Note: Descriptions are shown in the official language in which they were submitted.
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Description
STOMACH POLYPEPTIDE ZSIG28
BACKGROUND OF THE INVENTION
Proper control of the opposing processes of cell
proliferation versus terminal differentiation and
apoptotic programmed cell death is an important aspect of
normal development and homeostasis (Raff, M.C., Cell
86:173-175, 1996), and has been found to be altered in
many human diseases. See, for example, Sawyers, C.L. et
al., Cell 64:337-350, 1991; Meyaa.rd, L. et al., Science
257:217-219, 1992; Guo, Q. et al., Nature Med. 4:957-962,
1998; Barinaga, M., Science, 273:735-737, 1996; Solary, E.
et al., Eur. Respir. J., 9:1293-1305, 1996; Hamet, P. et
al., J. Hypertension, 14:565-570, 1996; Roy, N. et al.,
Cell, 80:167-178, 1995; and Ambrosini, G., Nature Med.,
8:917-921, 1997. Much progress has been made towards
understanding. the regulation of this balance. For
example, signaling cascades have been elucidated through
which extracellular stimuli, such as growth factors,
peptide hormones, and cell-cell interactions, control the
commitment of precursor cells to specific cell lineages
and their subsequent proliferative expansion (Morrison,
S.J. et al., Cell 88:287-298, 1997). Further, it has been
found that cell cycle exit and terminal differentiation
are coupled in most cell types. See, for example,
Coppola, J.A. et al., Nature 320:760-763, 1986; Freytag,
5.0, Mol. Cell. Biol. 8:1619-1624, 1988; Lee, E.Y. et al.,
Genes Dev. 8:2008-2021, 1999; Morgenbesser, S.D. et al.,
Nature 371:72-74, 1994; Casaccia-Bonnefil, P. et al.,
Genes Dev. 11:2335-2346, 1996; Zacksenhaus, E, et al.,
Genes Dev. 10:3051-3064, 1996; and Zhang, P. et al.,
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Nature 387:151-158, 1997. Apoptosis also plays an
important role in many developmental and homeostatic
processes (gaff, M.C., Nature 356:397-400, 1992; Raff,
M.C., supra.), and is often coordinately regulated with
terminal differentiation (Jacobsen, K.A. et al., Blood
84:2784-2794, 1999; Morgenbesser et al., supra.; Yan, Y.
et al., Genes Dev. 11:973-983, 1997; Zacksenhaus et al.,
supra.). Hence, it appears that the development of
individual lineages, tissues, organs, or even entire
multicellular organisms is the result of a finely tuned
balance between increased cell production due to
proliferation, and decreased numbers of cells resulting
from terminal differentiation and apoptosis. This balance
is most likely regulated coordinately by the convergence
of multiple regulatory pathways. The identification of
novel members of such networks can provide important
insights into both normal cellular processes, as well as
the etiology and treatment of human disease states.
Thus, there is a continuing need to discover new
proteins that regulate proliferation, differentiation, and
apoptotic pathways. The in vivo activities of both
inducers and inhibitors of these pathways illustrates the
enormous clinical potential of, and need for, novel
proliferation, differentiation, and apoptotic proteins,
their agonists and antagonists. The present invention
addresses this need by providing such polypeptides for
these and other uses that should be apparent to those
skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
Within one aspect, the present invention
provides an isolated polynucleotide that encodes a
polypeptide comprising a sequence of amino acid residues
that is at least 90% identical to an amino acid sequence
selected from the group consisting of: (a) the amino acid
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sequence as shown in SEQ ID N0:2 from amino acid number 24
(Ala), to amino acid number 261 (Val); and (b) the amino
acid sequence as shown in SEQ ID N0:2 from amino acid
number 1 (Met) to amino acid number 261 (Val), wherein the
amino acid percent identity is determined using a FASTA
program with ktup=1, gap opening penalty=10, gap extension
penalty=1, and substitution matrix=BLOSUM62, with other
parameters set as default. Within one embodiment, the
isolated polynucleotide disclosed above is selected from
the group consisting of: (a) a polynucleotide sequence as
shown in SEQ ID NO:1 from nucleotide 139 to nucleotide
853; and (b) a polynucleotide sequence as shown in SEQ ID
NO:1 from nucleotide 70 to nucleotide 853. Within another
embodiment, the isolated polynucleotide disclosed above
comprises nucleotide 1 to nucleotide 783 of SEQ ID NO:10.
Within another embodiment, the isolated polynucleotide
disclosed above comprises a sequence of amino acid
residues selected from the group consisting of: (a) the
amino acid sequence as shown in SEQ ID N0:2 from amino
acid number 24 (Ala), to amino acid number 261 (Val); and
(b) the amino acid sequence as shown in SEQ ID N0:2 from
amino acid number 1 (Met) to amino acid number 261 (Val).
Within a second aspect, the present invention
provides an expression vector comprising the following
operably linked elements: a transcription promoter; a DNA
segment encoding a zsig28 polypeptide as shown in SEQ ID
N0:2 from amino acid number 24 (Ala), to amino acid number
261 (Val); and a transcription terminator, wherein the
promoter is operably linked to the DNA segment, and the
DNA segment is operably linked to the transcription
terminator. Within one embodiment, the expression vector
disclosed above further comprises a secretory signal
sequence operably linked to the DNA segment.
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Within a third aspect, the present invention
provides a cultured cell comprising an expression vector
as disclosed above, wherein the cell expresses a
polypeptide encoded by the DNA segment.
Within another aspect, the present invention
provides a DNA construct encoding a fusion protein, the
DNA construct comprising: a first DNA segment encoding a
polypeptide comprising a sequence of amino acid residues
selected from the group consisting of: (a) the amino acid
sequence of SEQ ID N0:2 from amino acid number 1 (Met), to
amino acid number 23 (Ala); (b) the amino acid sequence of
SEQ ID N0:2 from amino acid number 24 (Ala) to amino acid
number 82 (Leu); (c) the amino acid sequence of SEQ ID
N0:2 from amino acid number 101 (Leu) to amino acid number
122 (Gly); (d) the amino acid sequence of SEQ ID N0:2 from
amino acid number 141 (Asn) to amino acid number 179
(Ala); (e) the amino acid sequencf= of SEQ ID N0:2 from
amino acid number 193 (Cys) to amino acid number 261
(Val); and (f) the amino acid sequence of SEQ ID N0:2 from
amino acid number 24 (Ala), to amino acid number 261
(Val}; and at least one other DNA segment encoding an
additional polypeptide, wherein the first and other DNA
segments are connected in-frame; and wherein the first and
other DNA segments encode the fusion protein.
Within another aspect, the present invention
provides an expression vector comprising the following
operably linked elements: a transcription promoter; a DNA
construct encoding a fusion protein as disclosed above;
and a transcription terminator, wherein the promoter is
operably linked to the DNA construct, and the DNA
construct is operably linked to the transcription
terminator.
Within another aspect, the present invention
provides a cultured cell comprising an expression vector
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as disclosed above, wherein the cell expresses a
polypeptide encoded by the DNA const:ruct.
Within another aspect, the present invention
provides a method of producing a fusion protein
5 comprising: culturing a cell as disclosed above; and
isolating the polypeptide produced by the cell.
Within another aspect, the present invention
provides an isolated polypeptide comprising a sequence of
amino acid residues that is at least 90o identical to an
amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as shown in SEQ ID N0:2 from
amino acid number 24 (Ala), to amino acid number 261
(Val) ; and (b) the amino acid sequence as shown in SEQ ID
N0:2 from amino acid number 1 (Met) to amino acid number
261 (Val), wherein the amino acid percent identity is
determined using a FASTA program with ktup=1, gap opening
penalty=10, gap extension penalty=1, and substitution
matrix=BLOSUM62, with other parameters set as default.
Within one embodiment, the isolated polypeptide disclosed
above further contains motifs 1 through 4 spaced apart
from N-terminus to C-terminus in a configuration selected
from the group consisting of: (a) Met-{47-50}-M1-{21-22}-
M2-{73-92}-M3; and (b) Met-{47-50}-M1-{21-22}-M2-{73-92}-
M3-{3}-M4, wherein M1 is "motif 1," a sequence of amino
acids as shown in amino acids 48 to 54 of SEQ ID N0:2, M2
is "motif 2, " a sequence of amino acids as shown in amino
acids 77 to 82 of SEQ ID N0:2, M3 is "motif 3," a sequence
of amino acids as shown in amino acids 174 to 180 of SEQ
ID N0:2, M4 is "motif 4," a sequence of amino acids as
shown in amino acids 184 to 189 of SEQ ID N0:2, and {#~
denotes the number of amino acids between the motifs.
Within another embodiment, the isolated polypeptide
disclosed above comprises a sequence of amino acid
residues selected from the group consisting of: (a) the
amino acid sequence as shown in SEQ ID N0:2 from amino
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acid number 24 (Ala), to amino acid number 261 (Val); and
(b) the amino acid sequence as shown in SEQ ID N0:2 from
amino acid number 1 (Met) to amino .acid number 261 (Val).
Within another aspect, the present invention
provides a method of producing a zsig28 polypeptide
comprising: culturing a cell as disclosed above; and
isolating the zsig28 polypeptide produced by the cell.
Within another aspect, the present invention
provides a method of producing an antibody to zsig28
ZO polypeptide comprising: inoculating an animal with a
polypeptide selected from the group consisting of: (a) a
polypeptide consisting of 9 to 238 amino acids, wherein
the polypeptide is identical to a contiguous sequence of
amino acids in SEQ ID N0:2 from amino acid number 24
(Ala), to amino acid number 261 (Val); (b) a polypeptide
consisting of the amino acid sequence of SEQ ID N0:2 from
amino acid number 24 (Ala) to amino acid number 82 (Leu};
(c) a polypeptide consisting of the amino acid sequence of
SEQ ID N0:2 from amino acid number 101 (Leu) to amino acid
number 122 (Gly); (d) a polypept.ide consisting of the
amino acid sequence of SEQ ID N0:2 from amino acid number
191 (Asn) to amino acid number 174 (Ala); (e) a
polypeptide consisting of the amino acid sequence of SEQ
ID N0:2 from amino acid number 193 (Cys) to amino acid
number 261 (Val); (f) a polypeptide as disclosed above;
(g) a polypeptide consisting of the amino acid sequence of
SEQ ID N0:2 from amino acid number 245 (Ala) to amino acid
number 250 (Glu); (h} a polypept.ide consisting of the
amino acid sequence of SEQ ID N0:2 from amino acid number
234 (Asn) to amino acid number 239 (Lys); (i) a
polypeptide consisting of the amino acid sequence of SEQ
ID N0:2 from amino acid number 2t)2 (Glu) to amino acid
number 207 (Lys); (j) a polypept.ide consisting of the
amino acid sequence of SEQ ID N0:2 from amino acid number
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254 (Lys) to amino acid number 259 (Asp); and (k) a
polypeptide consisting of the amino acid sequence of SEQ
ID N0:2 from amino acid number 110 (Glu) to amino acid
number 115 (Ala); and wherein the polypeptide elicits an
immune response in the animal to produce the antibody; and
isolating the antibody from the animal.
Within another aspect, the present invention
provides an antibody produced by the method disclosed
above, which binds to a zsig28 polypeptide. Within one
embodiment, the antibody disclosed above is a monoclonal
antibody.
Within another aspect, the present invention
provides an antibody which specifically binds to a
polypeptide disclosed above.
Within another aspect, the present invention
provides a method of detecting, in a test sample, the
presence of a modulator of zsig28 protein activity,
comprising: culturing a cell into which has been
introduced an expression vector as disclosed above,
wherein the cell expresses the zsig28 protein encoded by
the DNA segment in the presence and absence of a test
sample; and comparing levels of activity of zsig28 in the
presence and absence of a test sample, by a biological or
biochemical assay; and determining from the comparison,
the presence of modulator of zsig28 activity in the test
sample.
These and other aspects of the invention will
become evident upon reference to the following detailed
description of the invention and attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an alignment of zsig28 (SEQ ID
N0:2); murine claudin 1 (CLAUD1) (SEQ ID N0:3) (Furuse, M.
et al., J. Cell Biol. 141:1539-1550, 1998); Genbank
Accession No. AF072127); Murine CPE receptor (AB007) (SEQ
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ID N0:4) (Genbank Accession No. AB00713); human
oligodendrocyte-specific protein (OSP)-like protein
(HSU899) (SEQ ID N0:5) (Genbank Accession No. U89916);
human transmembrane protein deleted in Velo-Cardio-Facial
Syndrome (AF0009) (SEQ ID N0:6) (Genbank Accession No.
AF000959); human OSP (AF0688) (SEQ ID N0:7) (Genbank
Accession No. AF068863); murine claudin 2 (AF0721) (SEQ ID
N0:8) (Furuse, M. et al., supra.; Genbank Accession No.
AF072128); and rat androgen withdrawal protein RVP.l
(PIR A3) (SEQ ID N0:9) (Briehl, M.M. et al., Mol.
Endocrinol. 5:1381-1388, 1991).
Figure 2 is a hydrophobicity plot of zsig28
polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention in detail,
it may be helpful to the understanding thereof to define
the following terms:
The term "affinity tag" is used herein to denote
a polypeptide segment that can be attached to a second
polypeptide to provide for purification or detection of
the second polypeptide or provide sates for attachment of
the second polypeptide to a substrate . In principal , any
peptide or protein for which an antibody or other specific
binding agent is available can be used as an affinity tag.
Affinity tags include a poly-histidine tract, protein A
(Nilsson et al., EMBO J. 4:1075, :1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glut:athione S transferase
(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity
tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA
82:7952-4, 1985), substance P, FlagT'''' peptide (Hopp et al.,
Biotechnolocty 6:1204-10, 1988), streptavidin binding
peptide, 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
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are available from commercial suppliers (e. g., Pharmacia
Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to
denote 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 an 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 terms "amino-terminal" and "carboxyl
terminal" are used herein to denote positions within
polypeptides. Where the context allows, these terms are
used with reference to a particular sequence or portion of
a polypeptide to denote proximity or relative position.
For example, a certain sequence positioned carboxyl-
terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus
of the complete polypeptide.
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 (ar 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 term "complements of a polynucleotide
molecule'" denotes a polynucleotide molecule having a
complementary base sequence and reverse orientation as
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compared to a reference sequence. For example, the
sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT
3'.
The term "contig" denotes a polynucleotide that
5 has a contiguous stretch of identical or complementary
sequence to another polynucleotide. Contiguous sequences
are said to "overlap" a given stretch of polynucleotide
sequence either in their entirety or along a partial
stretch of the polynucleotide. For example,
10 representative contigs to the polynucleotide sequence 5'-
ATGGAGCTT-3' are 5'-AGCTTgagt-3' and 3'-tcgacTACC-5'.
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).
A ~~DNA segment" is a portion of a larger DNA
molecule having specified attributes. For example, a DNA
segment encoding a specified polypeptide is a portion of a
longer DNA molecule, such as a plasmid or plasmid
fragment, that, when read from the 5' to the 3' direction,
encodes the sequence of amino acids of the specified
polypeptide.
The term "expression vector" is used to denote 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 include promoter and terminator
sequences, and may also include one or more origins of
replication, one or more selectable markers, an enhancer,
a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may
contain elements of both.
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The term "isolated", when applied to a
polynucleotide, 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).
An "isolated" polypeptide or pratein is a
polypeptide or protein that is found in a condition other
than its native environment, such a.s apart from blood and
animal tissue. In a preferred form, the isolated
polypeptide is substantially free of other polypeptides,
particularly other polypeptides of animal origin. It is
preferred to provide the polypeptides in a highly purified
form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term
"isolated" does not exclude the presence of the same
polypeptide in alternative physical forms, such as dimers
or alternatively glycosylated or derivatized farms.
The term "operably linked", when referring to
DNA segments, indicates 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 "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.
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"Paralogs" are distinct but structurally related
proteins made by an organism. Paralogs are believed to
arise through gene duplication. For example, a-globin, (3-
globin, and myoglobin are paralogs of each other.
A "polynucleotide" is a single- or double-
stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. Pol.ynucleotides
include RNA and DNA, and may be isolated from natural
sources, synthesized in vitro, or prepared from a
combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated
"bp"), nucleotides ("nt"), or kilobases ("kb"). Where the
context allows, the latter twa terms may describe
polynucleotides that are single-stranded or double-
stranded. When the term is applied to double-stranded
molecules it is used to denote overall length and will be
understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the
two strands of a double-stranded palynucleotide may differ
slightly in length and that the ends thereof may be
staggered as a result of enzymatic cleavage; thus all
nucleotides within a double-stranded polynucleotide
molecule may not be paired.
A "polypeptide" is a polymer of amino acid
residues joined by peptide bonds, whether produced
naturally or synthetically. Polypeptides of less than
about 10 amino acid residues are commonly referred to as
"peptides".
The term "promoter" is used herein for its art
recognized meaning to denote 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.
A "protein" is a macromolecule comprising one or
more polypeptide chains. A protein may also comprise non-
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peptidic components, such as carbohydrate groups.
Carbohydrates and other non-peptidic substituents may be
added to a protein by the cell in which the protein is
produced, and will vary with the type of cell. Proteins
are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are
generally not specified, but may be present nonetheless.
The term "receptor" denotes a cell-associated
protein that binds to a bioacti.ve molecule (i.e., a
ligand) and mediates the effect of the ligand on the cell.
Membrane-bound receptors are characterized by a multi-
peptide 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 molecules) in the cell. This
interaction in turn leads to an alteration in the
metabolism of the cell. Metabolic events that are linked
to receptor-ligand interactions include gene
transcription, phosphorylation, dephosphorylation,
increases in cyclic AMP production, mobilization of
cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e. g., thyroid
stimulating hormone receptor, beta-adrenergic 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 "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
polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
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The term "splice variant" is used herein to
denote alternative forms of RNA transcribed from a gene.
Splice variation arises naturally through use of
alternative splicing sites within a transcribed RNA
molecule, or less commonly between separately transcribed
RNA molecules, and may result in several mRNAs transcribed
from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term
splice variant is also used herein to denote a protein
encoded by a splice variant of an mRNA transcribed from a
gene.
Molecular weights and lengths of polymers
determined by imprecise analytical methods (e.g., gel
electrophoresis) will be understood to be approximate
values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be
understood to be accurate to ~10%.
All references cited herein are incorporated by
reference in their entirety.
The present invention is based in part upon the
discovery of a novel DNA sequence that encodes a
polypeptide having homology to a diverse family of
receptor proteins, containing proteins such as human
claudin 1 and 2 (Furuse, M. et al., J. Cell Biol.
141:1539-1550, 1998), human and murine oligodendrocyte-
specific protein (OSP) (Bronstein, J.M. et al., Neurology
47:772-778, 1996), rat androgen.-withdrawal apoptosis
protein RVP.l (Briehl, M.M., and Miesfeld, R.L., Mol.
Endocrinol. 5:1381-1388, 1991), and others. Analysis of
the tissue distribution of the mRNA corresponding to this
novel DNA showed high expression in stomach, and low
expression in lung. The polypeptide has been designated
zsig28.
The novel zsig28 polypeptides of the present
invention were initially identified by querying an EST
database for proteins homologous to proteins having a
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secretory signal sequence. These proteins are
characterized by an upstream methioriine start site and a
hydrophobic region of approximately 13 amino acids,
followed by a peptide signal peptidase cleavage site. An
5 EST database was queried for novel DNA sequences whose
translations would meet these search criteria. An EST was
found and its corresponding cDNA was sequenced. The novel
polypeptide encoded by the cDNA showed homology with rat
RVP.1. Based on this homology, the zsig28 nucleotide
10 sequence encodes the entire coding sequence of the
predicted protein. Zsig28 may be a novel protein involved
in an apoptotic cellular pathway, cell-cell signaling
molecule, growth factor receptor, or extracellular matrix
associated protein with growth factor hormone activity, or
15 the like, and is a novel member of the claudin/OSP family
of proteins.
The sequence of the zsig28 polypeptide was
obtained from a single clone that contained its
corresponding polynucleotide sequence. The clone was
obtained from a lung library. Other libraries that might
also be searched for such sequences include stomach, fetal
lung, epithelial tissues, and the like.
The nucleotide sequence of a representative
zsig28-encoding DNA is described in SEQ ID NO:1, and its
deduced 261 amino acid sequence is described in SEQ ID
N0:2. In its entirety, the zsig28 polypeptide (SEQ ID
N0:2) represents a full-length polypeptide segment
(residue 1 (Met) to residue 261 (Val) of SEQ ID N0:2). The
domains and structural features of zsig28 are further
described below.
Analysis of the zsig28 polypeptide encoded by
the DNA sequence of SEQ ID N0:1 revealed an open reading
frame encoding 261 amino acids (SEQ ID N0:2) comprising a
predicted secretory signal peptide of 23 amino acid
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residues (residue 1 (Met) to residue 23 (Ala) of SEQ ID
N0:2), and a mature polypeptide of 238 amino acids
(residue 24 (Ala) to residue 261 (Val) of SEQ ID N0:2).
The zsig28 polypeptide contains three transmembrane
domains:
(1) the first transmembrane domain is from
amino acid 83 (Met) to amino acid 100 (Ala) of SEQ ID
N0:2;
(2) the second transmembrane domain is from
amino acid 123 (Ile) to amino acid 190 (Ala) of SEQ ID
N0:2; and
(3) the third transmembrane domain is from
amino acid 175 (Leu) to amino acid 192 (Met) of SEQ ID
N0:2.
These transmembrane domains are corroborated by
the zsig28 hydrophobicity plot (See, Figure 2). Between
and flanking these transmembrane domains lies regions of
the zsig28 molecule that may confer binding to a ligand,
cell-cell interactions, cellular signaling functions, and
the like. Moreover, such regions, and stretches of
hydrophilic amino acids within, would serve as suitable
antigenic epitopes for the production of antibodies, as
discussed herein. These regions include:
(1) "region 1," the amino-terminal region,
amino acid 24 (Ala) to amino acid 82 (Leu);
(2) "region 2," Amino acid 101 (Leu) to amino
acid 122 (Gly);
(3) "region 3," Amino acid 141 (Asn) to amino
acid 174 (Ala); and
(4) "region 4," t:he carboxy terminal
hydrophilic region, amino acid 193 (Cys) to amino acid 261
(Val) .
Within zsig28 are the several motifs of
conserved amino acids based comparison amongst family
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17
members (See Figure 1). Moreover, several regions of low
variance are also present within zsig28 polypeptide. For
determination of regions of low variance, see Sheppard, P.
et al., Gene 150:163-167, 1994. Examining a multiple
alignment of several known family members (For example,
see Figure 1) revealed the following motifs that are both
conserved and exhibit low degeneracy:
1) "motif 1" (a consensus motif pattern
encompassing information in figure 1 that corresponds to
amino acids 48 to 54 of SEQ ID N0:2;1;
2) "motif 2" (a consensus motif pattern
encompassing information in figure 1 that corresponds to
amino acids 77 to 82 of SEQ ID N0:2:1;
3) "motif 3" (a consensus motif pattern
encompassing information in figure 1 that corresponds to
amino acids 174 to 180 of SEQ ID N0:2);
Motifs 1 through 3 are spaced apart from N-
terminus to C-terminus in a configuration represented by
the following:
Met-{47-50}-M1-{21-22}-M2-{73-92}-M3,
where Met is the starting methionine residue
M# denotes the specific motif disclosed above
(e.g., M1 is motif l, etc.) and
{#} denotes the number of amino acids between
the motifs.
In addition, another conserved motif in the
third transmembrane domain of zsig28 is evident:
4) "motif 4" (a consensus motif pattern
encompassing information in figure 1 that corresponds to
amino acids 184 to 189 of SEQ ID N0:2);
Motifs 1 through 4 are spaced apart from N-
terminus to C-terminus in a configuration represented by
the following:
Met-{47-50}-M1-{21-22}-M2-{73-92}-M3-{3}-M4,
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where Met is the starting methionine residue
where M# denotes the specific motif disclosed
above (e.g., M4 is motif 4, etc.) and
{#) denotes the number of amino acids between
the motifs.
The presence of transmembrane regions, and
conserved and low variance motifs generally correlates
with or defines important structural regions in proteins.
Regions of low variance (e.g., hydrophobic clusters) are
generally present in regions of structural importance
(Sheppard, P. et al., supra.). Such regions of low
variance often contain rare or infrequent amino acids,
such as Tryptophan. The regions flanking and between such
conserved and low variance motifs may be more variable,
but are often functionally significant because they may
relate to or define important structures and activities
such as binding domains, biological and enzymatic
activity, signal transduction, cell-cell interaction,
tissue localization domains and the like. For example,
regions 1 through 4 described above may be functionally
significant.
In addition, there are several individual
conserved amino acids throughout the zsig28 polypeptide
located in SEQ ID N0:2 at the following amino acid
numbers: 30 (Trp) , 48 (Gly) , 49 (Leu) , 50 (Trp) , 53 (Cys) ,
59 (Gly) , 63 (Cys) , 72 (Leu) , 103 (C:ys) , and 124 (Lys) .
The regions of conserved amino acid residues in
zsig28, described above, can be used as tools to identify
new family members. For instance, reverse transcription-
polymerase chain reaction (RT-PCR) can be used to amplify
sequences encoding the conserved regions from RNA obtained
from a variety of tissue sources or cell lines. In
particular, highly degenerate primers designed from the
zsig28 sequences are useful for this purpose. Designing
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and using such degenerate primers may be readily performed
by one of skill in the art.
The corresponding polynucleotides encoding the
zsig28 polypeptide regions, domains, motifs, residues and
sequences described above are as shown in SEQ ID N0:1.
The present invention also provides
polynucleotide molecules, including DNA and RNA molecules,
that encode the zsig28 polypeptides disclosed herein.
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. SEQ ID N0:10 is a degenerate DNA sequence that
encompasses all DNAs that encode the zsig28 polypeptide of
SEQ ID N0:2. Those skilled in the art will recognize that
the degenerate sequence of SEQ ID NO:10 also provides all
RNA sequences encoding SEQ ID N0:2 by substituting U for
T. Thus, zsig28 polypeptide-encoding polynucleotides
comprising nucleotide 1 to nucleotide 783 of SEQ ID NO:10
and their RNA equivalents are contemplated by the present
invention. Table 1 sets forth the one-letter codes used
within SEQ ID N0:10 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.
TABLE 1
Nucleotide Resolution Complement Resolution
A A T T
C C G G
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G G C C
T T A A
R A~G Y CST
Y CST R A~G
M ABC K GET
K GET M ABC
S CMG S CMG
W ACT W ACT
H A~C~T D A~G~T
B C~G~T V A~C~G
U A~C~G B C~G~T
D A~G~T H A~C~T
N A~C~G~T N A~C~G~T
The degenerate codons used in SEQ ID NO:10,
encompassing all possible codons for a given amino acid,
are set forth in Table 2.
5
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TABLE 2
One
Amino Letter Colons Degenerate
Acid Code Colon
Cys C TGC TGT TGY
Ser S AGC AGTTCATCC TCG TCT WSN
Thr T ACA ACCACGACT ACN
Pro P CCA CCCCCGCCT CCN
Ala A GCA GCCGCGGCT GCN
Gly G GGA GGCGGGGGT 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 AGGCGACGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
Ile I ATA ATCATT ATH
Leu L CTA CTCCTGCTT TTA TTG YTN
Val U GTA GTCGTGGTT GTN
Phe F TTC TTT TTY
Tyr Y TAC TAT TAY
Trp W TGG TGG
Ter . TAA TAGTGA TRR
Asn~AspB RAY
Glu~GlnZ 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 N0:2. Variant sequences
can be readily tested for functionality as described
herein.
One of ordinary skill in the art will also
appreciate that different species can exhibit
"preferential codon usage." In general, see, Grantham, et
al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.
Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64,
1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol.
158:573-97, 1982. As used herein, the term "preferential
codon usage" or "preferential codons" is a term of art
referring to protein translation codons that are most
frequently used in cells of a certain species, thus
favoring one or a few representatives of the possible
codons encoding each amino acid (See Table 2). For
example, the amino acid Threonine (Thr) may be encoded by
ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the
most commonly used codon; in other species, for example,
insect cells, yeast, viruses or bacteria, different Thr
codons may be preferential. Preferential codons for a
particular species can be :introduced into the
polynucleotides of the present invention by a variety of
methods known in the art. Introduction of preferential
codon sequences into recombinant DNA can, for example,
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enhance production of the protein by making protein
translation more efficient within a particular cell type
or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID NO:10 serves as a template for
optimizing expression of polynucleotides in various cell
types and species commonly used in the art and disclosed
herein. Sequences containing preferential codons can be
tested and optimized for expression in various species,
and tested for functionality as disclosed herein.
Within preferred embodiments of the invention
the isolated polynucleotides will hybridize to similar
sized regions of SEQ ID N0:1, 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. Numerous equations for calculating Tm are
known in the art, and are specific for DNA, RNA and DNA-
RNA hybrids and polynucleotide probe sequences of varying
length (see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Edition (Cold Spring
Harbor Press 1989); Ausubel et al., (eds.), Current
Protocols in Molecular Biology (John Wiley and Sons, Inc.
1987); Berger and Kimmel (eds.), Guide to Molecular
Cloning Techniques, (Academic Press, Inc. 1987); and
Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)).
Sequence analysis software such as OLIGO 6.0 (LSR; Long
Lake, MN) and Primer Premier 4.0 (Premier Biosoft
International; Palo Alto, CA), as well as sites on the
Internet, are available tools for analyzing a given
sequence and calculating Tm based on user defined criteria.
Such programs can also analyze a given sequence under
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defined conditions and identify suitable probe sequences.
Typically, hybridization of :Longer polynucleotide
sequences (e.g., >50 base pairs) is performed at
temperatures of about 20-25°C below the calculated Tm. For
smaller probes (e.g., <50 base pairs) hybridization is
typically carried out at the Tm or 5-10°C below. This
allows for the maximum rate of hybridization for DNA-DNA
and DNA-RNA hybrids. Higher degrees of stringency at
lower temperatures can be achieved with the addition of
20 formamide which reduces the Tm of the hybrid about 1°C for
each 1% formamide in the buffer solution. Suitable
stringent hybridization conditions are equivalent to about
a 5 h to overnight incubation at about 42°C in a solution
comprising: about 40-50% formamide, up to about 6X-SSC,
about 5X Denhardt's solution, zero up to about 10% dextran
sulfate, and about 10-20 ~g/ml denatured commercially-
available carrier DNA. Genera_Lly, such stringent
conditions include temperatures of 20-70°C and a
hybridization buffer containing up to 6x SSC and 0-50%
formamide; hybridization is then followed by washing
filters in up to about 2X SSC. For example, a suitable
wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1%
SDS, at 55°C to 65°C. Different degrees of stringency can
be used during hybridization and washing to achieve
maximum specific binding to t:he target sequence.
Typically, the washes following hybridization are
performed at increasing degrees of: stringency to remove
non-hybridized polynucleotide probes from hybridized
complexes. Stringent hybridization and wash conditions
depend on the length of the probe, reflected in the Tm,
hybridization and wash solutions used, and are routinely
determined empirically by one of skill in the art.
As previously noted, the isolated
polynucleotides of the present invention include DNA and
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RNA. Methods for preparing DNA and RNA are well known in
the art. In general, RNA is isolated from a tissue or
cell that produces large amounts of zsig28 RNA. Such
tissues and cells are identified by Northern blotting
5 (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and
include stomach and lung. Total RNA can be prepared using
guanidinium isothiocyanate extraction followed by
isolation by centrifugation in a CsCl gradient (Chirgwin
et al., Biochemistry 18:52-94, 1979). Poly (A)+ RNA is
10 prepared from total RNA using the method of Aviv and Leder
(Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
Complementary DNA (cDNA) is prepared from poly(A)+ RNA
using known methods. In the alternative, genomic DNA can
be isolated. Polynucleotides encoding zsig28
15 polypeptides are then identified and isolated by, for
example, hybridization or polymerase chain reaction (PCR)
(Mullis, U.S. Patent No. 4,683,202).
A full-length clone encoding zsig28 can be
obtained by conventional cloning procedures.
20 Complementary DNA (cDNA) clones are preferred, although
for some applications (e. g., expression in transgenic
animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic
intron from the same or a different gene. Methods for
25 preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include
the use of the sequence disclosed herein, or parts
thereof, for probing or priming a library. Expression
libraries can be probed with antibodies to zsig28
polypeptide, receptor fragments, or other specific
binding partners.
The polynucleotides of the present invention can
also be synthesized using DNA synthesis machines.
Currently the method of choice is the phosphoramidite
method. If chemically synthesized double stranded DNA is
required for an application such as the synthesis of a
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26
gene or a gene fragment, then each complementary strand is
made separately. The production of short polynucleotides
(60 to 80 bp) is technically straightforward and can be
accomplished by synthesizing the complementary strands and
then annealing them. However, for producing longer
polynucleotides (>300 bp), special strategies are usually
employed, because the coupling efficiency of each cycle
during chemical DNA synthesis is seldom 1000. To overcome
this problem, synthetic genes (double-stranded) are
assembled in modular form from single-stranded fragments
that are from 20 to 100 nucleotides in length.
One method for building a synthetic gene
requires the-initial production of a set of overlapping,
complementary oligonucleotides, each of which is between
20 to 60 nucleotides long. Each internal section of the
gene has complementary 3' and 5' terminal extensions
designed to base pair precisely with an adjacent section.
Thus, after the gene is assembled, process is completed by
sealing the nicks along the backbones of the two strands
with T4 DNA ligase. In addition to the protein coding
sequence, synthetic genes can be designed with terminal
sequences that facilitate insertion into a restriction
endonuclease site of a cloning vector. Moreover, other
sequences should can be added that contain signals for
proper initiation and termination of transcription and
translation.
An alternative way to prepare a full-length gene
is to synthesize a specified set of overlapping
oligonucleotides (40 to 100 nucleotides). After the 3' and
5' short overlapping complementary regions (6 to 10
nucleotides) are annealed, large gaps still remain, but
the short base-paired regions are both long enough and
stable enough to hold the structure together. The are gaps
filled and the DNA duplex is completed via enzymatic DNA
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synthesis by E. coli DNA polymerase I. After the
enzymatic synthesis is completed, the nicks are sealed
with T4 DNA ligase. Double-stranded constructs are
sequentially linked to one another to form the entire gene
sequence which is verified by DNA sequence analysis. See
Glick and Pasternak, Molecular Biotechnology, Principles &
Applications of Recombinant DNA, (ASM Press, Washington,
D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323
56, 1989 and Climie et al., Proc. Natl. Acad. Sci. USA
87:633-7, 1990.
zsig28 polynucleotide sequences disclosed herein
can also be used as probes or primers to clone 5' non-
coding regions of a zsig28 gene. In view of the tissue-
specific expression observed for zsig28 by Northern
blotting, this gene region is expected to provide for
stomach-specific expression. Promoter elements from a
zsig28 gene could thus be used to direct the tissue-
specific expression of heterologous genes in, for example,
transgenic animals or patients treated with gene therapy.
Cloning of 5' flanking sequences also facilitates
production of zsig28 proteins by "gene activation" as
disclosed in U.S. Patent No. 5,641,670. Briefly,
expression of an endogenous zsig28 gene in a cell is
altered by introducing into the zsig28 locus a DNA
construct comprising at least a targeting sequence, a
regulatory sequence, an exon, and an unpaired splice donor
site. The targeting sequence is a zsig28 5' non-coding
sequence that permits homologous recombination of the
construct with the endogenous zsig28 locus, whereby the
sequences within the construct become operably linked with
the endogenous zsig28 coding sequence. In this way, an
endogenous zsig28 promoter can be replaced or supplemented
with other regulatory sequences to provide enhanced,
tissue-specific, or otherwise regulated expression.
The present invention further provides
counterpart polypeptides and polynucleotides from other
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28
species (orthologs). These species include, but are not
limited to mammalian, avian, amphibian, reptile, fish,
insect and other vertebrate and invertebrate species. Of
particular interest are zsig28 polypeptides from other
mammalian species, including murine, porcine, ovine,
bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human zsig28 can be cloned
using information and compositions provided by the present
invention in combination with conventional cloning
techniques . For example, a cDNA can be cloned using mRNA
obtained from a tissue or cell type that expresses zsig28
as disclosed herein. Suitable saurces 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 or cell line. A
zsig28-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 reactian, or PCR
(Mullis, supra.), using primers designed from the
representative human zsig28sequence 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
zsig28 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
Those skilled in the art will recognize that the
sequence disclosed in SEQ ID NO::L represents a single
allele of human zsig28 and that allelic variation and
alternative splicing are expected to occur. Allelic
variants of this sequence 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
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29
amino acid sequence changes, are within the scope of the
present invention, as are proteins which are allelic
variants of SEQ ID N0:2. cDNAs generated from
alternatively spliced mRNAs, which retain the properties
of the zsig28 polypeptide are included within the scope of
the present invention, as are polypeptides encoded by such
cDNAs and mRNAs. Allelic variants and splice variants of
these sequences can be cloned by probing cDNA or genomic
libraries from different individuals or tissues according
to standard procedures known in the art.
The present invention also provides isolated
zsig28 polypeptides that are substantially similar to the
polypeptides of SEQ ID N0:2 and their orthologs. The term
"substantially similar" is used herein to denote
polypeptides having at least 70%, more preferably at least
800, sequence identity to the sequences shown in SEQ ID
N0:2 or their orthologs. Such polypeptides will more
preferably be at least 90% identical, and most preferably
950 or more identical to SEQ ID N0:2 or its orthologs.
Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math.
Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc.
Natl. Acad. Sci. USA 89:10915-9, 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?.
The percent identity is then calculated as:
Total number of identical matches
x 100
[length of the longer sequence plus the
number of gaps introduced into the longer
sequence in order to align the two sequences]
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3 ,~ N M
r~ I
~nN N o
I I
d~ r~M N N
I I
L~r-Ir-~VI M N
w l0 d'N N rl M rl
I I I
L!1O N ~-Irir-Irif-I
1 1 I I I
.'~-~ L(1rlM rlO r-IM N N
I I I I I I 1
d~N N O M N rlN rlv-1
M I I I I I t
f"'I d~ N M rlO M N rlM v-1M
I 1 I I I I
'nTr' d0M M rl N ~-IN v-IN N N M
I I I 1 i 1 1 I 1 I
C,7 l0 N dl d~N M M N O N N M M
1 I i I I I I I I t I
W LI)N O M M rl N M r-1O .-1M N N
I I I I 1 I I 1 1 1
Lf1N N O M N r-IO M r-~O ~ N ~-1N
I I I I I I I 1 I
U 01M d~M M rl '-1M rlN M r-1r-IN N rd
i I I I 1 I 1 I I I I 1 I 1 I
M O N ~-1r-if'~1d~~rlM M r-IO r~ld~ M M
1 I I I I I I I I I I I I
l0rl M O O O riM M O N M N r-iO d~ N M
I I 1 I I I I I I
(Y, Lf1O N M r-1O N O M N N r-1M N ~ rlM N M
I I I I I I 1 I I I I I I
Q,' crrl N N O ~-IrlO N rl r~ri v-IN v-1r-iO M N O
I I 1 I I i I I I I I I I I
rx z a v a w ~ x H a x ~ w w ~n H 3 >-I
m o In o
N
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Sequence identity of polynucleotide molecules is
determined by similar methods using a ratio as disclosed
above.
Those skilled in the art appreciate that there
are many established algorithms available to align two
amino acid sequences. The "FASTA" similarity search
algorithm of Pearson and Lipman is a suitable protein
alignment method for examining the level of identity
shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant zsig28
polypeptide. The FASTA algorithm is described by Pearson
and Lipman, Proc. Nat'1 Acad. Sci. USA 85:2444 (1988), and
by Pearson, Meth. Enzymol. 183:63 (:1990).
Briefly, FASTA first characterizes sequence
similarity by identifying regions shared by the query
sequence (e. g., SEQ ID N0:2) and a test sequence that have
either the highest density of identities {if the ktup
variable is 1) or pairs of identities (if ktup=2), without
considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the
highest density of identities are then rescored by
comparing the similarity of all paired amino acids using
an amino acid substitution matrix, and the ends of the
regions are "trimmed" to include only those residues that
contribute to the highest score. If there are several
regions with scores greater than the "cutoff" value
(calculated by a predetermined formula based upon the
length of the sequence and the ktup value), then the
trimmed initial regions are examined to determine whether
the regions can be joined to form an approximate alignment
with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification
of the Needleman-Wunsch-Sellers a7.gorithm (Needleman and
Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J.
Appl. Math. 26:787 (1974)), which allows for amino acid
insertions and deletions. Preferred parameters for FASTA
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32
analysis are: ktup=1, gap opening penalty=10, gap
extension penalty=1, a nd substitution matrix=BLOSUM62.
These parameters introduced into FASTA program
can be a by
modifying the scoring matrix tile ("SMATRIX"),
as
explained in Appendix of Pearson, Meth. Enzymol. 183:63
2
(1990) .
FASTA can also be used to determine the sequence
identity of nucleic acid molecules using a ratio as
disclosed above. For nucleotide sequence comparisons, the
ktup value can range between one t.o six, preferably from
three to six, most preferably three, with other parameters
set as default.
The BLOSUM62 table (Table 3) is an amino acid
substitution matrix derived from about 2,000 local
multiple alignments of protein sequence segments,
representing highly conserved regions of more than 500
groups of related proteins (Henikoff and Henikoff, Proc.
Nat'1 Acad. Sci. USA 89:10915 (1992)). Accordingly, the
BLOSUM62 substitution frequencies can be used to define
conservative amino acid substitutions that may be
introduced into the amino acid sequences of the present
invention. Although it is possible to design amino acid
substitutions based solely upon chemical properties (as
discussed below), the language "conservative amino acid
substitution" preferably refers to a substitution
represented by a BLOSUM62 value of greater than -1. For
example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0,
1, 2, or 3. According to this system, preferred
conservative amino acid substitutions are characterized by
a BLOSUM62 value of at least 1 (e. g., 1, 2 or 3), while
more preferred conservative amino acid substitutions are
characterized by a BLOSUM62 value of at least 2 (e.g., 2
or 3 ) .
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Variant zsig28 polypeptides or substantially
homologous zsig28 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 polypeptide; small
deletions, typically of one to about 30 amino acids; and
amino- or carboxyl-terminal extensions, such as an amino-
terminal methionine residue, a small linker peptide of up
to about 20-25 residues, or an affinity tag. The present
invention thus includes polypeptides of from about 208 to
about 291 amino acid residues that comprise a sequence
that is at least 800, preferably at least 900, and more
preferably 95% or more identical to the corresponding
region of SEQ ID N0:2. Polypeptides comprising affinity
tags can further comprise a proteolytic cleavage site
between the zsig28 polypeptide and the affinity tag.
Suitable sites include thrombin cleavage sites and factor
Xa cleavage sites.
Table 4
Conservative amino acid substitutions
Basic: arginine
lysine
histidine
Acidic: glutamic acid
aspartic acid
Polar: glutamine
asparagine
Hydrophobic: leucine
isoleucine
valine
Aromatic: phenylalan:ine
tryptophan
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tyrosine
Small: glycine
alanine
serine
threonine
methionine
The present invention further provides a variety
of other polypeptide fusions and related multimeric
proteins comprising one or more polypeptide fusions. For
example, a zsig28 polypeptide can be prepared as a fusion
to a dimerizing protein as disclosed in U. S . Patents Nos .
5,155,027 and 5,567,584. Preferred dimerizing proteins in
this regard include immunoglobulin constant region
domains. Immunoglobulin-zsig28 polypeptide fusions can be
expressed in genetically engineered cells to produce a
variety of multimeric zsig28 analogs. Auxiliary domains
can be fused to zsig28 polypeptides to target them to
specific cells, tissues, or macromolecules (e. g.,
collagen). For example, a zsig28 polypeptide or protein
could be targeted to a predetermined cell type by fusing a
zsig28 polypeptide to a ligand that specifically binds to
a receptor on the surface of the target cell. In this
way, polypeptides and proteins can be targeted for
therapeutic or diagnostic purposes. A zsig28 polypeptide
can be fused to two or more moieties, such as an affinity
tag for purification and a targeting or dimerizing domain.
Polypeptide fusions can also comprise one or more cleavage
sites, particularly between domains. See, Tuan et al.,
Connective Tissue Research 34:1-9, 1996. Similarly, such
fusions can be constructed to enable the secretion of the
zsig28 polypeptide regions l, 2, 3 or 4, described herein,
or smaller fragments within those regions. Soluble zsig28
regions l, 2, 3 or 4 attached to dimerizing proteins have
can serve as antagonists of the natural ligand for zsig28
polypeptide, or to zsig28 polypeptide itself, for example
CA 02343001 2001-03-16
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by preventing dimerization or multimerization. Such
antagonists containing soluble zsig:?8 regions 1, 2, 3 or 4
can be tested for functionality as disclosed herein.
The proteins of the present invention can also
5 comprise non-naturally occurring amino acid residues.
Non-naturally occurring amino acids include, without
limitation, traps-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, traps-4-hydroxyproline, N-
methylglycine, allo-threonine, methylthreonine,
10 hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid,
thiazolidine carboxylic acid, dehydroproline, 3- and 4-
methylproline, 3,3-dimethylproline, tert-leucine,
norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-
15 azaphenylalanine, and 4-fluorophenylalanine. Several
methods are known in the art for incorporating non-
naturally occurring amino acid residues into proteins.
For example, an in vitro system can be employed wherein
nonsense mutations are suppressed using chemically
20 aminoacylated suppressor tRNAs. Methods for synthesizing
amino acids and aminoacylating tRNA are known in the art .
Transcription and translation of. plasmids containing
nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially
25 available enzymes and other reagents. Proteins are
purified by chromatography. See, for example, Robertson
et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al.,
Methods Enzymol. 202:301, 1991; Chung et al., Science
259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.
30 USA 90:10145-9, 1993). In a second method, translation is
carried out in Xenopus oocytes by microinjection of
mutated mRNA and chemically aminoacylated suppressor tRNAs
(Turcatti et al., J. Biol. Chem. 271:19991-8, 1996).
Within a third method, E. coli cells are cultured in the
35 absence of a natural amino acid that is to be replaced
CA 02343001 2001-03-16
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36
(e. g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acids) (e.g., 2-
azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine,
or 4-fluorophenylalanine). The non-naturally occurring
amino acid is incorporated into the protein in place of
its natural counterpart. See, Koide et al., Biochem.
33:7470-6, 1994. Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in
vitro chemical modification. Chemical modification can be
combined with site-directed mutagenesis to further expand
the range of substitutions (Wynn and Richards, Protein
Sci. 2:395-403, 1993).
A limited number of non-conservative amino
acids, amino acids that are not encoded by the genetic
code, non-naturally occurring amino acids, and unnatural
amino acids may be substituted for zsig28 amino acid
residues.
Essential amino acids in the 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
and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.
Natl. Acad. Sci. USA 88:4498-502, 1991). 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 as disclosed
below to identify amino acid residues that are critical to
the activity of the molecule. See also, Hilton et al. , J.
Biol. Chem. 271:4699-708, 1996. Sites of protein-protein
or other biological interaction can also be determined by
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-
12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992;
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37
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 proteins, such
as claudin 1 (SEQ ID N0:3), human GSP-like protein (SEQ ID
N0:5), and the like.
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 241:53-7, 1988) or Bowie and Sauer (Proc.
Natl. Acad. Sci. USA 86:2152-6, 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 pasition. Other methods
that can be used include phage display (e.g., Lowman et
al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. 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).
Variants of the disclosed zsigl8 DNA and
polypeptide sequences can be generated through DNA
shuffling as disclosed by Stemmer, Nature 370:389-91,
1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51,
1994 and WIPO Publication WO 97/20078. Briefly, variant
DNAs are generated by in vitro homologous recombination by
random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced
point mutations. This technique can be modified by using
a family of parent DNAs, such as allelic variants or DNAs
from different species, to introduce additional
variability into the process. Selection or screening for
the desired activity, followed by additional iterations of
mutagenesis and assay provides for rapid "evolution" of
sequences by selecting for desirable mutations while
simultaneously selecting against detrimental changes.
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38
Mutagenesis methods as disclosed herein 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., signal transduction, or binding
activities) 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 herein, one of
ordinary skill in the art can identify and/or prepare a
variety of polypeptide fragments or variants of SEQ ID
N0:2 or that retain, for example, binding, cell-cell
communication, or signal transduction activity of the
wild-type zsig28 protein. For example, using the methods
described above, one could identify a ligand binding
domain on zsig28; heterodimeric and homodimeric binding
domains; other functional or struct=ural domains; or other
domains important for protein-protein interactions, cell
cell interactions, or signal transduction. Such
polypeptides may also include additional polypeptide
segments, such as affinity tags, as generally disclosed
herein.
For any zsig28 polypeptide, including variants
and fusion proteins, one of ordinary skill in the art can
readily generate a fully degenerate polynucleotide
sequence encoding that variant using the information set
forth in Tables 1 and 2 above.
The zsig28 polypeptides of the present
invention, including full-length polypeptides,
biologically active fragments, and fusion polypeptides,
can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells
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39
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 Harbar Laboratory
Press, Cold Spring Harbor, NY, 1989, and Ausubel et al.,
eds., Current Protocols in Molecular Biology, John Wiley
and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a zsig28
polypeptide is operably linked to other genetic elements
required for its expression, generally including a
transcription promoter and terminator, within an
expression vector. Generally, the promoter is operably
linked to the DNA sequence or segment, and the DNA segment
is operably linked to the transcription terminator. 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 zsig28 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 zsig28, or
may be derived from another secreted protein (e.g., t-PA)
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or synthesized de novo. The secretory signal sequence is
operably linked to the zsig28 DNA sequence, i . a . , the two
sequences are joined in the correct reading frame and
positioned to direct the newly synthesized polypeptide
5 into the secretory pathway of the host cell. Secretory
signal sequences are commonly positioned 5~ to the DNA
sequence encoding the polypeptide of interest, although
certain secretory signal sequences may be positioned
elsewhere in the DNA sequence of interest (See, e.g.,
10 Welch et al., U.S. Patent No. 5,037,743; Holland et al.,
U.S. Patent No. 5,143,830).
Alternatively, the secretory signal sequence
contained in the polypeptides of the present invention is
used to direct other polypeptides into the secretory
15 pathway. The present invention provides for such fusion
polypeptides. A signal fusion polypeptide can be made
wherein a secretory signal sequence derived from amino
acid residue 1 (Met) to residue 23 (Ala) of SEQ ID N0:2 is
operably linked to a DNA sequence encoding another
20 polypeptide using methods known in the art and disclosed
herein. The secretory signal sequence contained in the
fusion polypeptides of the present invention is preferably
fused amino-terminally to an additional peptide to direct
the additional peptide into the secretory pathway. Such
25 constructs have numerous applications known in the art.
For example, these novel secretory signal sequence fusion
constructs can direct the secretion of an active component
of a normally non-secreted protein. Such fusions may be
used in vivo or in vitro to direct peptides through the
30 secretory pathway.
Cultured mammalian cells are suitable hosts
within the present invention. Methods for introducing
exogenous DNA into mammalian host cells include calcium
phosphate-mediated transfection (Wigler et al., Cell
35 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics
7:603, 1981: Graham and Van der Eb, Viroloay 52:456,
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41
1973), electroporation (Neumann et al., EMBO J. 1:841-5,
1982), DEAF-dextran mediated transf:ection (Ausubel et al.,
ibid.); and liposome-mediated transfection (Hawley-Nelson
et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,
1993, and viral vectors (Miller and Rosman, BioTechniques
7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6,
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.
Suitable cultured mammalian cells include the COS-1 (ATCC
No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No.
CRL 1632), 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 ( a . g . CHO-K1; ATCC No . CCL 61 ) cel l
lines. Additional suitable cell lines are known in the
art and available from public depositories such as the
American Type Culture Collection, Manassas, VA. 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 adenovirus 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
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 hike. Selection systems
can also be used to increase the expression level of the
gene of interest, a process referred to as
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42
"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. (Ban~alore) 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 Autographs californica nuclear
polyhedrosis virus (AcNPV). See, King, L.A. and Possee,
R.D., The Baculovirus Expression System: A Laboratory
Guide, London, Chapman & Hall; 0'Reilly, D.R. et al.,
Baculovirus Ex ression Vectors: A Laboratory Manual, New
York, Oxford University Press., 1999; and, Richardson, C.
D., Ed., Baculovirus Expression Protocols. Methods in
Molecular Biology, Totowa, NJ, Humans Press, 1995. The
second method of making recombinant zsig28 baculovirus
utilizes a transposon-based system described by Luckow
(Luckow, V.A, et al., J Virol 67:4566-79, 1993). This
system, which utilizes transfer vEactors, is sold in the
CA 02343001 2001-03-16
WO 00115659 PC'T/US99/2102~
43
Bac-to-BacT"" kit (Life Technologies, Rockville, MD) . This
system utilizes a transfer vector, pFastBaclT"" (Life
Technologies) containing a Tn7 transposon to move the DNA
encoding the zsig28 polypeptide into a baculovirus genome
maintained in E. coli as a large plasmid called a
"bacmid." The pFastBaclT"" transfer vector utilizes the
AcNPV polyhedrin promoter to drive the expression of the
gene of interest, in this case zsig28. However,
pFastBaclT"" 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. 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 zsig28 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 zsig28 secretory signal sequence.
In addition, transfer vectors ca.n include an in-frame
fusion with DNA encoding an epitope tag at the C- or N-
terminus of the expressed zsig28 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 zsig28 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
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44
baculovirus genome is isolated, using common techniques,
and used to transfect Spodoptera frugiperda cells, e.g.
Sf9 cells. Recombinant virus that expresses zsig28 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 FiveOT""
cell line (Invitrogen) derived from Trichoplusia ni (U. S.
Patent No. 5,300,435). Commercial:Ly available serum-free
media are used to grow and maintain the cells. Suitable
media are Sf900 IITM (Life Technologies) or ESF 921T""
(Expression Systems) for the Sf9 cells; and Ex-ce110405T""
(JRH Biosciences, Lenexa, KS) or Express F'iveOT"" (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.
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 zsig28 polypeptide from the
supernatant can be achieved using methods described
herein.
Fungal cells, including yeast cells, can also be
used within the present invention. Yeast species of
particular interest in this regard include Saccharomyces
cerevisiae, Pichia pastoris, and Pichia methanolica.
Methods for transforming S. cerevisiae cells with
exogenous DNA and producing recombinant polypeptides
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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
5 No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e. g., leucine). A preferred vector
system for use in Saccharomyces cerevisiae is the POTI
10 vector system disclosed by Kawasaki et 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.
15 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
20 Hansenula polymorpha, Schizosaccharomyces pombe,
Kluyveromyces Iactis, Kluyveromyces fragilis, Ustilago
maydis, Pichia pastoris, Pichia methanolica, Pichia
guillermondii and Candida maltosa are known in the art.
See, for example, Gleeson et al., J. Gen. Microbiol.
25 132:3459-65, 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
30 transforming Neurospora are disclosed by Lambowitz, U.S.
Patent No. 4,486,533.
The use of Pichia methanolica as host for the
production of recombinant proteins is disclosed in WIPO
Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO
35 98/02565. DNA molecules for use in transforming P.
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46
methanolica will commonly be prepared as double-stranded,
circular plasmids, which are preferably linearized prior
to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and
terminator in the plasmid be that of a P. methanolica
gene, such as a P. methano.Iica alcohol utilization gene
(AUGI or AUG2). Other useful promoters include those of
the dihydroxyacetone synthase (DHAS), formate
dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host
chromosome, it is preferred to have the entire expression
segment of the plasmid flanked at both ends by host DNA
sequences. A preferred selectable marker for use in
Pichia methanolica is a P. methanolica ADE2 gene, which
encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;
EC 4.1.1.21) , which allows ade2 host cells to grow in the
absence of adenine. For large-scale, industrial processes
where it is desirable to minimize the use of methanol, it
is preferred to use host cells in which both methanol
utilization genes (AUG1 and AUG2) are deleted. For
production of secreted proteins, host cells deficient in
vacuolar protease genes (PEP4 and PRBI) are preferred.
Electroporation is used to facilitate the introduction of
a plasmid containing DNA encoding a polypeptide of
interest into P. methanolica cells. It is preferred to
transform P. methanolica cells by electroporation using
an exponentially decaying, pulsed electric field having a
field strength of from 2.5 to 4.5 kV/cm, preferably about
3.75 kV/cm, and a time constant (i) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
Prokaryotic host cells, including strains of the
bacteria Escherichia coli, Bacillus and other genera are
also useful host cells within the present invention.
Techniques for transforming these hosts and expressing
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47
foreign DNA sequences cloned therein are well known in the
art (see, e.g., Sambrook et al., ibid.). When expressing
a zsig28 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic
space by a bacterial secretion sequence. In the former
case, the cells are lysed, and the granules are recovered
and denatured using, for example, guanidine isothiocyanate
or urea. The denatured polypeptide can then be refolded
and dimerized by diluting the denaturant, such as by
dialysis against a solution of urea and a combination of
reduced and oxidized glutathione, followed by dialysis
against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic
space in a soluble and functional form by disrupting the
cells (by, for example, sonication or osmotic shock) to
release the contents of the periplasmic space and
recovering the protein, thereby obviating the need for
denaturation and refolding.
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
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. methanoli.ca 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
CA 02343001 2001-03-16
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48
of small flasks or sparging of fermentors. A preferred
culture medium for P. methanolica is YEPD (2% D-glucose,
2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1%
BactoTM yeast extract (Difco Laboratories), 0.004% adenine
and 0.0060 L-leucine).
It is preferred to purify the polypeptides of
the present invention to >_80% purity, more preferably to
>_90% purity, even more preferably >_95% purity, 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. Preferably, a purified polypeptide is
substantially free of other polypeptides, particularly
other polypeptides of animal origin.
Expressed recombinant zsig28 polypeptides (or
chimeric zsig28 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
chromatographic media include derivatized dextrans,
agarose, cellulose, polyacrylamide, specialty silicas, and
the like. PEI, DEAE, QAE and Q derivatives are preferred.
Exemplary chromatographic media include those media
derivatized with phenyl, butyl, oz' octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Tayopearl 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
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49
they are to be used. These supports may be modified with
reactive 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 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 binding receptor
polypeptides to support media are well known in the art.
Selection of a particular method i.s 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 LKB
Biotechnology, Uppsala, Sweden, 1988.
The polypeptides of the present invention can be
isolated by exploitation of their biochemical, structural,
and biological properties. For example, immobilized metal
ion adsorption (IMAC} chromatography can be used to purify
histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a ge:L is first charged with
divalent metal ions to form a chelate (Sulkowski, Trends
in Biochem. 3:1-7, 1985}. Histidine- rich proteins will be
adsorbed to this matrix with differing affinities,
depending upon the metal ion used, and will be eluted by
competitive elution, lowering the pH, or use of strong
chelating agents. Other 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}. Within additional embodiments of the invention, a
fusion of the polypeptide of interest and an affinity tag
(e. g., maltose-binding protein, an immunoglobulin domain}
may be constructed to facilitate purification.
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Moreover, using methods described in the art,
polypeptide fusions, or hybrid zsig28 proteins, are
constructed using regions or domains of the inventive
zsig28 in combination with paralogs (e. g., human OSP-like
5 protein}, orthologs (e.g., murine OSP or RVP.1) or
heterologous proteins (Sambrook et al., ibid.; Altschul et
al., ibid.; Picard, Cur. Opin. BioloQV, 5:511-5, 1994,
and references therein). These methods allow the
determination of the biological importance of larger
10 domains or regions in a polypeptide of interest. Such
hybrids may alter reaction kinetics, binding, constrict or
expand the substrate specificity, or alter tissue and
cellular localization of a polypeptide, and can be applied
to polypeptides of unknown structure.
15 Fusion proteins can be prepared by methods known
to those skilled in the art by preparing each component of
the fusion protein and chemically conjugating them.
Alternatively, a polynucleotide encoding both components
of the fusion protein in the proper reading frame can be
20 generated using known techniques and expressed by the
methods described herein. For example, part or all of a
domains) conferring a biological function may be swapped
between zsig28 of the present invention with the
functionally equivalent domains) from another family
25 member. Such domains include, but are not limited to, the
secretory signal sequence, transmembrane domains, regions
1 through 4, and motifs 1 through 4, as described herein.
Such fusion proteins would be expected to have a
biological functional profile that is the same or similar
30 to polypeptides of the present invention or proteins to
which they are fused, depending on the fusion constructed.
Moreover, such fusion proteins may exhibit other
properties as disclosed herein.
Standard molecular biological and cloning
35 techniques can be used to swap the desired domains between
the zsig28 polypeptide and those to which they are fused.
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Generally, a DNA segment that encodes a domain of
interest, e.g., a domain described above, are operably
linked in frame, and inserted into an appropriate
expression vector, as described herein. Such fusion
proteins can be expressed, isolated, and assayed for
activity as described herein.
zsig28 polypeptides or fragments thereof may
also be prepared through chemical synthesis. zsig28
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; pegylated or non-pegylated; and may or
may not include an initial methionine amino acid residue.
Polypeptides of the present invention can also
be synthesized by exclusive solid phase synthesis, partial
solid phase methods, fragment condensation or classical
solution synthesis. Methods for synthesizing polypeptides
are well known in the art. See, for example, Merrifield,
J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal.
Biochem. 34:595, 1970. After the entire synthesis of the
desired peptide on a solid support,the peptide-resin is
with a reagent which cleaves the polypeptide from the
resin and removes most of the side-chain protecting
groups. Such methods are well established in the art.
The activity of molecules of the present
invention can be measured using a variety of assays that
measure proliferation and/or differentiation of specific
cell types, chemotaxis, adhesion, changes in ion channel
influx, pH flux, regulation of second messenger levels and
neurotransmitter release, cell motility, protein binding,
apoptosis, or the like. 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.
The activity of molecules of the present
invention can be measured using a variety of assays that
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measure stimulation of gastrointestinal cell
contractility, peristalsis, modulation of nutrient uptake
and/or secretion of digestive enzymes. Of particular
interest are changes in contractility of smooth muscle
cells. For example, the contractile response of segments
of mammalian duodenum or other gastrointestinal smooth
muscle tissue (Depoortere et al., J. Gastrointestinal
Motility 1:150-159, 1989, incorporated herein by
reference). An exemplary in vivo assay uses an ultrasonic
micrometer to measure the dimensional changes radially
between commissures and longitudinally to the plane of the
valve base (Hansen et al., Society of Thoracic Surgeons
60:5384-390, 1995).
Gastric motility is generally measured in the
clinical setting as the time required for gastric emptying
and subsequent transit time through the gastrointestinal
tract. Gastric emptying scans ar_e well known to those
skilled in the art, and briefly, comprise use of an oral
contrast agent, such as barium, or a radiolabeled meal.
Solids and liquids can be measured independently. A test
food or liquid is radiolabeled with an isotope (e. g.
99"'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 Supp.
5:56-10, 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. Moreover, these
assays can be used to test in vivo zsig28 agonists and
antagonists, discussed below.
High expression of zsig28 polypeptide in the
stomach suggests that modulators of zsig28 activity would
be therapeutically useful. Such modulators could be
agonists or antagonists that respectively stimulate or
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inhibit zsig28 polypeptide activity. Effects of
modulating zsig28 activity can be assayed by methods well
known in the art.
Zsig28 agonists or antagonists thereof may be
therapeutically useful for promoting wound healing, for
example, in the stomach. To verify the presence of
modulators of zsig28 polypeptides with these capabilities,
agonists or antagonists of the present invention are
evaluated with respect to their ability to facilitate
wound healing according to procedures known in the art.
If desired, zsig28 polypeptide performance in this regard
can be compared to growth factor receptors, such as those
for EGF, NGF, TGF-a, TGF-(3, insulin, IGF-I, IGF-II,
fibroblast growth factor (FGF) and the like. In addition,
zsig28 polypeptide agonists or antagonists may be
evaluated in combination with one or more growth factors
to identify synergistic effects on zsig28 activity.
In addition, zsig28 agonists or antagonists may
be therapeutically useful for anti-microbial applications.
To verify the presence of modulators of zsig28
polypeptides with these capabilities, agonists or
antagonists of the present invention are evaluated with
respect to their antimicrobial properties according to
procedures known in the art. See, for example, Barsum et
al., Eur. Respir. J. 8(5): 709-14, 1995; Sandovsky-Losica
et al., J. Med. Vet. Mycol. (England) 28(4): 279-87, 1990;
Mehentee et al., J. Gen. Microbiol (England) 135 (Pt. 8):
2181-8, 1989; Segal and Savage, J. Med. Vet. Mycol. 24:
477-479, 1986 and the like. In addition, zsig28 agonists
or antagonists thereof may be evaluated in. combination
with one or more antimicrobia:l agents to identify
synergistic effects on modulating the zsig28 polypeptide.
Zsig28 polypeptide or fragments thereof can act
as anti-microbial agents to block a pathogenic organism
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for adhering to zsig28 receptor or as a bacterial toxin
sink. Such anti-microbial agents operating via membrane
association or pore forming mechanisms of action directly
attach to the offending microbe. Anti-microbial agents
can also act via an enzymatic mechanism, breaking down
microbial protective substances or the cell wall/membrane
thereof. Anti-microbial agents, capable of inhibiting
microorganism proliferation or action or of disrupting
microorganism integrity by either. mechanism set forth
above, are useful in methods for preventing contamination
in cell culture by microbes susceptible to that anti-
microbial activity. Such techniques involve culturing
cells in the presence of an effective amount of said
zsig28 polypeptide, polypeptide fragment, or an agonist or
antagonist thereof. Alternatively, such anti-microbial
agents could directly attach to an offending bacterial
toxin, described below, and effe~~tively inactivate its
toxicity.
The zsig28 polypeptides of the present invention
may play a role in bacterial pathogenesis in human stomach
and intestinal infections. Zsig28 shares homology with
the Clostridium perfringens enterotoxin (CPE) receptor and
related proteins which bind CPE (Katahira, J. et al., J.
Cell Biol. 136:1239-1247, 1997; and, Katahira, J. et al.,
J. Biol. Chem. 272:26652-26658, 1997). Similarly, zsig28
may bind CPE or other bacterial enterotoxins or exotoxins,
herein collectively described as "bacterial toxins." For
example, such bacterial toxins are produced by bacteria
that cause diseases such as food poisoning, Botulism,
severe diarrhea, inflammation, cramping, or the like
(e. g., staphylococcus, enterotoxigenic E coli,
campylobacter, Clostridium botulinum and the like).
Moreover, as a receptor, the zsig28 may be a site for the
colonization and resultant pathogenic effects of
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pathogenic bacteria in the stomach. For example,
Helicobacter pylori, a causative agent in gastric ulcers,
may exert its effects by binding to zsig28 and inducing
cell lysis through apoptosis or other mechanisms. Such a
5 role for zsig28 can be elucidated by one of skill in the
art. For example, mammalian ce:Lls that are normally
insensitive to a bacterial toxin can be transfected with
zsig28 and tested for susceptibility to the bacterial
toxin (See Katahira, J. et al., supra.; and, Katahira, J.
10 et al., supra.;). When compared in parallel to
untransfected cells, the susceptible zsig28-expressing
cells can be measured by morphological changes associated
with cell death such as (blebbing), lysis, and depletion
of cell number when compared to the untransfected control.
15 Alternatively, assays measuring direct binding of a
radiolabeled or fluorescent labeled bacterial toxin can be
employed to measure direct binding to zsig28-expressing
cells. Similarly, using these assays disclosed above,
whole bacterial cells can be tested for their ability to
20 bind and or lyse cells expressing zsig28 polypeptide.
Thus, zsig28 polypeptides or agonists thereof
may be used as cell culture reagents in in vitro studies
of exogenous microorganism infection, such as bacterial,
viral or fungal infection. Such moieties may also be used
25 in in vivo animal models of infection. Also, the
microorganism-adherence properties of zsig28 polypeptides
or agonists thereof can be studied under a variety of
conditions in binding assays and the like.
As a receptor, the activity of zsig28
30 polypeptide can be measured by a silicon-based biosensor
microphysiometer which measures the extracellular
acidification rate or proton excretion associated with
receptor binding and subsequent physiologic cellular
responses. An exemplary device is the CytosensorT""
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56
Microphysiometer manufactured by Molecular Devices,
Sunnyvale, CA. A variety of cellular responses, such as
cell proliferation, ion transport, energy production,
inflammatory response, regulatory and receptor activation,
and the like, can be measured by this method. See, for
example, McConnell, H.M. et al., Science 257:1906-1912,
1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108,
1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,
1998; Van Liefde, I. Et al., Eur. ~7. Pharmacol. 346:87-95,
1998. The microphysiometer can be used for assaying
eukaryotic, prokaryotic, adherent or non-adherent cells.
By measuring extracellular acidification changes in cell
media over time, the microphysiometer directly measures
cellular responses to various stimuli, including agonists,
ligands, or antagonists of the zsig28 polypeptide.
Preferably, the microphysiometer is used to measure
responses of a zsig28-expressing eukaryotic cell, compared
to a control eukaryotic cell that does not express zsig28
polypeptide. Zsig28-expressing eukaryotic cells comprise
cells into which zsig28 has been transfected, as described
herein, creating a cell that is responsive to zsig28-
modulating stimuli, or are cells naturally expressing
zsig28, such as zsig28-expressing cells derived from
stomach tissue. Differences, measured by an increase or
decrease in extracellular acidification, in the response
of cells expressing zsig28, relative to a control, are a
direct measurement of zsig28-modulated cellular responses.
Moreover, such zsig28-modulated responses can be assayed
under a variety of stimuli. Also, using the
microphysiometer, there is provided a method of
identifying agonists and antagonists of zsig28
polypeptide, comprising providing cells expressing a
zsig28 polypeptide, culturing a first portion of the cells
in the absence of a test compound, culturing a second
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portion of the cells in the presence of a test compound,
and detecting an increase or a decrease in a cellular
response of the second portion of the cells as compared to
the first portion of the cells. Antagonists and agonists,
including the natural ligand for zsig28 polypeptide, can
be rapidly identified using this method.
An in vivo approach for assaying proteins of the
present invention involves viral delivery systems.
Exemplary viruses for this purpose include adenovirus,
herpesvirus, vaccinia virus, ret.roviruses, and adeno-
associated virus (AAV). Adenovirus, a double-stranded DNA
virus, is currently the best studied gene transfer vector
for delivery of heterologous nucleic acid (fox- review, see
T.C. Becker et al., Meth. Cell Biol. 43:161-89, 1994; and
J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53,
1997). The adenovirus system offers several advantages:
(i) adenovirus can accommodate relatively large DNA
inserts; (ii) can be grown to high-titer; (iii) infect a
broad range of mammalian cell types; and (iv) can be used
with many different promoters including ubiquitous, tissue
specific, and regulatable promoters. Also, because
adenoviruses are stable in the bloodstream, they can be
administered by intravenous injection.
Using adenovirus vectors where portions of the
adenovirus genome are deleted, inserts are incorporated
into the viral DNA by direct ligation or by homologous
recombination with a co-transfected plasmid. In an
exemplary system, the essential E1 gene has been deleted
from the viral vector, and the virus will not replicate
unless the El gene is provided by the host cell (the human
293 cell line is exemplary). GVhen intravenously
administered to intact animals, adenovirus primarily
targets the liver. If the adenoviral delivery system has
an E1 gene deletion, the virus cannot replicate in the
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host cells. However, the host's tissue (e. g., liver) will
express and process (and, if a secretory signal sequence
is present, secrete) the heterologous protein. Secreted
proteins will enter the circulation in the highly
vascularized liver, and effects on the infected animal can
be determined.
Moreover, adenoviral vectors containing various
deletions of viral genes can be used in an attempt to
reduce or eliminate immune responses to the vector. Such
adenoviruses are E1 deleted, and in addition contain
deletions of E2A or E4 (Lusky, M. et al., J. Virol.
72:2022-2032, 1998; Raper, S.E. et al., Human Gene Therapy
9:671-679, 1998). In addition, deletion of E2b is
reported to reduce immune responses (Amalfitano, A. et
al., J. Virol. 72:926-933, 1998). Moreover, by deleting
the entire adenovirus genome, very large inserts of
heterologous DNA can be accommodated. Generation of so
called "gutless" adenoviruses where all viral genes are
deleted are particularly advantageous for insertion of
large inserts of heterologous DNA. For review, see Yeh,
P. and Perricaudet, M., FASEB J. 11:615-623, 1997.
The adenovirus system can also be used for
protein production in vitro. By culturing adenovirus-
infected non-293 cells under conditions where the cells
are not rapidly dividing, the cells can produce proteins
for extended periods of time. For instance, BHK cells are
grown to confluence in cell factories, then exposed to the
adenoviral vector encoding the secreted protein of
interest. The cells are then grown under serum-free
conditions, which allows infected cells to survive for
several weeks without significant cell division.
Alternatively, adenovirus vector infected 293 cells can be
grown as adherent cells or in suspension culture at
relatively high cell density t:o produce significant
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59
amounts of protein (See Gamier et al., Cytotechnol.
15:145-55, 1994). With either protocol, an expressed,
secreted heterologous protein can be repeatedly isolated
from the cell culture supernatant, lysate, or membrane
fractions depending on the disposition of the expressed
protein in the cell. Within the infected 293 cell
production protocol, non-secreted proteins may also be
effectively obtained.
As a receptor, the activation of zsig28
polypeptide can be measured by a silicon-based biosensor
microphysiometer which measures the extracellular
acidification rate or proton excretion associated with
receptor binding and subsequent physiologic cellular
responses. An exemplary device is the CytosensorT""
Microphysiometer manufactured by Molecular Devices,
Sunnyvale, CA. A variety of cellular responses, such as
cell proliferation, ion transport, energy production,
inflammatory response, regulatory and receptor activation,
and the like, can be measured by this method. See, for
example, McConnell, H.M. et al., Science 257:1906-1912,
1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108,
1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,
1f98; Van Liefde, I. Et al., Eur. J. Pharmacol. 346:87-95,
1998. The microphysiometer can be used for assaying
adherent or non-adherent eukaryotic or prokaryotic cells.
By measuring extracellular acidification changes in cell
media over time, the microphysiometer directly measures
cellular responses to various stimuli, including agonists,
ligands, or antagonists of the zsig28 polypeptide.
Preferably, the microphysiometer is used to measure
responses of a zsig28-expressing eukaryotic cell, compared
to a control eukaryotic cell that does not express zsig28
polypeptide. ZSIG28-expressing eukaryotic cells comprise
cells into which zsig28 has been transfected, as described
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herein, creating a cell that is responsive to zsig28-
modulating stimuli; or cells naturally expressing zsig28,
such as zsig28-expressing cells derived from stomach
tissue. Differences, measured by a change in
5 extracellular acidification, for example, an increase or
diminution in the response of cells expressing zsig28,
relative to a control, are a direct. measurement of zsig28-
modulated cellular responses. Moreover, such zsig28-
modulated responses can be assayed under a variety of
10 stimuli. Also, using the microphysiometer, there is
provided a method of identifying agonists and antagonists
of zsig28 polypeptide, comprising providing cells
expressing a zsig28 polypeptide, culturing a first portion
of the cells in the absence of a test compound, culturing
15 a second portion of the cells in the presence of a test
compound, and detecting a change, for example, an increase
or diminution, in a cellular response of the second
portion of the cells as compared to the first portion of
the cells. The change in cellular response is shown as a
20 measurable change extracellular acidification rate.
Antagonists and agonists, including the natural ligand for
zsig28 polypeptide, can be rapidly identified using this
method.
A zsig28 polypeptide, or polypeptide fragment
25 thereof, can be expressed as a fusion with an
immunoglobulin heavy chain constant region, typically an
Fc fragment, which contains two constant region domains
and lacks the variable region. Methods for preparing such
fusions are disclosed in U.S. Patents Nos. 5,155,027 and
30 5,567,584. Such fusions are typically secreted as
multimeric molecules wherein the Fc portions are disulfide
bonded to each other and two non-Ig polypeptides are
arrayed in closed proximity to each other. Fusions of
this type can be used to affinity purify ligand, as an in
35 vitro assay tool. For use in assays, the chimeras are
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61
bound to a support via the Fc region and used in an enzyme
linked immunosorbent assay (ELISA} format.
A zsig28 polypeptide can also be used for
purification of ligand that binds it. The zsig28
polypeptide or a ligand-binding polypeptide fragment
thereof can be used. 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.
An assay system that uses a ligand-binding
receptor, such as zsig28, (or an antibody, one member of a
complement/ anti-complement pair) or a binding fragment
thereof, and a commercially available biosensor instrument
(BIAcore, 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
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62
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
245:821-25, 1991}.
Zsig28 polypeptides can also be used to prepare
antibodies that bind to zsig28 epitopes, peptides or
polypeptides. The zsig28 polypeptide or a fragment thereof
serves as an antigen (immunogen) to inoculate an animal
and elicit an immune response. One of skill in the art
would recognize that antigenic, epitope-bearing
polypeptides contain a sequence of at least 6, preferably
at least 9, and more preferably at least 15 to about 30
contiguous amino acid residues of a zsig28 polypeptide
(e. g., SEQ ID N0:2). Polypeptides comprising a larger
portion of a zsig28 polypeptide, i.e., from 30 to 10
residues up to the entire length of the amino acid
sequence are included. Antigens or immunogenic epitopes
can also include attached tags, adjuvants and carriers, as
described herein. Suitable antigens include the zsig28
polypeptide encoded by SEQ ID N0:2 from amino acid number
24 (Ala) to 261 (Val) of SEQ ID N0:2 or a contiguous 9 to
238 AA amino acid fragment thereof. Preferred peptides to
use as antigens are regions 1, 2, 3 and 4 disclosed
herein, and zsig28 hydrophilic peptides such as those
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63
predicted by one of skill in the art from a hydrophobicity
plot (See figure 2). Zsig28 hydraphilic peptides include
peptides comprising amino acid sequences selected from the
group consisting of: (1) amino acid number 245 (Ala) to
amino acid number 250 (Glu) of SEQ ID N0:2; (2) amino acid
number 234 (Asn) to amino acid number 239 (Lys) of SEQ ID
N0:2; (3) amino acid number 202 (Glu) to amino acid number
207 (Lys) of SEQ ID N0:2; (4) amino acid number 254 (Lys)
to amino acid number 259 (Asp) of SEQ ID N0:2; and (5)
amino acid number 110 (Glu) to amino acid number 115 (Ala)
of SEQ ID N0:2. In addition, conserved motifs, and
variable regions between conserved motifs of zsig28 are
suitable antigens. Antibodies from an immune response
generated by inoculation of an animal with these antigens
can be isolated and purified as described herein. Methods
for preparing and isolating polyclonal and monoclonal
antibodies are well known in the art. See, for example,
Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes of Health, John Wiley and Sons, Inc.,
1995; Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor, NY, 1989; and
Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:
Techniques and Applications, CRC Press, Inc., Boca Raton,
FL, 1982 .
As would be evident to one of ordinary skill in
the art, polyclonal antibodies ~~an be generated from
inoculating a variety of warm-blooded animals such as
horses, cows, goats, sheep, dogs, chickens, rabbits, mice,
and rats with a zsig28 polypeptide or a fragment thereof .
The immunogenicity of a zsig28 polypeptide may be
increased through the use of an adjuvant, such as alum
(aluminum hydroxide) or Freund's complete or incomplete
adjuvant. Polypeptides useful for immunization also
include fusion polypeptides, such as fusions of zsig28 or
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64
a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypepti.de immunogen
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.
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,
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 non-
human CDRs onto human framework and constant regions, 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
variable region framework domains to enhance proper
binding characteristics. Through humanizing antibodies,
biological half-life may be increased, and the potential
for adverse immune reactions upon administration to humans
is reduced.
Moreover, human antibodies can be produced in
transgenic, non-human animals that have been engineered to
contain human immunoglobulin genes as disclosed in WIPO
Publication WO 98/24893. It i.s preferred that the
endogenous immunoglobulin genes in these animals be
inactivated or eliminated, such as by homologous
recombination.
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Antibodies are considered to be specifically
binding if: 1) they exhibit a threshold level of binding
activity, and 2) they do not significantly cross-react
with related polypeptide molecules. A threshold level of
5 binding is determined if anti-zsig28 antibodies herein
bind to a zsig28 polypeptide, peptide or epitope with an
affinity at least 10-fold greater than the binding
affinity to control (non-zsig28) polypeptide. It is
preferred that the antibodies exhibit a binding affinity
10 (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 o.f ordinary skill in the
art, for example, by Scatchard araalysis (Scatchard, G.,
15 Ann. NY Acad. Sci. 51: 660-672, 1940 .
Whether anti-zsig28 antibodies do not
significantly cross-react with related polypeptide
molecules is shown, for example, by the antibody detecting
zsig28 polypeptide but not known related polypeptides
20 using a standard Western blot analysis (Ausubel et al.,
ibid.). Examples of known related polypeptides are those
disclosed in the prior art, such as known orthologs, and
paralogs, and similar known members of a protein family,
polypeptide or fragments, and the like. For example, a
25 zsig28-specific antibody would not bind to human OSP-like
protein, claudin l, claudin 2 murine CPE receptor or the
like. Screening can also be done using non-human zsig28,
and zsig28 mutant polypeptides. Moreover, antibodies can
be "screened against" known related polypeptides to
30 isolate a population that specifically binds to the
inventive polypeptides. For example, antibodies raised to
zsig28 are adsorbed to related polypeptides adhered to
insoluble matrix; antibodies specific to zsig28 will flow
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66
through the matrix under the proper buffer conditions.
Screening allows isolation of polyclonal and monoclonal
antibodies non-crossreactive to known closely related
polypeptides (Antibodies: A Laboratory Manual, Harlow and
Lane (eds.), Cold Spring Harbor Laboratory Press, 1988;
Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes of Health, John Wiley and Sons, Inc.,
1995). Screening and isolation of specific antibodies is
well known in the art. See, Fundamental Immunology, Paul
(eds.), Raven Press, 1993; Getzoff et al., Adv. in
Immunol. 43: 1-98, 1988; Monoclonal Antibodies:
Principles and Practice, Goding, J.W. (eds.), Academic
Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2:
67-101, 1984. Specifically binding anti-zsig28 antibodies
can be detected by a number of methods in the art, and
disclosed below.
A variety of assays known to those skilled in
the art can be utilized to detect antibodies which bind to
zsig28 proteins or polypeptides. 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 zsig28 protein or
polypeptide.
Alternative techniques for generating or
selecting antibodies useful herein include in vitro
exposure of lymphocytes to zsig28 protein or peptide, and
selection of antibody display libraries in phage or
similar vectors (for instance, through use of immobilized
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or labeled zsig28 protein or peptide). Genes encoding
polypeptides having potential zsig28 polypeptide binding
domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on
bacteria, such as E. coli. Nucleotide sequences encoding
the polypeptides can be obtained in a number of ways, such
as through random mutagenesis and random polynucleotide
synthesis. These random peptide display libraries can be
used to screen for peptides which interact with a known
target which can be a protein or polypeptide, such as a
ligand or receptor, a biological or synthetic
macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide
display libraries are known in the art (Ladner et al., US
Patent N0. 5,223,409; Ladner et. al., US Patent N0.
4,946,778; Ladner et al., US Patent NO. 5,403,484 and
Ladner et al., US Patent NO. 5,571,698) and random peptide
display libraries and kits for screening such libraries
are available commercially, for instance from Clontech
(Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New
England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB
Biotechnology Inc. (Piscataway, NJ). Random peptide
display libraries can be screened using the zsig28
sequences disclosed herein to identify proteins which bind
to zsig28. These "binding polypeptides" which interact
with zsig28 polypeptides can be used for tagging cells;
for isolating homolog polypeptides by affinity
purification; they can be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like.
These binding polypeptides can also be used in analytical
methods such as for screening expression libraries and
neutralizing activity, e.g., for blocking interaction
between ligand and receptor, or viral binding to a
receptor. The binding polypeptides can also be used for
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diagnostic assays for determining circulating levels of
zsig28 polypeptides; for detecting or quantitating soluble
zsig28 polypeptides as marker of underlying pathology or
disease. These binding polypeptides can also act as
zsig28 "antagonists" to block zsig28 binding and signal
transduction in vitro and in vivo. These anti-zsig28
binding polypeptides would be useful for inhibiting zsig28
activity or protein-binding.
Antibodies to zsig28 may be used for tagging
cells that express zsig28; for isolating zsig28 by
affinity purification; for diagnostic assays for
determining levels of zsig28 polypeptides in normal
tissues; for detecting or quantitating zsig28 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 or as antagonists to block zsig28 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.
Moreover, antibodies to zsig28 or fragments thereof may be
used in vitro to detect denatured zsig28 or fragments
thereof in assays, for example, Western Blots or other
assays known in the art.
Antibodies or polypeptides herein can also be
directly or indirectly conjugated to drugs, toxins,
radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. For
instance, polypeptides or antibodies or binding
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polypeptides which recognize zsig-28 of the present
invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary
molecule (e. g, a zsig28 receptor). More specifically,
anti-zsig28 antibodies, or bioactive fragments or portions
thereof, can be coupled to detectable or cytotoxic
molecules and delivered to a mammal having cells, tissues
or organs that express the zsig28 molecule.
Suitable detectable molecules may be directly or
indirectly attached to polypeptides that bind zsig28
("binding polypeptides"), antibodies, or bioactive
fragments or portions thereof. Suitable detectable
molecules include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like.
Suitable cytotoxic molecules may be directly or indirectly
attached to the polypeptide or antibody, and include
bacterial or plant toxins (for instance, diphtheria toxin,
Pseudomonas exotoxin, ricin, abrin and the like), as well
as therapeutic radionuclides, such as iodine-131, rhenium-
188 or yttrium-90 (either directly attached to the
polypeptide or antibody, or indirectly attached through
means of a chelating moiety, for instance). Binding
polypeptides or antibodies may also be conjugated to
cytotoxic drugs, such as adriamycin. For indirect
attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a
member of a complementary/ anticomplementary pair, where
the other member is bound to the binding polypeptide or
antibody portion. For these purposes, biotin/streptavidin
is an exemplary complementary/ anticomplementary pair.
In another embodiment, binding polypeptide-toxin
fusion proteins or antibody-toxin fusion proteins can be
used for targeted cell or tissue inhibition or ablation
(for instance, to treat cancer cells or tissues).
Alternatively, if the binding polypeptide has multiple
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functional domains (i.e., an activation domain or a ligand
binding domain, plus a targeting domain), a fusion protein
including only the targeting domain may be suitable for
directing a detectable molecule, a cytotoxic molecule or a
5 complementary molecule to a cell or tissue type of
interest. In instances where the domain only fusion
protein includes a complementary molecule, the anti-
complementary molecule can be conjugated to a detectable
or cytotoxic molecule. Such domain-complementary molecule
10 fusion proteins thus represent a generic targeting vehicle
for cell/tissue-specific delivery of generic anti-
complementary-detectable/ cytotoxic molecule conjugates.
In another embodiment, zsig28 binding
polypeptide-cytokine or antibody-cytokine fusion proteins
15 can be used for enhancing in vivo killing of target
tissues (for example, blood, stomach, colon, and bone
marrow cancers), if the binding polypeptide-cytokine or
anti-zsig28 antibody targets the hyperproliferative blood
or bone marrow cell (See, generally, Hornick et al., Blood
20 89:4437-47, 1997). For example, Hornick et al. described
fusion proteins that enable targeting of a cytokine to a
desired site of action, thereby providing an elevated
local concentration of cytokine. Suitable anti-zsig28
antibodies target an undesirable cell or tissue (i.e., a
25 tumor or a leukemia), and the fused cytokine mediated
improved target cell lysis by effector cells. Suitable
cytokines for this purpose include interleukin 2 and
granulocyte-macrophage colony-stimulating factor (GM-CSF),
for instance.
30 Alternatively, zsig28 binding polypeptide or
antibody fusion proteins described herein can be used for
enhancing in vivo killing of target tissues by directly
stimulating a zsig28-modulated apoptotic pathway,
resulting in cell death of hypoproliferative cells
35 expressing zsig28.
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The bioactive binding polypeptide or antibody
conjugates described herein can be delivered orally,
intravenously, intraarterially or intraductally, or may be
introduced locally at the intended site of action.
Molecules of the present invention. can be used
to identify and isolate l.igands involved in
gastrointestinal function, peristalsis, mucous secretion
and the like. For example, proteins and peptides of the
present invention can be immobilized on a column and
tissue preparations run over the column (Immobilized
Affinity Lictand 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, 721-37) or
photoaffinity labeled (Brunner et al., Ann. Rev. Biochem.
62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.
33:1167-80, 1984) and specific ligand proteins can be
identified.
The polypeptides, nucleic acid and/or antibodies
of the present invention can 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; reflux 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
gastroesophageal reflux disease. Additional uses include,
gastric emptying for radiological studies, stimulating
gallbladder contraction and antrectomy.
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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.
The polypeptides, nucleic acid, antagonists,
agonists and/or antibodies of the present invention can be
used in treatment, diagnosis or prevention of disorders
associated with gastric ulcers, bacterial diseases,
gastric emptying and function, mucosal repair and
secretion, stomach cancer, nausea (e. g., induced by cancer
therapy and opiate pain control), stomach acid secretion,
gastritis, trauma, diverticulitis, gastric mucositis or
appetite. The molecules of the present invention can be
used to isolate modulators of zsig28, including the
natural ligand, used in gene the=rapy, or to treat or
prevent development of pathological conditions in such
diverse tissue as stomach and lung.
In addition, zsig28 polypeptides or agonists or
antagonists thereof are expected to be useful in the
modulation of mucous production, composition or integrity
or in a mucous clearing role. Such modulation may be
useful in altering mucous composition or integrity for in
vitro study thereof, such as reducing integrity of mucous
to evaluate the implication thereof on bacterial-mucous
interaction. In addition, such modulation may be useful
in the treatment of disease states characterized by
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inappropriate mucous production, composition or integrity.
For example, cystic fibrosis is associated with
dehydration of the mucous, which results in mucous
thickening (reduction in viscosity). Other conditions,
such as chronic obstructive pulmonary disease, asthma, and
the like, are associated with chronic mucous
hypersecretion. See, for example, Prescott et al., Ugeskr
La- eger 158(45): 6456-60, 1996; Gordon, Ear Nose Throat J.
75(2): 97-101, 1996; and Jeffery, Am. J. Respir. Crit.
Care Med. 150(5 Pt 2): S6-13, 1994. Also, chronic
obstructive pulmonary disease and sinonasal inflammatory
disease are associated with changes in rhealogical
properties or thickening of mucous. See, for example,
Agliati, J. Int. Med. Res. 24(3): 302-10, 1996 and Wippold
et al., Allergy Proc. 16(4): 165-9, 1995. In addition,
mucous structural integrity is adversely impacted in
inflammatory bowel disease, possibly via increased
proteolysis. See, for example, Playford et al., Amer. J.
Pathol. 146(2): 310-6, 1995. Certain forms of chronic
obstructive pulmonary disease are associated with
increased acidic mucous. See, for example, Jeffery,
supra.. Mucous clearing may be useful in a number of
these conditions as well.
The zsig28 polypeptide is expressed in the
stomach. As a receptor, zsig28 can play important roles
in the maintenance of normal gastric epithelium and
function. Thus, zsig28 polypeptide pharmaceutical
compositions, agonists and antagonists of the present
invention may also be useful in prevention or treatment of
gastric mucositis. Mucositis is manifested by the damage
and loss of integrity of the oral and gastric epithelium.
Such damage often provides a microbial port of entry
leading to sepsis. Mucositis is often induced by
chemotherapy and radiation therapy, and is often a dose-
limiting side effect as well as a cause of mortality in
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cancer patients undergoing such treatment. The zsig28
polypeptides and agonists and antagonists of the present
invention may provide protection against gastric
mucositis, analogous to some growth factors and cytokines,
for example, interleukin-11 (Orazi, A. et al., Lab.
Invest. 75:33-42, 1996). The effect of zsig28, agonists
and antagonists in prevention or treatment of gastric
mucositis can be measured in in vivo animal models, for
example, the Syrian hamster model or in marine models
using methods described in the art (Sonis, S.T. et al.,
Oral Surg. Oral Med. Oral Pathol. 69:437-443, 1990;
Farrell, C.L. et al., Cancer Res. 58:933-939, 1998; Orazi,
A. et al., supra.). Moreover, zsig28 transgenic or
knockout mice may provide an addit-:ional in vivo model for
gastric mucositis.
To verify these capabilities in zsig28
polypeptides of the present invention, agonists, or
antagonists, the zsig28 polypeptides, agonists or
antagonists are evaluated fo.r mucosal integrity
maintenance activity according to procedures known in the
art. See, for example, Zahm et al., Eur. Respir. J. 8:
381-6, 1995, which describes methods for measuring
viscoelastic properties and surface properties of mucous
as well as for evaluating mucous transport by cough and by
ciliary activity. Other assays for evaluating the
properties of mucous are known to those of ordinary skill
in the art. Such assays include those for determining
mucin content, water content, carbohydrate content,
intrinsic buffering capacity, acidity, barrier properties,
ability to absorb water and the like.
Moreover, detection of zsig28 polypeptides in
the serum, mucous or tissue biopsy of a patient undergoing
evaluation for or disorders characterized by inappropriate
mucous deposition, composition o:r properties, such as
cystic fibrosis, asthma, bronchitis, inflammatory bowel
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disease, Crohn's disease, chronic obstructive pulmonary
disease or the like, can be employed in a diagnostic
application of the present invention. Such zsig28
polypeptides can be detected using immunoassay techniques
5 and antibodies capable of recognizing a zsig28 polypeptide
epitope. As an illustration, the present invention
contemplates methods for detecting zsig28 polypeptide
comprising:
exposing a sample possibly containing zsig28
10 polypeptide to an antibody attached to a solid support,
wherein said antibody binds to an epitope of a zsig28
polypeptide;
washing said immobilized antibody-polypeptide to
remove unbound contaminants;
15 exposing the immobilized antibody-polypeptide to
a second antibody directed to a second epitope of a zsig28
polypeptide, wherein the second antibody is associated
with a detectable label; and
detecting the detectable label. An increase or
20 decrease in the concentrations of zsig28 polypeptide (in
comparison to normal concentrations thereof) in the test
sample appears to be indicative of dysfunction. One of
skill in the art would appreciate that other assays known
in the' art can be used to detect zsig28 in a test sample,
25 for example, a simple solution assay with labeled anti-
zsig28 antibody.
In addition, pharmaceutical compositions
containing such mucosa-modulating agents may be employed
in the treatment of disorders associated with alterations
30 in mucosal production, composition or integrity, such as
those described above. Such patients will be given an
effective amount of zsig28 polypeptide or agonist or
antagonist thereof having mucosal-modulating activity to
achieve a therapeutic benefit, generally manifested in a
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change in mucosal production, composition or integrity in
the direction of the normal physiological state thereof.
Also, the zsig28 polypeptides of the present
invention are found in high abundance in digestive
tissues, such as stomach. Thus, expression of zsig28
polypeptides may serve as a marker for digestive function
or to promote digestive organ proliferation or
differentiation. Also, zsig28 polypeptides or agonists or
antagonists thereof may be useful in modulating the
lubrication or barrier properties of digestive organ
mucosa.
Zsig28 polypeptides of the present invention or
agonists or antagonists thereof may be used as anti-
microbial agents to protect against: pathological action of
microorganisms. Such anti-bacterial agents are preferably
active on mucosa-associated microorganisms, such as C.
albicans, pneumonus, hemophilus, Helicobacter pylori, and
the like. An example of a microbial-associated condition
with mucous involvement in humans is the diminution of the
defensive properties of the gastroduodenal mucosa by H.
pylori, potentially resulting in ulcer formation. See,
for example, Beligotskii et al., Klin. Khir. 8: 3-6, 1994.
These anti-microbial protective agents may be
directly acting or indirectly acting. Such agents
operating via membrane association or pore forming
mechanisms of action directly attach to the offending
microbe. Anti-microbial agents can also act via an
enzymatic mechanism, breaking down microbial protective
substances or the cell wall/membrane thereof. Anti-
microbial agents, capable of inhibiting microorganism
proliferation or action or of disrupting microorganism
integrity by either mechanism set forth above, are useful
in methods for preventing contamination in cell culture by
microbes susceptible to that anti-microbial activity.
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Such techniques involve culturing cells in the presence of
an effective amount of said zsig28 secreted region l, 2,
3, or 9 polypeptide fragment, o:r a zsig28 agonist or
antagonist. Assays to evaluate the efficacy of zsig28
polypeptides, agonists or antagonists thereof as anti-
microbial agents are known in the art.
Moreover, detection of zsig28 polypeptides in
the serum, mucous or tissue biopsy of a patient undergoing
evaluation for microbial disorders, particularly those
associated with mucosa, can be employed in a diagnostic
application of the present invention. Such zsig28
polypeptides can be detected using immunoassay techniques
and antibodies capable of recognizing a zsig28 polypeptide
epitope, as described herein. Altered levels of zsig28
polypeptides in a test sample, such as serum sweat,
saliva, biopsy, and the like, can be monitored as an
indication of digestive function, gastric ulcer or of
cancer or disease, when compared against a normal control.
In addition, pharmaceutical compositions
containing such anti-microbial agents may be employed in
the treatment of microbial disorders, particularly those
associated with mucosa. Such patients will be given an
effective amount of zsig28 soluble polypeptide fragment or
agonist or antagonist thereof having anti-microbial
activity to achieve a therapeutic benefit, generally
manifested in a decrease in proliferation or function of
the pathogenic microbe. Other conditions which may be
addressed in accordance with the present invention are
eye, nasal, oral and rectal conditions involving the
mucosa and/or pathological microbial agents, chemotherapy
side effects impacting the mucosa, AIDS complications
relating to mucosa or the like. The anti-microbial
activity of zsig28 soluble polypeptide fragment, agonists
or antagonists may be determined using known assays
therefore. See, for example, Barsum et al., Eur. Respir.
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J. 8(5): 709-14, 1995; Sandovsky-Losica et al., J. Med.
Vet. Mycol (England) 28(4): 279-87, 1990; Mehentee et al.,
J. Gen. Microbiol (England) 135 (Pt. 8): 2181-8, 1989;
Sepal and Savage, Journal of Medical and Veterinary
Mycology 24: 477-479, 1986, and the like.
Also, zsig28 polypepti.des of the present
invention may constitute a component of a known tissue
glue, imparting additional adhesive and/or anti-microbial
properties thereto. In such applications, purified zsig28
polypeptide would be used in combination with collagen or
a form of gelatin, muscle adhesion protein, fibrinogen,
thrombin, Factor XIII or the like. The different types of
tissue glues as well as the composition thereof are known
in the art.
Zsig28 can also be used to identify modulators
(e.g, antagonists) of its activity. Test compounds are
added to the assays disclosed herein to identify compounds
that inhibit the activity of zsig28. In addition to those
assays disclosed herein, samples can be tested for
inhibition of zsig28 activity within a variety of assays
designed to measure zsig28 binding, oligomerization, or
the stimulation/inhibition of zsig28-dependent cellular
responses. For example, zsig28-expressing cell lines can
be transfected with a reporter gene construct that is
responsive to a zsig28-stimulated cellular pathway.
Reporter gene constructs of this type are known in the
art, and will generally comprise a zsig28-DNA response
element operably linked to a gene encoding an assay
detectable protein, such as luciferase. DNA response
elements can include, but are not limited to, cyclic AMP
response elements (CRE), hormone response elements (HRE)
insulin response element (IRE) (Nasrin et al., Proc. Natl.
Acad. Sci. USA 87:5273-7, 1990) and serum response
elements (SRE) (Shaw et al. Ce:Ll 56: 563-72, 1989).
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Cyclic AMP response elements are reviewed in Roestler et
al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener,
Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone response
elements are reviewed in Beato, Cell 56:335-44; 1989.
Candidate compounds, solutions, mixtures or extracts are
tested for the ability to inhibit the activity of zsig28
on the target cells as evidenced by a decrease in zsig28
stimulation of reporter gene expression. Rssays of this
type will detect compounds that directly block zsig28
binding to cell-surface receptors, e.g., through
dimerization, as well as compounds that block processes in
the cellular pathway subsequent to such binding. As such,
there is provided a method of identifying antagonists of
zsig28 polypeptide, comprising providing cells responsive
to a zsig28 polypeptide, culturing a first portion of the
cells in the presence of zsig28 polypeptide, culturing a
second portion of the cells in the presence of the zsig28
polypeptide and a test compound, and detecting a decrease
in a cellular response of the second portion of the cells
as compared to the first portion of the cells. Moreover,
compounds or other samples can be tested for direct
blocking of zsig28, or blocking of zsig28 binding to other
cell surface molecules, using zsig28 tagged with a
detectable label (e. g., l2sl, biotin, horseradish
peroxidase, FITC, and the like). Within assays of this
type, the ability of a test sample to inhibit labeled
zsig28 binding to another protein can be indicative of
inhibitory activity, which can be confirmed through
secondary assays. Antagonists are therefore useful to
inhibit or diminish zsig28 polypeptide function.
Alternatively, there is provided a method of identifying
zsig28 polypeptide agonists, comprising providing cells
expressing a zsig28 polypeptide as disclosed above,
culturing the cells in the presence of a test compound and
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comparing the cellular response with the cell cultured in
the presence of the zsig28 polypeptide, and selecting the
test compounds for which the cellular response is of the
same type. Agonists are therefore useful to mimic a
5 zsig28 ligand and/or to augment the function of zsig28
polypeptides.
In view of the tissue distribution observed for
zsig28, agonists (including the natural ligand/ substrate/
cofactor/ etc.) and antagonists have enormous potential in
10 both in vitro and in vivo applications. Compounds
identified as zsig28 agonists are useful for promoting
apoptosis in cells over-expressing sig58, in vitro and in
vivo, such as tumor cells. Compounds identified as zsig28
agonists are also useful for and stimulating cell growth
15 or differentiation, of various cell types. For example,
zsig28 agonist compounds are useful as components of
defined cell culture media, and may be used alone or in
combination with other cytokines and hormones to replace
serum that is commonly used in cell culture. Also, zsig28
20 polypeptide can be hydrolyzed to provide a source of amino
acids to cultured cells. Moreover, the zsig28
polypeptides and zsig28 agonist polypeptides are useful as
a research reagent, such as for the expansion of stomach-
derived, or intestinal cells. Zsig28 agonists can be
25 added to tissue culture media for cell types expressing
zsig28 polypeptide.
Inhibitors of zsig28 activity (zsig28
antagonists) include anti-zsig28 antibodies and
polypeptide binding fragments, as well as other peptidic
30 and non-peptidic agents (including ribozymes). Zsig28 can
also be used to identify inhibitors (antagonists) of its
activity. Test compounds are added to the assays
disclosed herein to identify compounds that inhibit the
activity of zsig28. In addition to those assays disclosed
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herein, samples can be tested for inhibition of zsig28
activity within a variety of assays designed to measure
receptor binding or the stimulation/inhibition of zsig28-
dependent cellular responses. For example, zsig28-
responsive cell lines can be transfected with a reporter
gene construct that is responsive to a zsig28-stimulated
cellular pathway. Reporter gene constructs of this type
are known in the art, and will. generally comprise a
zsig28-DNA response element operably linked to a gene
encoding an assayable protein, such as luciferase. DNA
response elements can include, but are not limited to,
cyclic AMP response elements (CRE), hormone response
elements (HRE) insulin response e_Lement (IRE) (Nasrin et
al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum
response elements (SRE) (Shaw et al. Cell 56: 563-72,
1989). Cyclic AMP response elements are reviewed in
Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and
Habener, Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone
response elements are reviewed in Beato, Cell 56:335-44;
1989. Candidate compounds, solutions, mixtures or
extracts are tested for the ability to inhibit the
activity of zsig28 polypeptide on the target cells as
evidenced by a decrease in zsig28 stimulation of reporter
gene expression. Assays of this type will detect
compounds that directly block zsig28 ligands from binding
to zsig28 polypeptide receptors, or receptor
multimerization, as well as compounds that block processes
in the cellular pathway subsequent to receptor-ligand
binding.
Molecules of the present invention can be used
to identify and isolate zsig28 receptors involved or
present in cancer metastases. Thus, the zsig28
polypeptide can serve as a diagnostic for cancer
metastasis. For example, proteins and peptides of the
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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). (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 zsig28 cell-surface proteins can
be identified. Moreover, using methods known in the art,
antibodies to zsig28 can also be radiolabeled, fluorescent
or chemically labeled and used in histological assays to
detect elevated zsig28 present in biopsies. Polypeptides
of the present invention are useful for measuring changes
in levels of expression of zsig28 polypeptides. Because
zsig28 expression is restricted to specific tissues (i.e.,
stomach and lung), changes in expression levels could be
used to monitor metabolism within these tissues. For
example, increases in expression and/or transcription of
zsig28 polypeptides and polynucleotides, may be predictive
for increased cell proliferation of tumor cells.
Furthermore, expression of zsig28 in tissue not normally
expressing zsig28, may be indicative of metastasis of
tumor cells.
Zsig28 may be demonstrated to be expressed
differentially in certain epithelial tissues and
carcinomas, particularly in stomach, colon, esophagus, or
intestine. Differential expression is the transient
expression, or lack thereof, of specific genes, proteins
or other phenotypic properties (known as differentiation
markers) that occur during the progress of maturation in a
cell or tissue. A set of differentiation markers is
defined as one or more phenotypic properties that can be
identified and are specific to a particular cell type.
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Thus, pluripotent stem cells that can regenerate without
commitment to a lineage express a set of differentiation
markers that are lost when commitment to a cell lineage is
made. Precursor cells express a set of differentiation
markers that may or may not continue to be expressed as
the cells progress down the cell lineage pathway toward
maturation. Differentiation markers that are expressed
exclusively by mature cells are usually functional
properties such as cell products, enzymes to produce cell
products and receptors.
Zsig28 expression can be used as a
differentiation marker in normal and tumor tissues to
determine the stage of the tumor or maturity of a cell.
Zsig28 will be particularly valuable as a marker for
epithelial cells and tumor of epithelial origin, and more
particularly epithelial cells and epithelial-derived
tumors from stomach tissues.
A set of differentiation markers is defined as
one or more phenotypic properties that can be identified
and are specific to a particular cell type.
Differentiation markers are transiently exhibited at
various stages of cell lineage. Pluripotent stem cells
that can regenerate without commitment to a lineage
express a set of differentiation markers that are lost
when commitment to a cell lineage is made. Precursor
cells express a set of differentiation markers that may or
may not continue to be expressed as the cells progress
down the cell lineage pathway toward maturation.
Differentiation markers that are expressed exclusively by
mature cells are usually functional properties such as
cell products, enzymes to produce cell products and
receptors. The activity of molecules of the present
invention can be measured using a variety of assays that
measure proliferation and/or differentiation of specific
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cell types, chemotaxis, adhesion, changes in ion channel
influx, regulation of second messenger levels and
neurotransmitter release. Such assays are well known in
the art and described herein.
Additional methods using probes or primers
derived, for example, from the nucleotide sequences
disclosed herein can also be used to detect zsig28
expression in a patient sample, such as a tumor biopsy,
stomach, lung, blood, saliva, tissue sample, or the like.
For example, probes can be hybridized to tumor tissues and
the hybridized complex detected by in situ hybridization.
Zsig28 sequences can also be detected by PCR amplification
using cDNA generated by reverse translation of sample mRNA
as a template (PCR Primer A Laboratory Manual, Dieffenbach
and Dveksler, eds., Cold Spring Harbor Press, 1995). When
compared with a normal control., both increases or
decreases of zsig28 expression in a patient sample,
relative to that of a control, can be monitored and used
as an indicator or diagnostic for disease.
Polynucleotides encoding zsig28 polypeptides are
useful within gene therapy applications where it is
desired to increase or inhibit zsig28 activity. If a
mammal has a mutated or absent zsig28 gene, the zsig28
gene can be introduced into the cells of the mammal.
Moreover, using gene therapy applications zsig28 can also
be used directly as a chemotherapeutic agent. For
example, using methods disclosed herein, zsig28 can be
directly introduced into cancer cells to trigger apoptosis
and cell death. In one embodiment, a gene encoding a
zsig28 polypeptide is introduced in vivo in a viral
vector. Such vectors include an attenuated or defective
DNA virus, such as, but not limited to, herpes simplex
virus (HSV), papillomavirus, Epstein Barr virus (EBV),
adenovirus, adeno-associated virus (AAV), and the like.
Defective viruses, which entirely or almost entirely lack
viral genes, are preferred. A defective virus is not
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infective after introduction into a cell. Use of
defective viral vectors allows for administration to cells
in a specific, localized area, without concern that the
vector can infect other cells. Examples of particular
5 vectors include, but are not limited to, a defective
herpes simplex virus 1 (HSV1) vector (Kaplitt et al.,
Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated
adenovirus vector, such as the vector described by
Stratford-Perricaudet et al., J. C:lin. Invest. 90:626-30,
10 1992; and a defective adeno-associated virus vector
(Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-8, 1989).
In another embodiment, a zsig28 gene can be
introduced in a retroviral vector, e.g., as described in
15 Anderson et al., U.S. Patent No. 5,399,346; Mann et al.
Cell 33:153, 1983; Temin et al., U.S. Patent No.
4,650,764; Temin et al., U.S. Patent No. 4,980,289;
Markowitz et al., J. Virol. 62:1120, 1988; Temin et al.,
U.S. Patent No. 5,124,263; International Patent
20 Publication No. WO 95/07358, published March 16, 1995 by
Dougherty et al.; and Kuo et al., Blood 82:845, 1993.
Alternatively, the vector can be introduced by lipofection
in vivo using liposomes. Synthetic cationic lipids can be
used to prepare liposomes for in vivo transfection of a
25 gene encoding a marker (Felgner et al., Proc. Natl. Acad.
Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad.
Sci. USA 85:8027-31, 1988). The use of lipofection to
introduce exogenous genes into specific organs in vivo has
certain practical advantages. Molecular targeting of
30 liposomes to specific cells represents one area of
benefit. More particularly, directing transfection to
particular cells represents one area of benefit. For
instance, directing transfection to particular cell types
would be particularly advantageous in a tissue with
35 cellular heterogeneity, such as the pancreas, liver,
kidney, and brain. Lipids may be chemically coupled to
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other molecules for the purpose of targeting. Targeted
peptides (e. g., hormones or neurotransmitters), proteins
such as antibodies, or non-peptide molecules can be
coupled to liposomes chemically.
It is possible to remove the target cells from
the body; to introduce the vector as a naked DNA plasmid;
and then to re-implant the transformed cells into the
body. Naked DNA vectors for gene therapy can be
introduced into the desired host cells by methods known in
the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran,
calcium phosphate precipitation, use of a gene gun or use
of a DNA vector transporter. See, e.g., Wu et al., J.
Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem.
263:14621-4, 1988.
Antisense methodology can be used to inhibit
zsig28 gene transcription, such as to inhibit cell
proliferation in vivo. Polynucleotides that are
complementary to a segment of a zsig28-encoding
polynucleotide (e.g., a polynucleotide as set froth in SEQ
ID N0:1) are designed to bind to zsig28-encoding mRNA and
to inhibit translation of such mRNA. Such antisense
polynucleotides are used to inhibit expression of zsig28
polypeptide-encoding genes in cell culture or in a
subject.
In addition, as a cell surface molecule, zsig28
polypeptide can be used as a target to introduce gene
therapy into a cell. This application would be
particularly appropriate for introducing therapeutic genes
into cells in which zsig28 is normally expressed, such as
stomach tissue and lung, or cancer cells which express
zsig28 polypeptide. For example, viral gene therapy, such
as described above, can be targeted to specific cell types
in which express a cellular receptor, such as zsig28
polypeptide, rather than the viral receptor. Antibodies,
or other molecules that recognize zsig28 molecules on the
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target cell's surface can be used to direct the virus to
infect and administer gene therapeutic material to that
target cell. See, Woo, S.L.C, Nature Biotech. 14:1538,
1996; Wickham, T.J. et al, Nature Biotech. 14:1570-1573,
1996; Douglas, J.T et al., Nature Biotech. 14:1574-1578,
1996; Rihova, B., Crit. Rev. Biotechnol. 17:149-169, 1997;
and Vile, R.G. et al., Mol. Med. Today 4:84-92, 1998. For
example, a bispecific antibody containing a virus-
neutralizing Fab fragment coupled to a zsig28-specific
antibody can be used to direct. the virus to cells
expressing the zsig28 receptor and allow efficient entry
of the virus containing a genetic element into the cells.
See, for example, Wickham, T.J., et al., J. Virol.
71:7663-7669, 1997; and Wickham, T.J., et al., J. Virol.
70:6831-6838, 1996.
The present invention also provides reagents
which will find use in diagnostic applications. For
example, the zsig28 gene, a probe comprising zsig28 DNA or
RNA or a subsequence thereof can be used to determine if
the zsig28 gene is present on chromosome 3 or if a
mutation has occurred. Zsig28 is located at the 3q22.1-
3q22.2 region of chromosome 3 (See, Example 3).
Detectable chromosomal aberrations at the zsig28 gene
locus include, but are not limited to, aneuploidy, gene
copy number changes, insertions, deletions, restriction
site changes and rearrangements. Such aberrations can be
detected using polynucleotides of the present invention by
employing molecular genetic techniques, such as
restriction fragment length polymorphism (RFLP) analysis,
short tandem repeat (STR) analysis employing PCR
techniques, and other genetic linkage analysis techniques
known in the art (Sambrook et al., ibid.; Ausubel et. al.,
ibid.; Marian, Chest 108:255-65, 1995).
The precise knowledge of a gene's position can
be useful for a number of purposes, including: 1)
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determining if a sequence is part of an existing contig
and obtaining additional surrounding genetic sequences in
various forms, such as YACs, BACs or cDNA clones; 2)
providing a possible candidate gene for an inheritable
disease which shows linkage to the same chromosomal
region; and 3) cross-referencing model organisms, such as
mouse, which may aid in determining what function a
particular gene might have.
The zsig28 gene is located at the 3q22.1-3q22.2
region of chromosome 3. Several genes of known function
map to this region. For example, angiotensin receptor 1
is mapped to 3q21-q25 and is related to hypertension in
humans since it controls blood pressure. In addition,
zsig28 polynucleotide probes can be used to detect
abnormalities or genotypes associated with tumor
associated antigen L6, which maps to 3q21-q25, and is
highly expressed in lung breast and colon cancers (Marken
et al, Proc. Nat. Acad. Sci. 89:3503-3507, 1992).
Moreover, amongst other genetic loci, those for
alkaptoneuria (3q21-q23), Moebius 2 syndrome (3q21-q22),
open angle glaucoma (3q21-q24), calcium sensing receptor
(3q21-q24) all manifest themselves in human disease states
as well as map to this region of the human genome. See
the Online Mendellian Inheritance of Man (OMIM) gene map,
and references therein, for this region of chromosome 3 on
a publicly available WWW server
(http://www3.ncbi.nlm.nih.gov/htbin-
post/Omim/getmap?chromosome=3q22.1). All of these serve
as possible candidate genes for an inheritable disease
which show linkage to the same chromosomal region as the
zsig28 gene.
Moreover, a gene related to zsig28, the human
transmembrane protein deleted in velo-cardio-facial
syndrome (TMVCF) (Sirotkin H. et al., Genomics 42:245-251,
1997) is a marker for a human autosomal dominant disorder
velo-cardio-facial syndrome (VCFS) characterized amongst
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other symptoms by facial deformation, mental retardation,
and heart defects and is deleted in 80-85% of patients
with this syndrome. Moreover, mutations in another
zsig28-related gene, peripheral myelin protein-22 (PMP-
22), are present in the human Charcot-Marie-Tooth
neuropathy (CMT) with a phenotypically similar neuropathy
in a PMP-22 transgenic murine mouse model (Erdem, S. et
al., J Neuropathol. Exp. Neurol. 57:635-642, 1998;
Fabbretti, E. et al., Genes Dev. 15:1846-1856, 1995;
Magyar, J.P. et al., J Neurosci. 16:5351-5360, 1996;
Adlkofer, K. et al., Nat. Genet. 11:274-280, 1995).
Similarly, defects in the zsig28 locus itself may result
in a heritable human disease state. Molecules of the
present invention, such as the polypeptides, antagonists,
agonists, polynucleotides and antibodies of the present
invention would aid in the detection, diagnosis
prevention, and treatment associated with a zsig28 genetic
defect .
Mice engineered to express the zsig28 gene,
referred to as "transgenic mice, " and mice that exhibit a
complete absence of zsig28 gene function, referred to as
"knockout mice," may also be generated (Snouwaert et al.,
Science 257:1083, 1992; Lowell et al., Nature 366:740-42,
1993; Capecchi, M.R., Science 244: 1288-1292, 1989;
Palmiter, R.D. et al. Annu Rev Genet. 20: 465-499, 1986) .
For example, transgenic mice that over-express zsig28,
either ubiquitously or under a tissue-specific or tissue-
restricted promoter can be used to ask whether over-
expression causes a phenotype. For example, over-
expression of a wild-type zsig28 polypeptide, polypeptide
fragment or a mutant thereof may alter normal cellular
processes, resulting in a phenotype that identifies a
tissue in which zsig28 expression is functionally relevant
and may indicate a therapeutic target for the zsig28,its
agonists or antagonists. For example, a preferred
transgenic mouse to engineer is one that over-expresses
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the zsig28 mature polypeptide (approximately amino acids
24 (Ala) to 261 (Val) of SEQ ID N0:2). Moreover, such
over-expression may result in a phenotype that shows
similarity with human diseases. Similarly, knockout
5 zsig28 mice can be used to determine where zsig28 is
absolutely required in vivo. The phenotype of knockout
mice is predictive of the in vivo effects of that a zsig28
antagonist, such as those described herein, may have. The
human zsig28 cDNA can be used to isolate murine zsig28
10 mRNA, cDNA and genomic DNA, which are subsequently used to
generate knockout mice. These mice may be employed to
study the zsig28 gene and the protein encoded thereby in
an in vivo system, and can be used as in viva models for
corresponding human diseases. Moreover, transgenic mice
15 expression of zsig28 antisense polynucleotides or
ribozymes directed against zsig28, described herein, can
be used analogously to transgenic mice described above.
The invention is further illustrated by the
20 following non-limiting examples.
EXAMPLES
Example 1
25 Identification of zsig28
A. Using an EST Sequence to Obtain Full-length zsig28
Scanning of a translated lung library DNA
database using a signal trap as a query resulted in
identification of an expressed sequence tag (EST) sequence
30 found to be homologous to a human secretory signal
sequence.
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
35 sequenced using the following primers: ZC497(SEQ ID
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NO:11), ZC12501 (SEQ ID N0:12), ZC 12502 (SEQ ID N0:13),
and ZC 976 (SEQ ID N0:14). The clone appeared to be full
length.
Example 2
Tissue Distribution
Northern blot analysis was performed using Human
Multiple Tissue NorthernT"" Blots (MTN I, MTN II, and MTN
III) (Clontech). An insert from the full length clone,
described in Example l, was excised using EcoRI and NotI
(Boehringer) and gel purified using a commercially
available kit (QiaexIITM; Qiagen) and then radioactively
labeled with 32P-dCTP using RediprimeT"' (Amersham), a random
prime labeling system, according to the manufacturer's
specifications. The probe was then purified using a Nuc-
TrapTM column (Stratagene) according to the manufacturer's
instructions. ExpressHybTM (Clontech) solution was used
for prehybridization and as a hybridizing solution for the
Northern blots. Hybridization took place overnight at 92°C
using 3 x 106 cpm/ml of labeled probe. The blots were then
washed in 2X SSC/lo SDS at room temperature, followed by a
wash in O.1X SSC/O.lo SDS at 65°C. An approximately 4 kb
transcript was strongly detected in stomach and weakly
detected in lung. No signals were apparent in other
tissues represented on the blots.
Dot Blots were also performed using Human RNA
Master BlotsTM (Clontech). The methods and conditions for
the Dot Blots are the same as for the Multiple Tissue
Blots described above. Strong signal intensity was
present in stomach, with detectable but low expression in
adult and fetal lung.
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Example 3
PCR-Based Chromosomal Mapping of the zsig28 Gene
Zsig28 was mapped to c:hromosome 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 r.ecipient). A publicly
available WWW server (http://www-genome.wi.mit.edu/cgi-
bin/contig/rhmapper.pl) 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.
For the mapping of Zsig28 with the "GeneBridge 4
RH Panel", 20 ul reactions were set up i.n 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 ul lOX KlenTaq PCR
reaction buffer (Clontech Laboratories, Inc., Palo Alto,
CA), 1.6 ul dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster
City, CA), 1 ul sense primer, ZC19,410 (SEQ ID N0:15), 1
ul antisense primer, ZC19,411 (SEQ ID N0:16), 2 ul
"RediLoad" (Research Genetics), 0.4 ul 50X Advantage
KlenTaqT"" Polymerase Mix (Clontech), 25 ng of DNA from an
individual hybrid clone or contro7_ and ddHZO for a total
volume of 20 ul. 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 60°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
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electrophoresis on a 2% agarose gel (Life Technologies,
Gaithersburg, MD).
The results showed that Zsig28 maps 4.40 cR 3000
from the framework marker D3S1576 on the WICGR chromosome
3 radiation hybrid map. Proximal and distal framework
markers were D3S1576 and WI-3522, respectively. The use of
surrounding markers positions Zsig28 in the 3q22.1-3q22.2
region on the integrated LDB chromosome 3 map (The Genetic
Location Database, University of Southhampton, WWW server:
http://cedar.genetics. soton.ac.uk/public html/).
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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1
SEQUENCE LISTING
<110> ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle. Washington 98102
United States of America
<120> Stomach polypeptide zsig28
<130> 98-47
<150> US 60/100.656
<151> 1998-09-16
<160> 16
<170> FastSEQ for Windows Version 3.0
<210>1
<211>982
<212>DNA
<213>Homo Sapiens
<220>
<221> CDS
<222> (70)...(853)
<400> 1
ttagcttcac tccttcggca gcaggagggc ggcagcttct cgcaggcggc agggcgggcg 60
gccagtatc atg tcc acc acc aca tgc caa gtg gtg gcg ttc ctc ctg tcc 111
Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu L.eu Ser
1 5 10
atc ctg ggg ctg gcc ggc tgc atc gcg gcc acc ggg atg gac atg tgg 159
Ile Leu Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp
15 20 25 30
agc acc cag gac ctg tac gac aac ccc gtc acc tcc gtg ttc cag tac 207
Ser Thr Gln Asp Leu Tyr Asp Asn Pro Ual Thr Ser Val Phe Gln Tyr
35 40 45
gaa ggg ctc tgg agg agc tgc gtg agg cag agt tca ggc ttc acc gaa 255
Glu Gly Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu
50 55 60
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2
tgc agg ccc tat ttc acc atc ctg gga ctt cca gcc atg ctg cag gca 303
Cys Arg Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala
65 70 75
gtg cga gcc ctg atg atc gta ggc atc gtc ctg ggt gcc att ggc ctc 351
Val Arg Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu
80 85 90
ctg gta tcc atc ttt gcc ctg aaa tgc atc cgc att ggc agc atg gag 399
Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu
95 100 105 110
gac tct gcc aaa gcc aac atg aca ctg acc tcc ggg atc atg ttc att 447
Asp Ser Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile
115 120 125
gtc tca ggt ctt tgt gca att get gga gtg tct gtg ttt gcc aac atg 495
Ual Ser Gly Leu Cys Ala Ile Ala Gly Val Ser Ual Phe Ala Asn Met
130 135 140
ctg gtg act aac ttc tgg atg tcc aca get aac atg tac acc ggc atg 543
Leu Val Thr .Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met
145 150 155
ggt ggg atg gtg cag act gtt cag acc agg tac aca ttt ggt gcg get 591
Gly Gly Met Val Gln Thr Ual Gln Thr Arg Tyr Thr Phe Gly Ala Ala
160 165 170
ctg ttc gtg ggc tgg gtc get gga ggc ctc aca cta att ggg ggt gtg 639
Leu Phe Val Gly Trp Ual Ala Gly Gly Leu Thr Leu Ile Gly Gly Val
175 180 185 190
atg atg tgc atc gcc tgc cgg ggc ctg gca cca gaa gaa acc aac tac 687
Met Met Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr
195 200 205
aaa gcc gtt tct tat cat gcc tca ggc cac agt gtt gcc tac aag cct 735
Lys Ala Ual Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro
210 215 220
gga ggc ttc aag gcc agc act ggc ttt ggg tcc aac acc aaa aac aag 783
Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys
225 230 235
aag ata tac gat gga ggt gcc cgc aca gag gac gag gta caa tct tat 831
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3
Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr
240 245 250
cct tcc aag cac gac tat gtg t aatgctctaa gacctctcag cacgggcgga 883
Pro Ser Lys His Asp Tyr Ual
255 260
agaaactccc ggagaactca cccaaaaaac aaggagatcc catctagatt cttcttgctt 943
ttgactcaca gctggaagtt agaaaacctc gaattcatc 982
<210>2
<211>261
<212>PRT
<213>Homo Sapiens
<400> 2
Met Ser Thr Thr Thr Cys Gln Ual Val Ala Phe Leu Leu Ser Ile Leu
1 5 10 15
Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr
20 25 30
Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Ual Phe Gln Tyr Glu Gly
35 40 45
Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg
50 55 6G
Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg
65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Ual
85 90 95
Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110
Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125
Gly Leu Cys Ala Ile Ala Gly Val Ser Ual Phe Ala Asn Met Leu Val
130 135 140
Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly
145 150 155 160
Met Ual Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe
165 170 175
Val Gly Trp Ual Ala Gly Gly Leu Thr Leu Ile Gly Gly Ual Met Met
180 185 190
Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala
195 200 205
Ual Ser Tyr His Ala Ser Gly His Ser Ual Ala Tyr Lys Pro Gly Gly
210 215 220
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Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser
245 250 255
Lys His Asp Tyr Val
260
<210>3
<211>211
<212>PRT
<213>Mus musculus
<400> 3
Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Ser Leu
1 5 10 15
Gly Trp Ile Gly Ser Ile Ual Ser Thr Ala Leu Pro Gln Trp Lys Ile
20 25 30
Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Ile Tyr Glu
35 40 45
Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile Gln Cys
50 55 60
Lys Ual Phe Asp Ser Leu Leu Asn Leu Asn Ser Thr Leu Gln Ala Thr
65 70 75 80
Arg Ala Leu Met Val Ile Gly Ile Leu Leu Gly Leu Ile Ala Ile Phe
85 90 95
Val Ser Thr Ile Gly Met Lys Cys Met Arg Cys Leu Glu Asp Asp Glu
100 105 110
Val Gln Lys Met Trp Met Ala Val Ile Gly Gly Ile Ile Phe Leu Ile
115 120 125
Ser Gly Leu Ala Thr Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile
130 135 140
Val Gln Glu Phe Tyr Asp Pro Leu Thr Pro Ile Asn Ala Arg Tyr Glu
145 150 155 160
Phe Gly Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu
165 170 175
Leu Gly Gly Val Leu Leu Ser Cys Ser Cys Pro Arg Lys Thr Thr Ser
180 185 190
Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Thr Pro Ser Ser Gly Lys
195 200 205
Asp Tyr Val
210
<210> 4
<211> 210
CA 02343001 2001-03-16
WO 00/15659 PCT/US99/Z10Z3.
<212> PRT
<213> Mus musculus
<400> 4
Met Ala Ser Met Gly Leu Gln Ual Leu Gly Ile Ser Leu Ala Val Leu
1 5 10 15
Gly Trp Leu Gly Ile Ile Leu Ser Cys Ala Leu Pro Met Trp Arg Ual
20 25 30
Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ala Gln Thr Ser Trp Glu
35 40 45
Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys
50 55 60
Lys Met Tyr Asp Ser Met Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala
65 70 75 80
Arg Ala Leu Met Val Ile Ser Ile Ile Val Gly Ala Leu Gly Met Leu
85 90 95
Leu Ser Val Ual Gly Gly Lys Cys Thr Asn Cys Met Glu Asp Glu Thr
100 105 110
Val Lys Ala Lys Ile Met Ile Thr Ala Gly Ala Val Phe Ile Val Ala
115 120 125
Ser Met Leu Ile Met Ual Pro Ual Ser Trp Thr Ala His Asn Val Ile
130 135 140
Arg Asp Phe Tyr Asn Pro Met Val Ala Ser Gly Gln Lys Arg Glu Met
145 150 155 160
Gly Ala Ser Leu Tyr Ual Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu
165 170 175
Gly Gly Gly Leu Leu Cys Cys Ser Cys Pro Pro Arg Ser Asn Asp Lys
180 185 190
Pro Tyr Ser Ala Lys Tyr Ser Ala Ala Arg Ser Val Pro Ala Ser Asn
195 200 205
Tyr Val
210
<210>5
<211>219
<212>PRT
<213>Homo Sapiens
<400> 5
MetAla Thr AlaSer Glu IleAla Phe Val SerIle
Ser Ile Met Ser
1 5 10 15
GlyTrp Leu UalSer Ser LeuPro Thr Tyr TrpLys
Val Thr Asp Ual
20 25 30
SerThr Asp GlyThr Ual ThrThr Ala Tyr TrpAla
Ile Ile Thr Asn
35 40 45
CA 02343001 2001-03-16
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Leu Trp Lys Ala Cys Ual Thr Asp Ser Thr Gly Val Ser Asn Cys Lys
50 55 60
Asp Phe Pro Ser Met Leu Ala Leu Asp Gly Tyr Ile Gln Ala Cys Arg
65 70 75 80
Gly Leu Met Ile Ala Ala Val Ser Leu Gly Phe Phe Gly Ser Ile Phe
85 90 95
Ala Leu Phe Gly Met Lys Cys Thr Lys Val Gly Gly Ser Asp Lys Ala
100 105 110
Lys Ala Lys Ile Ala Cys Leu Ala Gly Ile Ual Phe Ile Leu Ser Gly
115 120 125
Leu Cys Ser Met Thr Gly Cys Ser Leu Tyr Ala Asn Lys Ile Thr Thr
130 135 140
Glu Phe Phe Asp Pro Leu Phe Val Glu Gln Lys Tyr Glu Leu Gly Ala
145 150 155 160
Ala Leu Phe Ile Gly Trp Ala Gly Ala Ser Leu Cys Ile Ile Gly Gly
165 170 175
Val Ile Phe Cys Phe Ser Ile Ser Asp Asn Asn Lys Thr Pro Arg Tyr
180 185 190
Thr Tyr Asn Gly Ala Thr Ser Val Met Ser Ser Arg Thr Lys Tyr His
195 200 205
Gly Gly Glu Asp Phe Lys Thr Thr Asn Pro Ser
210 215
<210>6
<211>218
<212>PRT
<213>Homo Sapiens
<400> 6
Met Gly Ser Ala Ala Leu Glu Ile Leu Gly Leu Val Leu Cys Leu Ual
1 5 10 15
Gly Trp Gly Gly Leu Ile Leu Ala Cys Gly Leu Pro Met Trp Gln Ual
20 25 30
Thr Ala Phe Leu Asp His Asn Ile Ual Thr Ala Gln Thr Thr Trp Lys
35 40 45
Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly His Met Gln Cys
50 55 6U
Lys Val Tyr Asp Ser Val Leu Ala Leu Ser Thr Glu Val Gln Ala Ala
65 70 75 80
Arg Ala Leu Thr Ual Ser Ala Val Leu Leu Ala Phe Val Ala Leu Phe
85 90 95
Val Thr Leu Ala Gly Ala Gln Cys Thr Thr Cys Val Ala Pro Gly Pro
100 105 110
Ala Lys Ala Arg Val Ala Leu Thr Gly Gly Val Leu Tyr Leu Phe Cys
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115 120 125
Gly Leu Leu Ala Leu Val Pro Leu Cys Trp Phe Ala Asn Ile Val Val
130 135 140
Arg Glu Phe Tyr Asp Pro Ser Ual Pro Val Ser Gln Lys Tyr Glu Leu
145 150 155 160
Gly Ala Ala Leu Tyr Ile Gly Trp Ala Ala Thr Ala Leu Leu Met Val
165 170 I75
Gly Gly Cys Leu Leu Cys Cys Gly Ala Trp Val Cys Thr Gly Arg Pro
180 185 190
Asp Leu Ser Phe Pro Val Lys Tyr Ser Ala Pro Arg Arg Pro Thr Ala
195 200 205
Thr Gly Asp Tyr Asp Lys Lys Asn Tyr Val
210 215
<210>7
<211>218
<212>PRT
<213>Homo sapiens
<400> 7
Met Val Ala Thr Cys Leu Gln Val Ual Gly Phe Ual Thr Ser Phe Val
1 5 10 15
Gly Trp Ile Gly Ual Ile Val Thr Thr Ser Thr Asn Asp Trp Ual Val
20 25 30
Thr Cys Gly Tyr Thr Ile Pro Thr Cys Arg Lys Leu Asp Glu Leu Gly
35 40 45
Ser Lys Gly Leu Trp Ala Asp Cys Ual Met Ala Thr Gly Leu Tyr His
50 55 60
Cys Lys Pro Leu Val Asp Ile Leu Ile Leu Pro Gly Tyr Val Gln Ala
65 70 75 80
Cys Arg Ala Leu Met Ile Ala Ala Ser Val Leu Gly Leu Pro Ala Ile
85 90 95
Leu Leu Leu Leu Thr Val Leu Pro Cys Ile Arg Met Gly Gln Glu Pro
100 105 110
Gly Ual Ala Lys Tyr Arg Arg Ala Gln Leu Ala Gly Val Leu Leu Ile
115 120 125
Leu Leu Ala Leu Cys Ala Leu Val Ala Thr Ile Trp Phe Pro Val Cys
130 135 140
Ala His Arg Glu Thr Thr Ile Val Ser Phe Gly Tyr Ser Leu Tyr Ala
145 150 155 160
Gly Trp Ile Gly Ala Val Leu Cys Leu Val Gly Gly Cys Val Ile Leu
165 170 175
Cys Cys Ala Gly Asp Ala Gln Ala Phe Gly Glu Asn Ual Ser Thr Thr
180 185 190
Leu Arg Ala Leu Ala Pro Arg Leu Met Arg Arg Val Pro Thr Tyr Lys
CA 02343001 2001-03-16
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195 200 205
Arg Ala Ala Arg Leu Pro Thr Glu Ual Leu
210 215
<210>8
<211>228
<212>PRT
<213>Mus musculus
<400> 8
Met Ala Ser Leu Gly Ual Gln Leu Val Gly Tyr Ile Leu Gly Leu Leu
1 5 10 15
Gly Leu Leu Gly Thr Ser Ile Ala Met Leu Leu Pro Asn Trp Arg Thr
20 25 30
Ser Ser Tyr Val Gly Ala Ser Ile Val Thr Ala Ual Gly Phe Ser Lys
35 40 45
Gly Leu Trp Met Glu Cys Ala Thr His Ser Thr Gly Ile Thr Gln Cys
50 55 60
Asp Ile Tyr Ser Thr Leu Leu Gly Leu Pro Ala Asp Ile Gln Ala Ala
65 70 75 80
Gln Ala Met Met Val Thr Ser Ser Ala Met Ser Ser Leu Ala Cys Ile
85 90 95
Ile Ser Val Val Gly Met Arg Cys Thr Val Phe Cys Gln Asp Ser Arg
100 105 110
Ala Lys Asp Arg Ual Ala Val Val Gly Gly Ual Phe Phe Ile Leu Gly
115 120 125
Gly Ile Leu Gly Phe Ile Pro Val Ala Trp Asn Leu His Gly Ile Leu
130 135 140
Arg Asp Phe Tyr Ser Pro Leu Val Pro Asp Ser Met Lys Phe Glu Ile
145 150 155 160
Gly Glu Ala Leu Tyr Leu Gly Ile Ile Ser Ala Leu Phe Ser Leu Val
165 170 175
Ala Gly Val Ile Leu Cys Phe Ser Cys Ser Pro Gln Gly Asn Arg Thr
180 185 190
Asn Tyr Tyr Asp Gly Tyr Gln Ala Gln Pro Leu Ala Thr Arg Ser Ser
195 200 205
Pro Arg Ser Ala Gln Gln Pro Lys Ala Lys Ser Glu Phe Asn Ser Tyr
210 215 220
Ser Leu Thr Gly
225
<210>9
<211>280
<212>PRT
<213>Ratus norvegicus
CA 02343001 2001-03-16
WO 00/15659 PCT/US99/21023_
9
<400> 9
Met Ser Met Ser Leu Glu Ile Thr Gly Thr Ser Leu Ala Val Leu Gly
1 5 10 15
Trp Leu Cys Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser
20 25 30
Ala Phe Ile Gly Ser Ser Ile Ile Thr Ala Gln Ile Thr Trp Glu Gly
35 40 45
Leu Trp Met Asn Cys Val Gln Ser Thr Gly Gln Met Gln Cys Lys Met
50 55 60
Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg Ala
65 70 75 80
Leu Ile Val Val Ser Ile Leu Leu Ala Ala Phe Gly Leu Leu Val Ala
85 90 95
Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Glu Thr Ala Lys
100 105 110
Ala Lys Ile Thr Ile Val Ala Gly Val Leu Phe Leu Leu Ala Ala Val
115 120 125
Leu Thr Leu Ual Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg Asp
130 135 140
Phe Tyr Asn Pro Leu Val Pro Glu Ala Gln Lys Arg Glu Met Gly Thr
145 150 155 160
Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly Gly
165 170 175
Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Tyr Ala Pro Thr
180 185 190
Lys Ile Leu Tyr Ser Ala Pro Arg Ser Thr Giy Pro Gly Thr Gly Thr
195 200 205
Gly Thr Ala Tyr Asp Arg Lys Thr Thr Ser Glu Arg Pro Gly Ala Arg
210 215 220
Thr Pro His His His His Tyr Gln Pro Ser Met Tyr Pro Thr Arg Pro
225 230 235 240
Ala Cys Ser Leu Ala Ser Glu Thr Thr Pro Pro Ser Arg Arg Leu Gln
245 250 255
Thr Pro Arg Ser Leu Leu Ala Arg Leu Glu Glu Asp Arg Gln Pro Gly
260 265 270
Val Pro Phe Ser Pro Val Ala Thr
275 280
<210> 10
<211> 783
<212> DNA
<213> Artificial Sequence
CA 02343001 2001-03-16
WO 00/15659 PCT/US99/21023.
<220>
<223> Degenerate nucleotide sequence of zsig28
polypeptide
<221> misc_feature
<222> (1). .(783)
<223> n = A,T,C or G
<400> 10
atgwsnacnacnacntgycargtngtngcnttyytnytnwsnathytnggnytngcnggn 60
tgyathgcngcnacnggnatggayatgtggwsnacncargayytntaygayaayccngtn 120
acnwsngtnttycartaygarggnytntggmgnwsntgygtnmgncarwsnwsnggntty 180
acngartgymgnccntayttyacnathytnggnytnccngcnatgytncargcngtnmgn 240
gcnytnatgathgtnggnathgtnytnggngcnathggnytnytngtnwsnathttygcn 300
ytnaartgyathmgnathggnwsnatggargaywsngcnaargcnaayatgacnytnacn 360
wsnggnathatgttyathgtnwsnggnytntgygcnathgcnggngtnwsngtnttygcn 420
aayatgytngtnacnaayttytggatgwsnacngcnaayatgtayacnggnatgggnggn 480
atggtncaracngtncaracnmgntayacnttyggngcngcnytnttygtnggntgggtn 540
gcnggnggnytnacnytnathggnggngtnatgatgtgyathgcntgymgnggnytngcn 600
ccngargaracnaaytayaargcngtnwsntaycaygcnwsnggncaywsngtngcntay 660
aarccnggnggnttyaargcnwsnacnggnttyggnwsnaa,yacnaaraayaaraarath 720
taygayggnggngcnmgnacngargaygargtncarwsnta,yccnwsnaarcaygaytay 780
Stn 783
<210> 11
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer ZC447
<400> 11
taacaatttc acacagg 17
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer ZC12501
<400> 12
CA 02343001 2001-03-16
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11
gtatccatct ttgccctgaa 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> 0ligonucleotide primer ZC12502
<400> 13
gctaggatta caggcgtgag 20
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer ZC976
<400> 14
cgttgtaaaa cgacggcc lg
<210> 15
<2I1> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer ZC19410
<400> 15
gcgggcggcc agtatcat 18
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide primer ZC19411
<400> 16
cggaggtgac ggggttgt 18
