tBoc peptide cleavage
and deprotection include trifluoromethanesulfonic acid
(TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).
The zsig33 polypeptides of the present invention
are capable of stimulating gastric motility. The activity
20 of molecules of the present invention can be measured
using a variety of assays that measure stimulation of
gastrointestinal cell contractility, modulation of
nutrient uptake and/or secretion of digestive enzymes. Of
particular interest are changes in contractility of smooth
25 muscle cells. For example, the contractile response of
segments of mammalian duodenum or other gastrointestinal
smooth muscles tissue (Depoortere et al., J.
Gastrointestinal Motility 1:150-159, 1989). An exemplary
in vivo assay uses an ultrasonic micrometer to measure the
30 dimensional changes radially between commissures and
longitudinally to the plane of the valve base (Hansen et
al.,Society of Thoracic Surgeons 60:S384-390,1995).
Gastric motility is generally measured in the
clinical setting as the time required for gastric emptying
35 and subsequent transit time through the gastrointestinal
tract. Gastric emptying scans are well known to those
CA 02284733 2003-12-02
36
skilled in the art, and briefly, comprise use of an oral
contrast agent, such as barium, or a radiolabeled meal.
Solids and liquids ca: be measured independently. A test
food or liquid is radiolabeled with an isotope (e.g.
99m
Tc), and after ingestion or administration, transit time
through the gastrointestinal tract and gastric emptying
are measured by visualization using gamma cameras (Meyer
et al-., Am. J. Dig. Dis. 21:296, 1976; Collins et al., Gut
24:1117, 1983; Maughan et al., Diabet. Med. 13 9 Sugp.
5:S6-10, 1996 and Horowitz et al., Arch. Intern. Med.
145:1467-1472, 1985). These studies may be performed
before and after the administration of a promotility agent
to quantify the efficacy of the drug.
Assays measuring zsig33 polypeptides ability to
affect cell proliferation or differentiation are well
known in the art. For example, assays measuring
proliferation include such assays as chemosensitivity to
neutral red dye (Cavanaugh et al., Investigational New
Drugs 8:347-354, 1990
incorporation of radiolabelled nucleotides (Cook et al.,
Analytical Biochem. 179:1-7, 1989
incorporation of 5-bromo-2'-deoxyuridine
(BrdU) in the DNA of proliferating cells (Porstmann et
al., J. Immunol. Methods 82:169-179, 1985
), and use of tetrazolium salts
(Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et
al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth
Rea. 5:69-84, 1995; and Scudiero et al., Cancer Res.
48:4827-4833, 1988
Assays measuring differentiation include, for example,
measuring cell-surface markers associated with stage-
specific expression of a tissue, enzymatic activity,
functional activity or morphological changes (Watt, FASEB,
5:281-284, 1991; Francis, Differentiation 57:63-75, 1994;
Raes, Adv. Anim. Cell Biol. Technol. Bioorocesses, 161-
171, 1989
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37
Assays can be used to measure other cellular
responses, that include, chemotaxis, adhesion, changes in
ion channel influx, regulation of second messenger levels
and neurotransmitter release. Such assays are well known
in the art. See, for example, in "Basic & Clinical
Endocrinology Ser., Vol. Vol. 3," Cytochemical Bioassays:
Techniques & Applications, Chayen; Chayen, Bitensky, eds.,
Dekker, New York, 1983.
In view of the tissue distribution observed for
zsig33, agonists (including the natural ligand/ substrate/
cofactor/ etc.) and antagonists have enormous potential in
both in vitro and in vivo applications. Compounds
identified as zsig33 agonists are useful for promoting
stimulation of gastrointestinal cell contractility,
modulation of nutrient uptake and/or secretion of
digestive enzymes in vivo and in vitro. For example,
agonist compounds are useful as components of defined cell
culture media and regulate the uptake of nutrients, and
thus are useful in specifically promoting the growth
and/or development of gastrointestinal cells such as G
cells, enterochromaffin cells and the epithelial mucosa of
the stomach, duodenum, proximal jejunum, antrum and
fundus.
The family of gut-brain peptides has been
associated with neurological and CNS functions. For
example, NPY, a peptide with receptors in both the brain
and the gut has been shown to stimulate appetite when
administered to the central nervous system (Gehlert, Life
Sciences 55 6 :551-562, 1994). Motilin immunoreactivity
has been identified in different regions of the brain,
particularly the cerebellum, and in the pituitary
(Gasparini et al., Hum. Genetics 94 6 :671-674, 1994).
Motilin has been found to coexist with neurotransmitter 7-
aminobutyric acid in cerebellum (Chan-Patay, Proc. Svm.
50th Anniv. Meet. Br. Pharmalog. Soc.:1-24, 1982).
Physiological studies have provided some evidence that
CA 02284733 1999-09-21
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38
motilin has an affect on feeding behavior (Rosenfield et
al., Phys. Behav. 39(6):735-736, 1987), bladder control,
pituitary growth hormone release. Other gut-brain
peptides, such as CCK, enkephalin, VIP and secretin have
been shown to be involved in control of blood pressure,
heart rate, behavior, and pain modulation, in addition to
be active in the digestive system. Therefore, zsig33, or
some portion thereof, could be expected to have some
neurological association.
Using site-specific changes in the amino acid
and DNA sequences of the present invention analogs can be
made that are either antagonists, agonists or partial
agonists (Macielay et al., Peptides: Chem. Struct. Biol.
pp.659, 1996). Antagonists are useful for clinical
conditions associated with gastrointestinal hypermotility
such as diarrhea and Crohn's disease. Antagonists are
also useful as research reagents for characterizing sites
of ligand-receptor interaction.
A zsig33 ligand-binding polypeptide can also be
used for purification of ligand. The polypeptide is
immobilized on a solid support, such as beads of agarose,
cross-linked agarose, glass, cellulosic resins, silica-
based resins, polystyrene, cross-linked polyacrylamide, or
like materials that are stable under the conditions of
use. Methods for linking polypeptides to solid supports
are known in the art, and include amine chemistry,
cyanogen bromide activation, N-hydroxysuccinimide
activation, epoxide activation, sulfhydryl activation, and
hydrazide activation. The resulting medium will generally
be configured in the form of a column, and fluids
containing ligand are passed through the column one or
more times to allow ligand to bind to the receptor
polypeptide. The ligand is then eluted using changes in
salt concentration, chaotropic agents (guanidine HC1), or
pH to disrupt ligand-receptor binding.
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39
An assay system that uses a ligand-binding
receptor (or an antibody, one member of a complement/
anti-complement pair) or a binding fragment thereof, and a
commercially available biosensor instrument (BIAcoreTM,
Pharmacia Biosensor, Piscataway, NJ) may be advantageously
employed. Such receptor, antibody, member of a
complement/anti-complement pair or fragment is immobilized
onto the surface of a receptor chip. Use of this
instrument is disclosed by Karlsson, J. Immunol. Methods
145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-63, 1993. A receptor, antibody, member or
fragment is covalently attached, using amine or sulfhydryl
chemistry, to dextran fibers that are attached to gold
film within the flow cell. A test sample is passed
through the cell. If a ligand, epitope, or opposite
member of the complement/anti-complement pair is present
in the sample, it will bind to the immobilized receptor,
antibody or member, respectively, causing a change in the
refractive index of the medium, which is detected as a
change in surface plasmon resonance of the gold film.
This system allows the determination of on- and off-rates,
from which binding affinity can be calculated, and
assessment of stoichiometry of binding.
Ligand-binding receptor polypeptides can also be
used within other assay systems known in the art. Such
systems include Scatchard analysis for determination of
binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:
660-72, 1949) and calorimetric assays (Cunningham et al.,
Science 253:545-48, 1991; Cunningham et al., Science
24,:821-25, 1991).
zsig33 polypeptides can also be used to prepare
antibodies that specifically bind to zsig33 epitopes,
peptides or polypeptides. Methods for preparing
polyclonal and monoclonal antibodies are well known in the
art (see, for example, Sambrook et al., Molecular Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor,
CA 02284733 2003-12-02
NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hvbridoma
Antibodies: Techniaues and Aonlications, CRC Press, Inc.,
Boca Raton, FL, 1982.
As would be evident to one of ordinary skill
5 in the art, polyclonal antibodies can be generated from a
variety of warm-blooded animals, such as horses, cows,
goats, sheen, dogs, chickens, rabbits, mice, and rats.
The immunogenicity of a zsig33 polypeptide may
be increased through the use of an adjuvant, such as alum
0 (aluminum hydroxide) or Freund's complete or incomplete
adjuvant. Polypeptides useful for immunization also
include fusion polypeptides, such as fusions of zsig33 or
a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen
15 may be a full-length molecule or a portion thereof. If
the polypeptide portion is "hapten-like", such portion may
be advantageously joined or linked to a macromolecular
carrier (such as keyhole limpet hemocyanin (KLH), bovine
serum albumin (BSA) or tetanus toxoid) for immunization.
20 As used herein, the term "antibodies" includes
polyclonal antibodies, affinity-purified polyclonal
antibodies, monoclonal antibodies, and antigen-binding
fragments, such as F(ab')2 and Fab proteolytic fragments.
Genetically engineered intact antibodies or fragments,
25 such as chimeric antibodies, Fv fragments, single chain
antibodies and the like, as well as synthetic antigen-
binding peptides and polypeptides, are also included.
Non-human antibodies may be humanized by grafting only
non-human CDRs onto human framework and constant regions,
30 or by incorporating the entire non-human variable domains
(optionally "cloaking" them with a human-like surface by
replacement of exposed residues, wherein the result is a
"veneered" antibody). In some instances, humanized
antibodies may retain non-human residues within the human
35 variable region framework domains to enhance proper
binding characteristics. Through humanizing antibodies,
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41
biological half-life may be increased, and the potential
for adverse immune reactions upon administration to humans
is reduced. Alternative techniques for generating or
sellacting antibodies useful herein include in vitro
exposure of lymphocytes to zsig33 protein or peptide, and
selection of antibody display libraries in phage or
similar vectors (for instance, through use of immobilized
or labeled zsig33 protein or peptide).
Antibodies are defined to be specifically
binding if they bind to a zsig33 polypeptide with a
binding affinity (Ka) of 106 M-1 or greater, preferably 107
M-1 or greater, more preferably 108 M-1 or greater, and most
preferably 109 M-1 or greater. The binding affinity of an
antibody can be readily determined by one of ordinary
skill in the art (for example, by Scatchard analysis).
A variety of assays known to those skilled in
the art can be utilized to detect antibodies which
specifically bind to zsig33 proteins or peptides.
Exemplary assays are described in detail in Antibodies: A
Laboratory Manual, Harlow and Lane (Eds.), Cold Spring
Harbor Laboratory Press, 1988. Representative examples of
such assays include: concurrent immunoelectrophoresis,
radioimmunoassay, radioimmuno-precipitation, enzyme-linked
immunosorbent assay (ELISA), dot blot or Western blot
assay, inhibition or competition assay, and sandwich
assay. In addition, antibodies can be screened for
binding to wild-type versus mutant zsig33 protein or
peptide.
Antibodies to zsig33 may be used for tagging
cells that express zsig33 for isolating zsig33 by affinity
purification; for diagnostic assays for determining
circulating levels of zsig33 polypeptides; for detecting
or quantitating soluble zsig33 as marker of underlying
pathology or disease; in analytical methods employing
FACS; for screening expression libraries; for generating
anti-idiotypic antibodies; and as neutralizing antibodies
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42
or as antagonists to block zsig33 activity in vitro and in
vivo. Suitable direct tags or labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic
particles and the like; indirect tags or labels may
feature use of biotin-avidin or other complement/anti-
complement pairs as intermediates. Antibodies herein may
also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates
used for in vivo diagnostic or therapeutic applications.
Molecules of the present invention can be used
to identify and isolate receptors that mediate the
function of zsig33. For example, proteins and peptides of
the present invention can be immobilized on a column and
membrane preparations run over the column (Immobilized
Affinity Ligand Techniques, Hermanson et al., eds.,
Academic Press, San Diego, CA, 1992, pp.195-202).
Proteins and peptides can also be radiolabeled (Methods in
Enzymol., vol. 182, "Guide to Protein Purification", M.
Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or
photoaffinity labeled (Brunner et al., Ann. Rev. Biochem.
62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.
33:1167-1180, 1984) and specific cell-surface proteins can
be identified.
The polypeptides, nucleic acid and/or antibodies
of the present invention may be used in treatment of
disorders associated with gastrointestinal cell
contractility, secretion of digestive enzymes and acids,
gastrointestinal motility, recruitment of digestive
enzymes; inflammation, particularly as it affects the
gastrointestinal system; ref lux disease and regulation of
nutrient absorption. Specific conditions that will
benefit from treatment with molecules of the present
invention include, but are not limited to, diabetic
gastroparesis, post-surgical gastroparesis, vagotomy,
chronic idiopathic intestinal pseudo-obstruction and
CA 02284733 2003-12-02
43
gastroesophageal ref lux disease. Additional uses include,
gastric emptying for radiological studies, stimulating
gallbladder contraction and antrectomy.
The motor and neurological affects of molecules
of the present invention make it useful for treatment of
obesity and other metabolic disorders where neurological
feedback modulates nutritional absorption. The molecules
of the present invention are useful for regulating
satiety, glucose absorption and metabolism, and
neuropathy-associated gastrointestinal disorders.
Molecules of the present invention are also
useful as additives to anti-hypoglycemic preparations
containing glucose and as adsorption enhancers for oral
drugs which require fast nutrient action. Additionally,
molecules of the present invention can be used to
stimulate glucose-induced insulin release.
For pharmaceutical use, the proteins of the
present invention are formulated for parenteral, nasal
inhalation, particularly intravenous or subcutaneous,
delivery according to conventional methods. Intravenous
administration will be by bolus injection or infusion over
a typical period of one to several hours. In general,
pharmaceutical formulations will include a zsig33 protein
in combination with a pharmaceutically acceptable vehicle,
such as saline, buffered saline, 5o dextrose in water or
the like. Formulations may further include one or more
excipients, preservatives, solubilizers, buffering agents,
albumin to prevent protein loss on vial surfaces, etc.
Methods of formulation are well known in the art and are
disclosed, for example, in Remington's Pharmaceutical
Sciences, Gennaro, ed., Mack Publishing Co., Easton PA,
1990.
Therapeutic doses will generally be in the range of 0.1 to
100 pg/kg of patient weight per day, preferably 0.5-20
}gig/kg per day, with the exact dose determined by the
clinician according to accepted standards, taking into
CA 02284733 2003-12-02
44
account the nature and severity of the condition to be
treated, patient traits, etc. Determination of dose is
within the level of ordinary skill in the art. The
proteins may be administered for acute treatment, over one
week or less, often over a period of one to three days or
may be used in chronic treatment, over several months or
years. For example, a therapeutically effective amount of
zsig33 is an amount sufficient to produce a clinically
significant change in gastric motility and parameters used
to measure changes in nutritional absorption. Specific
tests, for making such measurements are known to these
ordinarily skilled in the art.
Examples
Example I
Scanning of a cDNA database for cDNAs containing
a secretion sequence revealed an expressed sequence tag
(EST) that has homology to motilin. The cDNA is from a
human fetal pancreatic cDNA library.
Confirmation of the EST sequence was made by
sequence analyses of the cDNA from which the EST
originated. This cDNA was contained in a plasmid, and was
excised using cloning sites. The analyses revealed that
the cDNA encompassed the entire coding region of the DNA
encoding zsig33.
Example 2
Northerns were performed using Human Multiple
Tissue Blots and Human RNA Master dot blots from Clontech
(Palo Alto, CA). The probe was approximately 40 bp
oligonucleotide ZC12,494 (SEQ ID NO: 7). The probe was
end labeled using T4 Polynucleotide Kinase (Life
Technologies, Inc., Gaithersburg, MD) and T4
Polynucleotide Kinase Forward Buffer (Life Technologies,
Inc.). The probe was purified using a NUCTRAP* push
* trade-mark
CA 02284733 2003-12-02
columns (Stratagene, La Jolla, CA). EXPRESSITYB* (Clontech)
solution was used for prehybridization and as a
hybridizing solution for the Northern blots.
Hybridization took place at 42 C, and the blots were washed
5 in 2X SSC and 0.05% SDS at RT, followed by a wash in 1 X
SSC and 0.1% SDS at 71 C. An approximately 600 bp
transcript was observed as a strong signal in stomach,
with weaker signals seen in pancreas and small intestine.
10 Example 3
Two male Sprague-Dawley rats, approximately 12
weeks old (Harlan, Indianapolis, IN) were anesthetized
with urethane and their stomachs were exposed through a
small abdominal incision. Two 2.4 mm transducing crystals
15 (Sonometrics, Ontario, Canada) were placed on the antral
portion of the stomach such that circular contractions
could be monitored as a change in the distance between the
two crystals. The crystals were attached with VETBOND*
TISSUE ADHESIVE (3M, St. Paul, MN).
20 10 .il of 1 .LM acetylcholine was applied
topically to the stomach between the two crystals, and
resulted in a rapid, but transient increase in the
distance between two crystals. 10 l of norepinephrine
(NE) at 1 M caused a reduction in the distance between
25 the two crystals. The amplitude of the NE-induced
decrease was approximately 50% of the acetylcholine-
induced increase in distance. Both responses were
transient.
A negative control of 10 l of phosphate buffer
30 solution (PBS) applied topically between the crystals had
no effect.
A 14 amino acid zsig33 peptide (from amino acid
residue 24 (Gly) to amino acid residue 37 (Gln) of SEQ ID
NO: 2) was dissolved in PBS) and 10 .il was applied
35 topically for a final concentration of 1 g,
10 g or 100 g. The zsig33 at 1 g induced a sustained,
* trade-mark
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46
rhythmic increase and decrease in crystal distance. This
effect appeared to be dose-dependent, with enhanced
responses in both rate and amplitude when of the
contractions 10 g and 100 g were tested.
Example 4
Eight female ob/ob mice, approximately 6 weeks
old (Jackson Labs, Bar Harbor, ME) were adapted to a 4
hour daily feeding schedule for two weeks. After two
weeks on the feeding schedule, the mice were give 100 g
of a 14 amino acid amino zsig33 peptide (from amino acid
residue 24 (Gly) to amino acid residue 37 (Gln) of SEQ ID
NO: 2) in 100 gl sterile 0.1% BSA by oral gavage,
immediately after their eating period (post-prandially).
Thirty minutes later, the mice were challenged orally with
a 0.5 ml volume of 25% glucose. Retroorbital bleeds were
done to determine serum glucose levels. Blood was drawn
prior to zsig33 dosing, prior to oral glucose challenge,
and at 1, 2, 4, and 20 hours following the glucose
challenge.
When zsig33 peptide was given orally at 100 g,
minutes prior to an oral glucose challenge, an enhanced
post-prandial glucose absorption was seen.
25 Example 5
zsig33-1, a peptide corresponding to amino acid
residue 24 (Gly) to amino acid residue 37 (Gln) of SEQ ID
NO: 2, was synthesized by solid phase peptide synthesis
using a model 431A Peptide Synthesizer (Applied
30 Biosystems/Perkin Elmer, Foster City, CA) . Fmoc-Glutamine
resin (0.63 mmol/g; Advanced Chemtech, Louisville, KY) was
used as the initial support resin. 1 mmol amino acid
cartridges (Anaspec, Inc. San Jose, CA) were used for
synthesis. A mixture of 2(1-Hbenzotriazol-y-yl 1,1,3,3-
tetrahmethylhyluronium hexafluorophosphate (HBTU), 1-
hydroxybenzotriazol (HOBt), 2m N,N-Diisolpropylethylamine,
CA 02284733 2003-12-02
47
N-Methylpyrrolidone, Dichloromethane (all from Applied
Biosystems/Perkin Elmer) and piperidine (Aldrich Chemical
Co., St. Louis, MO), and used for synthesis reagents.
The Peptide Companion software ('Pep t_ ides
S International, Louisville, KY) was used to predict the
aggregation potential and difficulty level for synthesis
for the zsig33-1 peptide. Synthesis was performed using
single coupling programs, according to the manufacturer's
specifications.
The peptide was cleaved from the solid phase
following standard TFA cleavage procedure (according to
Peptide Cleavage manual, Applied Biosystems/Perkin Elmer)
Purification of the peptide was done by RP-HPLC using a
C18, 10 m semi-peparative column (Vydac, Hesperial, CA).
Eluted fractions from the column were collected and
analyzed for correct mass and purity by electrospray mass
spectrometry. Two pools of the eluted material were
collected. The mass spectrometry analysis results
indicated that both pools contained the purified form of
zsig33 with a mass of 1600 Daltons. This was the expected
mass, so the pools were combined, frozen and lyophilized.
Example 6
zsig33 was mapped to chromosome 3 using the
commercially available "GeneBridge 4 Radiation Hybrid
Panel" (Research Genetics, Inc., Huntsville, AL). The
GeneBridge 4 Radiation Hybrid Panel contains DNAs from
each of 93 radiation hybrid clones, plus two control DNAs
(the HFL donor and the A23 recipient). A publicly
available WWW server
allows mapping relative to the
Whitehead Institute/MIT Center for Genome Research's
radiation hybrid map of the human genome (the "WICGR"
radiation hybrid map) which was constructed with the
GeneBridge 4*Radiation Hybrid Panel.
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For mapping of zsig33 with the "GeneBridge 4* RH
Panel", 20 l reactions were set up in a 96-well
microtiter plate (Stratagene, La Jolla, CA) and used in a
"RoboCycler* Gradient 96" thermal cycler (Stratagene).
Each of the 95 PCR reactions consisted of 2 gl lOX KlenTaq*
PCR reaction buffer (CLONTECH Laboratories, Inc., Palo
Alto, CA), 1.6 Al dNTPs mix (2.5 mM each, Perkin-Elmer,
Foster City, CA), 1 l sense primer, ZC13,166 (SEQ ID NO:
8), 1 pl antisense primer, ZC13,167 (SEQ ID NO: 9), 2 gl
"RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4
g1 50X Advantage KlenTaq* Polymerase Mix (Clontech
Laboratories, Inc.), 25 ng of DNA from an individual
hybrid clone or control and ddH2O for a total volume of 20
l. The reactions were overlaid with an equal amount of
mineral oil and sealed. The PCR cycler conditions were as
follows: an initial 1 cycle 5 minute denaturation at 95 C,
35 cycles of a 1 minute denaturation at 95 C, 1 minute
annealing at 64 C and 1.5 minute extension at 72 C,
followed by a final 1 cycle extension of 7 minutes at 72 C.
The reactions were separated by electrophoresis on a 30
NuSieve*GTG agarose gel (FMC Bioproducts, Rockland, ME).
The results showed that. zsig33 maps 10.43
cR3000 from the framework marker AFMA216ZG1* on the WICGR
chromosome 3 radiation hybrid map. Proximal and distal
framework markers were AFMA216ZG1* and D3S1263,
respectively. The use of surrounding markers positions
zsig33 in the 3p26.1 region on the integrated LD3
chromosome 3 map (The Genetic Location Database,
University of Southhampton, WWW server
) .
Example 7
The effect of topically applied zsig33 peptide
(amino acid '24 to 37 of SEQ ID NO: 2) on the transit of
phenol red through the stomachs of fasted male Sprague-
Dawley rats (Harlan, Indianapolis, IN) was evaluated. The
* trade-mark
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rats (6 animals, 8 weeks old) were fasted 24 hrs prior to
being anesthetized with urethane(0.5 ml/100 grams of 25%
solution). After anesthetizing, the animals were orally
gavaged with 1 ml of Phenol Red solution (50 mg/ml in 2%
methylcellulose solution).
The stomach of each animal was exposed through a
small abdominal incision and either 1 g zsig33 peptide or
a 14 amino acid control of a scrambled sequence peptide
was applied topically to the stomach five minutes
following the gavage. The amount of Phenol Red remaining
in the stomach was determined by measuring optical density
of the extracted stomach contents 30 minutes after the
gavage.
The zsig33 peptide reduced the amount of Phenol
Red remaining in the stomach by approximately 25% compared
to a scrambled peptide, indicating that the zsig33 peptide
enhanced gastric emptying in these rats.
Example 8
Sixteen female ob/ob mice, 8 weeks old, (Jackson
Labs, Bar Harbor, ME) were adapted to a special 4 hour
daily feeding schedule for two weeks. The were fed ad
libitum from 7:30-11:30 am daily. After two weeks on the
feeding schedule, the mice were divided into two groups of
8. One group was given 1.0 g/mouse of zsig33-1 (14 amino
acid peptide) and the other vehicle (a 14 amino acid
scrambled sequence peptide) in 100 l sterile 0.1% BSQA by
oral gavage just prior to receiving food, and at the end
of the 4 hour feeding period. The mice were injected
twice daily for fourteen days, during which time food
intake and body weight was measured daily. One day 14,
immediately after the second oral gavage of the zsig33-1
peptide, the mice were challenged orally with an 0.5 ml
volume of 25% glucose. Retro-orbital bleeds were done to
determine serum glucose levels immediately prior to
administration of the zsig33-1 peptide or vehicle (t=30
CA 02284733 2003-12-02
min.), and also at 0, 1, 2, and 4 hours following the
glucose challenge.
Results indicated that when zsig33-1 given
orally at 1 .ig/mouse had no affect on daily body weight or
5 food intake measurements, or on glucose clearance as
determined on day 14.
Example 9
A. Gut Northern Tissue Blot
10 A Northern blot was prepared using mRNA from the
following sources:
1. RNA from Human Colorectal Andenocarcinoma
cell line SW480 (Clontech, Palo Alto, CA)
2. RNA from human small intestine tissue
15 (Clontech)
3. RNA from human stomach tissue (Clontech)
4. Human Intestinal Smooth Muscle cell line
(Hism; ATCC No.CRL-1692; American Type Culture Collection,
12301 Parklawn Drive, Rockville, MD)
20 S. Normal Human Colon cell line (FHC; ATCC No.
CRL-1831; American Type Culture Collection)
6. Human Normal Fetal Small Intestine cell
line (FHs74 Int.; ATCC No. CCL241; American Type Culture
Collection).
25 Total RNAs were isolated from Hism, FHC and
FHs74 Int. by acid guanidium method (Chomczynski et al.,
Anal. Biochem. 162:156-159, 1987) . The polyA' RNAs were
selected by eluting total RNA through a column that
retains polyA' RNAs (Aviv et al., Proc. Nat. Acad. Sci.
30 69:1408-1412, 1972). 2 g of polyA' RNA from each sample
was separated out in a 1.5% agarose gel in 2.2 M
formaldehyde and phosphate buffer. The RNAs were
transferred onto Nytran* membrane (Schleicher and Schuell,
Keene, NH) in 20X SSC overnight. The blot was treated in
35 the LTV Stratalinker* 2400 (Stratagene, La Jolla, CA) at
0.12 Joules. The bolt was then baked at 80 C for one hour.
* trade-mark
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Using the full length cDNA (shown in SEQ ID NC:
1) amplified by PCR approximately 50 ng of zsig33 DNA and
42.5 l of water was radiolabeled with 3`P dCTP using a
Rediprime pellet kit (Amersham, Arlington Heights, IL)
according to the manufacturer's specifications. The blot
was hybridized in EXPRESSITYB*(Clontech) at 55 C overnight.
The blot was washed at room temp. in 2X SSC and 0. 1 o SDS,
then in 2X SSC and 0.1v SDS at 65 C, and finally at 65 C in
0.1X SSC and 0.1% SDS. Results showed that zsig33
hybridized to stomach RNA and not to other RNAs from other
tissue origins.
B. Tumor Northern Blot
A Northern TerritoryT"' -Human Tumor Panel Blot II
(Invitrogen, San Diego, CA) and a Northern Territory1"" -
Human Stomach Tumor Panel Blot (Invitrogen) were analyzed
for expression patterns of zsig33 RNA.
The Human Tumor Panel Blot contained 20 g of
total RNA per lane and was run on a la denaturing
formaldehyde gel. The blot contained RNA from: esophageal
tumor, normal esophagus, stomach tumor, normal stomach,
colon tumor, normal colon, rectal tumor and normal rectum.
The Stomach Tumor Panel Blot contained total RNA isolated
human and normal tissues of four separate donors. 20 g
of RNA was used for each sample lane and the lanes
alternated a normal and tumor set from each donor.
Probes that were approximately 40 by
oligonucleotide ZC12,494 (SEQ ID NO: 7) were prepared.
The probes were end labeled using T4 Polynucleotide Kinase
(Life Technologies, Inc., Gaithersburg, MD) and T4
Polynucleotide Kinase Forward Buffer (Life Technologies,
Inc.). The probes were purified using a NUCTRAP* push
columns (Stratagene, La Jolla, CA). The tumor blot and
the stomach blot were both treated in the same way.
EXPRESSHYB* (Clontech) solution was used for
prehybridization and as a hybridizing solution for the
Northern blots. Hybridization took place at 42 C, and the
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blots were washed in 0.1X SSC and 0.01% SDS at 60 C,
followed by a wash in 0.1X SSC and 0.1% SDS at 70 C. The
results clearly indicate that zsig33 is exclusively
expressed in normal stomach tissue in both the Human Tumor
Panel and the Human Stomach Tumor Panel.
From the foregoing, it will be appreciated that,
although specific embodiments of the invention have been
described herein for purposes of illustration, various
modifications may be made without deviating from the
spirit and scope of the invention. Accordingly, the
invention is not limited except as by the appended claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: ZymoGenetics. Inc.
1201 Eastlake Avenue East
Seattle
WA
USA
98102
(ii) TITLE OF THE INVENTION: MOTILIN HOMOLOGS
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ZymoGenetics. Inc.
(3) STREET: 1201 Eastlake Avenue East
(C) CITY: Seattle
(D) STATE: WA
(E) COUNTRY: USA
(F) ZIP: 98102
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM*Compatible
(C) OPERATING SYSTEM: DOS*
(D) SOFTWARE: FastSEQ*for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sawislak, Deborah A
(?) REGISTRATION NUMBER: 37,438
(C) REFERENCE/DOCKET NUMBER: 97-04PC
* trade-mark
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54
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 206-442-6672
(B) TELEFAX: 206-442-6678
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 351 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...351
(D) OTHER INFORMATION:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 1...69
(D) OTHER INFORMATION:
(A) NAME/KEY: mat-peptide
(B) LOCATION: 70...351
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATG CCC TCC CCA GGG ACC GTC TGC AGC CTC CTG CTC CTC GGC ATG CTC 48
Met Pro Ser Pro Gly Thr Val Cys Ser Leu Leu Leu Leu Gly Met Leu
1 5 10 15
TGG CTG GAC TTG GCC ATG GCA GGC TCC AGC TTC CTG AGC CCT GAA CAC 96
Trp Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro Glu His
20 25 30
CAG AGA GTC CAG CAG AGA AAG GAG TCG AAG AAG CCA CCA GCC AAG CTG 144
Gln Arg Val Gln Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu
35 40 45
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CAG CCC CGA GCT CTA GCA GGC TGG CTC CGC CCG GAA GAT GGA GGT CAA 192
Gin Pro Arg Ala Leu Ala Gly Trp Leu Arg Pro Glu Asp Gly Gly Gln
50 55 60
GCA GAA GGG GCA GAG GAT GAA CTG GAA GTC CGG TTC AAC GCC CCC TTT 240
Ala Glu Giy Ala Glu Asp Glu Leu Glu Val Arg Phe Asn Ala Pro Phe
70 75 80
GAT GTT GGA ATC AAG CTG TCA GGG GTT CAG TAC CAG CAG CAC AGC CAG 288
Asp Val Gly Ile Lys Leu Ser Gly Val Gin Tyr Gln Gln His Ser Gin
85 90 95
GCC CTG GGG AAG TTT CTT CAG GAC ATC CTC TGG GAA GAG GCC AAA GAG 336
Ala Leu Gly Lys Phe Leu Gln Asp Ile Leu Trp Glu Glu Ala Lys Glu
100 105 110
GCC CCA GCC GAC AAG 351
Ala Pro Ala Asp Lys
115
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Pro Ser Pro Gly Thr Val Cys Ser Leu Leu Leu Leu Gly Met Leu
1 5 10 15
Trp Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro Glu His
20 25 30
Gln Arg Val Gin Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu
35 40 45
Gln Pro Arg Ala Leu Ala Gly Trp Leu Arg Pro Glu Asp Gly Gly Gin
50 55 60
Ala Glu Gly Ala Glu Asp Glu Leu Glu Val Arg Phe Asn Ala Pro Phe
65 70 75 80
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Asp Val Gly Ile Lys Leu Ser Gly Val Gln Tyr Gln Gin His Ser Gln
85 90 95
Ala Leu Gly Lys Phe Leu Gln Asp Ile Leu Trp Glu Glu Ala Lys Glu
100 105 110
Ala Pro Ala Asp Lys
115
(2) INFORMATION FOR SEQ ID NO:3:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 546 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 40...396
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GGGCAGAGAC ACACACGCGC CCAGTTGTCC AGCTCCAGG ATG GTG TCC CGC AAG 54
Met Val Ser Arg Lys
1 5
GCT GTG GTC GTC CTG CTG GTG GTG CAC GCA GCT GCC ATG CTG GCC TCC 102
Ala Val Val Val Leu Leu Val Val His Ala Ala Ala Met Leu Ala Ser
15 20
CAC ACG GAA GCC TTT GTT CCC AGC TTT ACC TAC GGG GAA CTT CAG AGG 150
His Thr Glu Ala Phe Val Pro Ser Phe Thr Tyr Gly Glu Leu Gln Arg
25 30 35
ATG CAG GAA AAG GAG CGG AAT AAA GGG CAA AAG AAA TCC CTG AGT GTC 198
Met Gln Glu Lys Glu Arg Asn Lys Gly Gln Lys Lys Ser Leu Ser Val
40 45 50
CAG CAG GCG TCG GAG GAG CTC GGC CCT CTG GAC CCC TCG GAG CCC ACG 246
Gin Gin Ala Ser Glu Glu Leu Gly Pro Leu Asp Pro Ser Glu Pro Thr
55 60 65
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AAG GAA GAA GAA AGG GTG GTT ATC AAG CTG CTC GCG CCT GTG GAC ATT 294
Lys Glu Glu Glu Arg Val Val Ile Lys Leu Leu Ala Pro Val Asp Ile
70 75 80 85
GGA ATC AGG ATG GAC TCC AGG CAG CTG GAA AAG TAC CGG GCC ACC CTG 342
Gly Ile Arg Met Asp Ser Arg Gln Leu Glu Lys Tyr Arg Ala Thr Leu
90 95 100
GAA AGG CTG CTG GGC CAG GCG CCG CAG TCC ACC CAG AAC CAG AAT GCC 390
Glu Arg Leu Leu Gly Gin Ala Pro Gln Ser Thr Gln Asn Gln Asn Ala
105 110 115
GCC AAG TAACAGGCCG CTGGGGGAGA AGGAGGACAC AGCTCGGACC CCCCTCCCAC GC 448
Ala Lys
AGGGAGGGCC TAGAAATCCG CTGGGCTTGG AAGGAAAACA CCCTCTCCCA AACAGCCCTC 508
AGCCCCCCTC CCCCAGCAAA TAAAGCGTGG AAATAGGC 546
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Val Ser Arg Lys Ala Val Val Vai Leu Leu Val Val His Ala Ala
1 5 10 15
Ala Met Leu Ala Ser His Thr Glu Ala Phe Val Pro Ser Phe Thr Tyr
20 25 30
Gly Glu Leu Gln Arg Met Gln Glu Lys Glu Arg Asn Lys Gly Gln Lys
35 40 45
Lys Ser Leu Ser Val Gln Gln Ala Ser Glu Glu Leu Gly Pro Leu Asp
50 55 60
Pro Ser Glu Pro Thr Lys Glu Glu Glu Arg Val Vai Ile Lys Leu Leu
65 70 75 80
Ala Pro Val Asp Ile Gly Ile Arg Met Asp Ser Arg Gln Leu Glu Lys
85 90 95
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Tyr Arg Ala Thr Leu Glu Arg Leu Leu Gly Gln Ala Pro Gln Ser Thr
100 105 110
Gln Asn Gin Asn Ala Ala Lys
115
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(vii) IMMEDIATE SOURCE:
(B) CLONE: ZC12494
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
TTCTTCGACT CCTTTCTCTG CTGGACTCTC TGGTGTTCAG 40
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Glu Xaa Gln Arg Xaa Gln
1 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Pro Xaa Asp Xaa Gly Ile
1 5
