U.S. patent application number 09/757908 was filed with the patent office on 2002-05-02 for disulfide core polypeptides.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Conklin, Darrell C..
Application Number | 20020052468 09/757908 |
Document ID | / |
Family ID | 26778316 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052468 |
Kind Code |
A1 |
Conklin, Darrell C. |
May 2, 2002 |
Disulfide core polypeptides
Abstract
The present invention relates to polynucleotide and polypeptide
molecules for a disulfide core protein (Zdsc1). The polypeptides,
and polynucleotides encoding them, are serine proteinase
inhibitors. Also disclosed are expression vectors containing
polynucleotides which encode a Zdsc1 polypeptide, antibodies which
specifically bind to Zdsc1 polypeptides and anti-idiotypic
antibodies which neutralize the antibodies which specifically bind
to Zdsc1 polypeptides.
Inventors: |
Conklin, Darrell C.;
(Seattle, WA) |
Correspondence
Address: |
Paul G. Lunn, Esq.
Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26778316 |
Appl. No.: |
09/757908 |
Filed: |
January 10, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09757908 |
Jan 10, 2001 |
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09326039 |
Jun 4, 1999 |
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6239254 |
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60088136 |
Jun 4, 1998 |
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Current U.S.
Class: |
530/300 ;
530/387.2; 530/388.1; 536/23.5 |
Current CPC
Class: |
C07K 14/811
20130101 |
Class at
Publication: |
530/300 ;
530/388.1; 530/387.2; 536/23.5 |
International
Class: |
C07K 016/42; C07K
016/18; C07K 014/435; C07H 021/04 |
Claims
What is claimed is:
1. An isolated polypeptide comprised of the amino acid sequence of
SEQ ID NO:2, or SEQ ID NO:3.
2. A isolated polypeptide comprised of an amino acid sequence of
SEQ ID NO:5.
3. An isolated polynucleotide which is at least 90% homologous to a
polynucleotide which encodes a polypeptide comprised of an amino
acid sequence selected from the group consisting of SEQ ID NO: 2,
SEQ ID NO:3 and SEQ ID NO:5.
4. The isolated polynucleotide of claim 3 wherein the
polynucleotide encodes a polypeptide comprised of an amino acid
sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO:3 and SEQ ID NO:5.
5. An antibody which binds specifically to a polypeptide selected
from the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID
NO:5.
6. An anti-idiotypic antibody which binds to and neutralizes an
antibody which binds specifically to a polypeptide selected from
the group consisting of SEQ ID NO: 2, SEQ ID NO:3 and SEQ ID
NO:5.
7. An expression vector which contains a polynucleotide which
encodes a polypeptide selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:3 and SEQ ID NO:5.
Description
[0001] The present application claims the benefit under 35 U1.C.
.sctn.119(e) of U.S. Provisional Application Ser. No. 60/088,136
filed Jun. 4, 1998.
BACKGROUND OF THE INVENTION
[0002] Protein inhibitors are classified into a series of families
based on extensive sequence homologies among the family members and
the conservation of intrachain disulfide bridges, see Laskowski and
Kato, Ann. Rev. Biochem. 49: 593-626 (1980). An example of a serine
proteinase inhibitor is the serine proteinase inhibitor aprotinin
which is used therapeutically in the treatment of acute
pancreatitis, various states of shock syndrome, hyperfibrinolytic
hemorrhage and myocardial infarction. Administration of aprotinin
in high doses significantly reduces blood loss in connection with
cardiac surgery, including cardiopulmonary bypass operations.
[0003] However, when administered in vivo, aprotinin has been found
to have a nephrotoxic effect in rats, rabbits and dogs after
repeated injections of relatively high doses. The nephrotoxicity
(appearing, i.e., in the form of lesions) observed for aprotinin
might be ascribed to the accumulation of aprotinin in the proximal
tubulus cells of the kidneys as a result of the high positive net
charge of aprotinin, which causes it to be bound to the negatively
charged surfaces of the tubuli. This nephrotoxicity makes aprotinin
less suitable for clinical purposes, particularly in those uses
requiring administration of large doses of the inhibitor (such as
cardiopulmonary bypass operations). Furthermore, aprotinin is a
bovine protein, which may induce an immune response upon
administration to humans.
[0004] Thus there is a need for serine proteinase inhibitors which
are not toxic for the treatment of acute pancreatitis, various
states of shock syndrome, hyperfibrinolytic hemorrhage and
myocardial infarction.
SUMMARY OF THE INVENTION
[0005] The present invention fills this need by providing for a new
class of proteinase inhibitors called disulfide core proteinase
inhibitors (hereinafter referred to as a Zdsc1 polypeptide). Murine
Zdsc1, SEQ ID NOs: 1 and 2 has a signal sequence extending from the
methionine at position 1 through and including the alanine at
position 24 of SEQ ID NO:2. The mature murine Zdsc1 polypeptide is
also depicted by SEQ ID NO:3. SEQ ID NO:4 and 5 are examples of a
mature human Zdsc1 polypeptide and polynucleotide which encodes it.
A generic Zdsc1 polypeptide is exemplified by SEQ ID NO:6.
[0006] Within one aspect of the invention there is provided an
isolated polypeptide. The polypeptide being comprised of a sequence
of amino acids containing the sequence of SEQ ID NO:2, SEQ ID NO:3
or SEQ ID NO:5.
[0007] Within another aspect of the invention there is provided an
isolated polynucleotide which encodes a polypeptide comprised of a
sequence of amino acids containing the sequence of SEQ ID NO:2, SEQ
ID NO:3 or SEQ ID NO:5.
[0008] Within an additional aspect of the invention there is
provided a polynucleotide sequence which hybridizes under stringent
conditions to either SEQ ID NO:1 or SEQ ID NO:4 or to a
complementary sequence of SEQ ID NO:1 or to a complementary
sequence of SEQ ID NO:4.
[0009] Within an additional aspect of the invention there is
provided a polynucleotide sequence which is at least 90%, 95%, or
99% homologous to a polynucleotide sequence which encodes the
polypeptide of SEQ ID NO:3 or SEQ ID NO:4.
[0010] Within another aspect of the invention there is provided an
expression vector comprising (a) a transcription promoter; (b) a
DNA segment encoding a Zdsc1 polypeptide, containing an amino acid
sequence as described above.
[0011] Within another aspect of the invention there is provided a
cultured eukaryotic, bacterial, fungal or other cell into which has
been introduced an expression vector as disclosed above, wherein
said cell expresses a mammalian Zdsc1 polypeptide encoded by the
DNA segment.
[0012] Within another aspect of the invention there is provided a
chimeric polypeptide consisting essentially of a first portion and
a second portion joined by a peptide bond. The first portion of the
chimeric polypeptide consists essentially of a Zdsc1 polypeptide as
described above. The invention also provides expression vectors
encoding the chimeric polypeptides and host cells transfected to
produce the chimeric polypeptides.
[0013] Within an additional aspect of the invention there is
provided an antibody that specifically binds to a polypeptide as
disclosed above and an anti-idiotypic antibody of an antibody which
specifically binds to a Zdsc1 antibody.
[0014] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0015] All references cited herein are incorporated in their
entirety herein by reference.
[0016] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms.
[0017] 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 sites 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), glutathione 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,
Flag.TM. peptide (Hopp et al., Biotechnology 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 are
available from commercial suppliers, (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0018] 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 altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0019] 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.
[0020] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0021] The term "complements of a polynucleotide molecule" is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the
[0022] sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT
3'.
[0023] The term "contig" denotes a polynucleotide that 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.
[0024] 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).
[0025] 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.
[0026] 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).
[0027] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as 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 forms.
[0028] 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.
[0029] 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.
[0030] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0031] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two 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 polynucleotide 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. Such unpaired ends will
in general not exceed 20 nt in length.
[0032] 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".
[0033] 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.
[0034] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-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.
[0035] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-domain (Frank Grant suggests
"multi-peptide" in that subunit binding and signal transduction can
be separate subunits) structure comprising an extracellular
ligand-binding domain and an intracellular effector domain that is
typically involved in signal transduction. Binding of ligand to
receptor results in a conformational change in the receptor that
causes an interaction between the effector domain and other
molecule(s) in the cell. This interaction in turn leads to an
alteration in the 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).
[0036] 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.
[0037] 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.
[0038] 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%.
[0039] Serine proteinase inhibitors regulate the proteolytic
activity of target proteinases by occupying the active site and
thereby preventing occupation by normal substrates. Although serine
proteinase inhibitors fall into several unrelated structural
classes, they all possess an exposed loop (variously termed an
"inhibitor loop", a "reactive core", a "reactive site", a "binding
loop") which is stabilized by intermolecular interactions between
residues flanking the binding loop and the protein core. See Bode,
W. and Huber, R. "Natural protein proteinase inhibitors and their
interactions with proteinases", Eur. J. Biochem., 204: 433-451
(1992). Interaction between inhibitor and enzyme produces a stable
complex which disassociates very slowly, producing either virgin or
a modified inhibitor which is cleaved at the scissile bond of the
binding loop.
[0040] Serine proteinase inhibitors fall into various structural
families, for example, the Kunitz family, the Kazal family, and the
Hirudin family. The protein Zdsc1 is a member of a new subfamily,
which appears to be closely related the Chelonianin family. The
Chelonianin family is characterized by a common structural motif
which comprises two adjacent beta-hairpin motifs, each consisting
of two antiparallel beta strands connected by a loop region. The
secondary structure of this motif is depicted by beta-sheet
topology K (Branden, C. and Tooze, J. Introduction to Protein
Structure. p. 28 (GarlandPublishing, Inc., 1991). The beta strands
are linked by intra-chain hydrogen bonding and by a network of four
disulfide bonds. These disulfide bonds-stabilize the structure of
the proteinase inhibitor and render it less susceptible to
degradation. This structural feature has caused the Chelonianin
family to be referred to as the "four-disulfide core", family of
proteinase inhibitors. This family includes human
antileukoproteinase, human elafin, guinea pig caltrin-like protein,
human kallman syndrome protein, sea turtle chelonianin, and human
epididymal secretory protein E4, and trout TOP-2, and C. Elegans
C08G9. Several of these family members contain several copies of
this structural motif.
[0041] Imbalances between native proteinases and a proteinase
inhibitor is seen in patients where levels of human
antileukoproteinase inhibitor are compromised by genetic background
or by air contamination. In these patients, severe lung damage can
result due to unmitigated activity of proteinases. The elastase
inhibitory domain of antileukoproteinase inhibitor falls into the
four-disulfide core family, which is related to the three-disulfide
core family of zdsc1. As another example, human elafin (also in the
four-disulfide core family) is a specific inhibitor of leukocyte
elastase and pancreatic elastase. These proteinases have the
ability to cleave the connective tissue protein elastin and
therefore elafin may prevent excessive elastase-mediated tissue
proteolysis and damage.
[0042] Serine proteinase inhibitor activity can be measured using
the method essentially described by Norris et al., Biol. Chem.
Hoppe-Seyler 371: 37-42 (1990). Briefly, various fixed
concentrations of the Kunitz-type inhibitor are incubated in the
presence of serine proteinases at the concentrations listed in
Table 1 in 100 mM NaCl, 50 mM Tris HCl, 0.01% TWEEN80
(Polyoxyethylenesorbitan monoleate) (pH 7.4) at 25.degree. C. After
a 30 minute incubation, the residual enzymatic activity is measured
by the degradation of a solution of the appropriate substrate as
listed in Table 1 in assay buffer. The samples are incubated for 30
minutes after which the absorbance of each sample is measured at
405 nm. An inhibition of enzyme activity is measured as a decrease
in absorbance at 405 nm or fluorescence Em at 460 nm. From the
results, the apparent inhibition constant K.sub.i is
calculated.
1TABLE 1 Protease (concentration) Substrate (concentration) Source
Source Trypsin (8 nM) H-D-Val-Leu-Lys-pNA (0.6 mM) Novo Nordisk
A/S, Kabi Bagsvaerd, Denmark Chymotrypsin (2.5 nM)
MeO-Suc-Arg-Pro-Tyr-pNA (0.6 mM) Novo Nordisk A/S Kabi GL
Kallikrein (1 U/ml) H-D-Val-Leu-Arg-pNA (0.6 mM) Sigma, St Louis,
MO Kabi Plasmin (10 nM) H-D-Val-Leu-Lys-pNA (0.6 mM) Kabi Kabi
Urokinase (5 nM) <Glu-Gly-Arg-pNA (0.6 mM) Serono Kabi Freigurg,
Germany rec. Protein Ca (5 nM) <Glu-Pro-Arg-pNA (0.6 mM) Novo
Nordisk A/S Kabi PL Kallikrein (3 nM) H-D-Pro-Phe-Arg-pNA (0.6 mM)
Kabi Kabi human Factor Xlla (30 nM) H-D-Pro-Phe-Arg-pNA (0.6 mM)
Dr. Walt Kisiel Kabi University of New Mexico, Albuquerque, NM
human Factor Xla (1 nM) Boc-Glu(OBzI)-Ala-Arg-MCA (0.12 Dr. Kazuo
Fulikawa mM) University of Washington, Peptide Institute Seattle,
WA Osaka, Japan human Factor Xa (3 nM) MeO-CO-CHA-Gly-Arg-pNA (0.3
mM) Dr. I. Schousboe NycoMed Copenhagen, Denmark Oslo, Norway rec.
human Factor Vhs (300 nM) H-D-lIe-Pro-Arg-pNA (0.6 mM) Novo Nordisk
A/S Kabi Leukocyte Elastase MeO-Suc-Ala-Ala-Pro-Val-pNA (0.6 mM)
purified at Novo Nordisk A/S (SEQ ID NO:14) using the method of
Sigma Chemical Co. Baugh and Travis St. Louis, MO (Biochemistry 15:
836-843, 1976) Cathepsin G Suc-Ala-Ala-Pro-Phe-pNA (0.6 mM)
purified at Novo Nordisk A/S (SEQ ID NO:15 using the method of
Sigma Chemical Co. Baugh and Travis (Biochemistry 15: 836-843,
1976) Abbreviations in Table 1: rec. refers to recombinant, GL
kallikrein refers to glandular kallikrein, and PL kallikrein refers
to plasma kahhikrein.
[0043] Inhibition assays were performed in microtiter wells in a
total volume of 300 .mu.l in 10 mM NaCl, 50 mM Tris-HCl (pH 7.4),
0.01% TWEEN80 (Polyoxyethylenesorbitan monoleate). Each reaction
contained 1 .mu.M of the sample inhibitor and one of the proteases
at the concentration listed in Table 1. The reactions were
incubated at 25.degree. C. for ten minutes after which the
appropriate chromogenic substrate was added to the final
concentration listed in Table 1 and the final reaction was
incubated for thirty minutes at 25.degree. C. Amidolytic activity
was measured at 405 nm or by fluorescence Em at 460 nm. Percent
inhibition was determined relative to reactions carried out in the
absence of inhibitor representing 100% activity or 0%
inhibition.
[0044] The serine proteinase inhibitors of the present invention
may be used in the disclosed methods for the treatment of, inter
alia, acute pancreatitis, various states of shock syndrome,
hyperfibrinolytic hemorrhage and myocardial infarction. The amyloid
protein precursor homologues of the present invention may be used,
inter alia, to generate antibodies for use in demonstrating tissue
distribution of the precursor or for use in purifying such
proteins.
[0045] Cysteines 3-8 in members of the four disulfide core family
occur according to the motif:
2 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Cys (SEQ ID
NO:17) Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys
[0046] The residue Xaa can by any amino acid residue except for
cysteine.
[0047] The spacing between cysteines 1-2 and between cysteines 2-3
in this family is variable. Cysteines 1-3 have been observed to
occur according to one of the following motifs:
3 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa (SEQ ID
NO:18) Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
Xaa (SEQ ID NO:19) Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Cys (SEQ ID NO:20) Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys (SEQ ID NO:21) Cys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa (SEQ ID NO:22) Cys
[0048] The 8 cysteines in the four-disulfide core are bonded
according to the pattern:
[0049] 1-6, 2-7, 3-5, 4-8
[0050] Zdsc1
[0051] The protein Zdsc1 is a member of a new related subfamily,
which will be referred to as the "three-disulfide core" family.
This family is distinct from the four-disulfide core family due to
the absence of cysteines 1 and 6. The remaining 6 cysteines occur
according to the pattern:
4 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa (SEQ ID
NO:23) Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
[0052] Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys (SEQ ID NO:23).
[0053] Zdsc1 is related by sequence homology to most of the
four-disulfide core proteins, having the highest similarity to
trout TOP-2 and mouse WDMN1 protein. See Garczynski, M. and Goetz,
F. Molecular characterization of a RNA transcript that is highly
up-regulated at the time of ovulation in the brook trout ovary,
Biology of Reproduction, 57: 856-864 (1997).
[0054] To further characterize the three-dimensional structure of
Zdsc1, including the disulfide bonding pattern and binding loop, we
have constructed a homology model based on the NMR structure for
porcine elafin, FLE, Francart, C. et al "Solution structure of
R-elafin, a specific inhibitor of elastase", J. Mol. Biol. 268:
666-677 (1997). The multiple alignment between the three proteins
is given below. By analogy with the known and predicted
structure/function relationships in elafin and the crystal
structure of antileukoproteinase complexed with chymotrypsin
certain features of Zdsc1/2 can be predicted. See Grutter, M. et
al., "The 2.5A X-ray crystal structure of the acid-stable
proteinase inhibitor from human mucous secretions analyzed in its
complex with bovine alpha-chymotrypsin", EMBO J., 7: 345-351
(1988). The 6 cysteines in Zdsc1 are bonded according to the
pattern:
[0055] 2-7, 3-5, 4-8
[0056] The reactive binding loop of Zdsc1 includes the sequence
LQLLGT (SEQ ID NO: 9). Their active binding loop of human Zdsc
includes the sequence DRLLGT (SEQ ID NO: 10). In Zdsc1 flanking
residues around this binding loop are expected to interact with the
target proteinase. The scissile bond is in the reactive binding
loop between the two Leucines. Substitution at the P1 position (the
second Leucine) is not tolerated as this residue is predicted to
influence specificity towards the target proteinase, Bode, W. and
Huber, R. "Natural protein proteinase inhibitors and their
interactions with proteinases", Eur. J. Biochem., 204: 433-451
(1992). Substitution of any cysteine residue is not tolerated as
this is predicted to significantly destabilize the structure.
[0057] To predict the variation acceptable from positions Gln30
through Cys60 in Zdsc1 we have created a generalized motif which
enumerates the permissible substitutions at each position.
5 MKLGAFLLLILVSLITLSLEVQELQA (SEQ ID NO:8) (The predicted signal
sequence for Zdscl) 3 4 56 7 8 FLE:
IILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NO:7) ZDSC1 (m):
AVRPLQLLGTCAELCRGDWDCGPEEQCVSIGCSHICTTN (SEQ ID NO:3) ZDSC1 (h):
AGDRLLGTCVELCTGDWDCNPGDHCVSNGCGHECVAG (SEQ ID NO:5) 2 3 4 5 7 8
[0058] Multiple alignment between porcine elafin, and the predicted
mature peptide for Zdsc1. Cysteines 3-8 of FLE are labeled on the
top of the alignment. Cysteines 1-6 of Zdsc1 are labeled on the
bottom of the alignment, using the standard numbering for
four-disulfide core proteins. Based upon the analysis of Zdsc1 and
Zdsc2 the following generic protein has been deduced as shown below
in SEQ ID NO: 6.
6 Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Gly Thr Cys Xaa Glu Leu SEQ ID
NO:6 Cys Xaa 5 10 15 Gly Asp Trp Asp Cys Xaa Pro Xaa Xaa Xaa Cys
Val Ser Xaa Gly Cys 20 25 30 Xaa His Xaa Cys Xaa Xaa Xaa 35 wherein
Xaa at amino acid position 1 is Ala or is absent; Xaa at amino acid
position 2 is Val or is absent; Xaa at amino acid position 3 is Arg
or Ala; Xaa at amino acid position 4 is Pro or Gly; Xaa at amino
acid position 5 is Leu or Asp; Xaa at amino acid position 6 is Gln,
Arg, Lys or Glu; Xaa at amino acid position 12 is Val, Ala, Ile,
Leu, Met or Ser; Xaa at amino acid position 16 is Thr, Arg, Ala,
Asn, Ser, Val, Gln, Glu, His or Lys; Xaa at amino acid position 22
is Asn, Gly, Asp, His or Ser; Xaa at amino acid position 24 is Ala,
Arg, Asn, Asp, Glu, Gln, Gly, His, Lys, Pro, Ser, or Thr; Xaa at
amino acid position 25 is Asp or Glu Xaa at amino acid position 26
is His, Gln Tyr or Glu; Xaa at amino acid position 30 is Ala, Arg,
Asn, Asp, Gln, Glu, Gly His, Ile, Leu, Lys, Met, Phe, Ser, Thr,
Tyr, or Val; Xaa at amino acid position 33 is Gly, Ser, Ala, Asn,
Thr; Xaa at amino acid position 35 is Ala, Arg, Asn, Asp, Glu, Gln,
Gly, His, Ile, Leu, Lys, Met Phe, Pro, Ser, Thr, Trp, Tyr or Val;
Xaa at amino acid position 37 is Val or Thr; Xaa at amino acid
position 38 is Ala or Thr; and Xaa at amino acid position 39 is Asn
or Gly.
[0059] Any resultant polypeptide based upon SEQ ID NO: 8 must be at
least 80%, preferably 90 or 95% and most preferably 99% identical
to SEQ ID NO: 3, SEQ ID NO: 5 or to SEQ ID NO:7.
[0060] Polynucleotides
[0061] The present invention also provides polynucleotide
molecules, including DNA and RNA molecules, that encode the Zdsc
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. Polynucleotides, generally a cDNA
sequence, of the present invention encode the described
polypeptides herein. A cDNA sequence which encodes a polypeptide of
the present invention is comprised of a series of codons, each
amino acid residue of the polypeptide being encoded by a codon and
each codon being comprised of three nucleotides. The amino acid
residues are encoded by their respective codons as follows.
7 Alanine (Ala) is encoded by GCA, GCC, GCG or GCT; Cysteine (Cys)
is encoded by TGC or TGT; Aspartic acid (Asp) is encoded by GAC or
GAT; Glutamic acid (Glu) is encoded by GAA or GAG; Phenylalanine
(Phe) is encoded by TTC or TTT; Glycine (Gly) is encoded by GGA,
GGC, GGG or GGT; Histidine (His) is encoded by CAC or CAT;
Isoleucine (Ile) is encoded by ATA, ATC or ATT; Lysine (Lys) is
encoded by AAA, or AAG; Leucine (Leu) is encoded by TTA, TTG, CTA,
CTC, CTG or CTT; Methionine (Met) is encoded by ATG; Asparagine
(Asn) is encoded by AAC or AAT; Proline (Pro) is encoded by CCA,
CCC, CCG or CCT; Glutamine (Gln) is encoded by CAA or CAG; Arginine
(Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT; Serine (Ser) is
encoded by AGC, AGT, TCA, TCC, TCG or TCT; Threonine (Thr) is
encoded by ACA, ACC, ACG or ACT; Valine (Val) is encoded by GTA,
GTC, GTG or GTT; Tryptophan (Trp) is encoded by TGG; and Tyrosine
(Tyr) is encoded by TAC or TAT.
[0062] Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;
[0063] Cysteine (Cys) is encoded by TGC or TGT;
[0064] Aspartic acid (Asp) is encoded by GAC or GAT;
[0065] Glutamic acid (Glu) is encoded by GAA or GAG;
[0066] Phenylalanine (Phe) is encoded by TTC or TTT;
[0067] Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;
[0068] Histidine (His) is encoded by CAC or CAT;
[0069] Isoleucine (Ile) is encoded by ATA, ATC or ATT;
[0070] Lysine (Lys) is encoded by AAA, or AAG;
[0071] Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or
CTT;
[0072] Methionine (Met) is encoded by ATG;
[0073] Asparagine (Asn) is encoded by AAC or AAT;
[0074] Proline (Pro) is encoded by CCA, CCC, CCG or CCT;
[0075] Glutamine (Gln) is encoded by CAA or CAG;
[0076] Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or
CGT;
[0077] Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or
TCT;
[0078] Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
[0079] Valine (Val) is encoded by GTA, GTC, GTG or GTT;
[0080] Tryptophan (Trp) is encoded by TGG; and
[0081] Tyrosine (Tyr) is encoded by TAC or TAT.
[0082] It is to be recognized that according to the present
invention, when a polynucleotide is claimed as described herein, it
is understood that what is claimed are both the sense strand, the
anti-sense strand, and the DNA as double-stranded having both the
sense and antisense strand annealed together by their respective
hydrogen bonds. Also claimed is the messenger RNA (mRNA) which
encodes the polypeptides of the president invention, and which mRNA
is encoded by the cDNA described herein. Messenger RNA (mRNA) will
encode a polypeptide using the same codons as those defined herein,
with the exception that each thymine nucleotide (T) is replaced by
a uracil nucleotide (U).
[0083] 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. 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, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Sequences containing preferential codons can
be tested and optimized for expression in various species, and
tested for functionality as disclosed herein.
[0084] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO:1, SEQ ID NO:4, or a sequence complementary thereto, under
stringent conditions. In general, stringent conditions are selected
to be about 5.degree. C. lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH. The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe. Typical stringent conditions are those in
which the salt concentration is up to about 0.03 M at pH 7 and the
temperature is at least about 60.degree. C.
[0085] As previously noted, the isolated polynucleotides of the
present invention include DNA and 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 Zdsc1 RNA. Such
tissues and cells are identified by Northern blotting, Thomas,
Proc. Natl. Acad. Sci. USA 77:5201 (1980), and include high
expression of human Zdsc1 in the liver. Total RNA can be prepared
using guanidine HCl extraction followed by isolation by
centrifugation in a CsCl gradient, Chirgwin et al., Biochemistry
18:52-94, 1979).
[0086] Poly (A).sup.+ RNA is prepared from total RNA using the
method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412
(1972). Complementary DNA (cDNA) is prepared from poly(A).sup.+ RNA
using known methods. In the alternative, genomic DNA can be
isolated. Polynucleotides encoding Zdsc polypeptides are then
identified and isolated by, for example, hybridization or PCR.
[0087] A full-length clone encoding Zdsc1 polypeptide can be
obtained by conventional cloning procedures. 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. Methods for 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 Zdsc, receptor fragments, or other
specific binding partners.
[0088] The polynucleotides of the present invention can also be
synthesized using gene 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 gene or a gene fragment, then each complementary strand is
made separately. The production of short genes (60 to 80 bp) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 bp), however, special
strategies must be invoked, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. 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. The double-stranded constructs are
sequentially linked to one another to form the entire gene
sequence. Because it is absolutely essential that a chemically
synthesized gene have the correct sequence of nucleotides, each
double-stranded fragment and then the complete sequence is
characterized 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 (1984) and Climie et al., Proc.
Natl. Acad. Sci. USA 87:633-637 (1990).
[0089] The present invention further provides counterpart
polypeptides and polynucleotides from other 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 Zdsc polypeptides
from other mammalian species, including murine, porcine, ovine,
bovine, canine, feline, equine, and other primate polypeptides.
Orthologs of human Zdsc 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 Zdsc
as disclosed herein. Suitable sources of mRNA can be identified by
probing Northern blots with probes designed from the sequences
disclosed herein. A library is then prepared from mRNA of a
positive tissue or cell line. A Zdsc-encoding cDNA can then be
isolated by a variety of methods, such as by probing with a
complete or partial human cDNA or with one or more sets of
degenerate probes based on the disclosed sequences. A cDNA can also
be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.
Pat. No. 4,683,202), using primers designed from the representative
human Zdsc sequence 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 Zdsc1 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
[0090] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NO:1 and SEQ ID NO:4 represent a single alleles
of murine Zdsc1 and human Zdsc1 respectively, 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 amino acid sequence changes, are within
the scope of the present invention, as are proteins which are
allelic variants of SEQ ID NO:2. cDNAs generated from alternatively
spliced mRNAs, which retain the properties of the Zdsc1 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.
[0091] The present invention also provides isolated Zdsc1
polypeptides that are substantially identical to the polypeptides
of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5 and their orthologs.
The term "substantially identical" is used herein to denote
polypeptides having 50%, preferably 60%, more preferably at least
80%, sequence identity to the sequences shown in SEQ ID NO:2 or
their orthologs. Such polypeptides will more preferably be at least
90% identical, and most preferably 95% or more identical to SEQ ID
NO:2 or its orthologs.) Percent sequence identity is determined by
conventional methods. See, for example, Altschul et al., Bull.
Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc.
Natl. Acad. Sci. USA 89:10915-10929 (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 2 (amino acids are indicated by the standard
one-letter codes). The percent identity is then calculated as: 1
Total number of identical matches [length of the longer sequence
plus the numberof gaps introduced into the longer sequence inorder
to align the two sequences] .times. 100
8 TABLE 2 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0092] Those skilled in the art appreciate that there are many
established algorithms 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 and the amino acid
sequence of a putative variant. The FASTA algorithm is described by
Pearson and Lipman, Proc. Nat'l 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 NO: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-Seller- s algorithm (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. Illustrative
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=l, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0093] 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 to six, preferably from four to six.
[0094] The present invention includes nucleic acid molecules that
encode a polypeptide having one or more conservative amino acid
changes, compared with the amino acid sequence of SEQ ID NO:3 or
with the amino acid sequence of SEQ ID NO:5. The BLOSUM62 table 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'l 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. As used herein, the language
"conservative amino acid substitution" 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. 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 substitutions are characterized by a BLOSUM62 value of
at least 2 (e.g., 2 or 3). Accordingly the present invention claims
those polypeptides which are at least 90%, preferably 95% and most
preferably 99% identical to SEQ ID NO:3 and which are able to
stimulate antibody production in a mammal, and said antibodies are
able to bind the native sequence of SEQ ID NO:3.
[0095] Variant Zdsc1 polypeptides or substantially identical Zdsc1
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 3) 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 small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue, a small linker peptide of up to about 20-25 residues, or
an affinity tag. Polypeptides comprising affinity tags can further
comprise a proteolytic cleavage site between the Zdsc polypeptide
and the affinity tag. Preferred such sites include thrombin
cleavage sites and factor Xa cleavage sites.
9TABLE 3 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serane threonine methionine
[0096] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev.
60:512 (1996). 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. 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, enhance production of the protein by making
protein translation more efficient within a particular cell type or
species. Sequences containing preferential codons can be tested and
optimized for expression in various species, and tested for
functionality as disclosed herein.
[0097] The present invention further provides variant polypeptides
and nucleic acid molecules that represent counterparts from other
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
Zdsc1 polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human Zdsc1 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 Zdsc1 as disclosed herein. Suitable sources of mRNA
can be identified by probing northern blots with probes designed
from the sequences disclosed herein. A library is then prepared
from mRNA of a positive tissue or cell line.
[0098] An Zdsc1-encoding cDNA can then be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction with primers designed from the representative human
Zdsc1 sequences disclosed herein. Within an additional method, the
cDNA library can be used to transform or transfect host cells, and
expression of the cDNA of interest can be detected with an antibody
to Zdsc1 polypeptide. Similar techniques can also be applied to the
isolation of genomic clones.
[0099] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human Zdsc1,
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 nucleotide sequences
shown in SEQ ID NO:1 or SEQ ID NO:4, including those containing
silent mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as
are proteins which are allelic variants of SEQ ID NO:2, SEQ ID NO:3
or SEQ ID NO:5. cDNA molecules generated from alternatively spliced
mRNAs, which retain the properties of the Zdsc1 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.
[0100] In general, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (T.sub.m) for the
specific sequence at a defined ionic strength and pH. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched
probe.
[0101] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m hof the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0102] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polypeptide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
which influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m 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, Calif.), as
well as sites on the Internet, are available tools for analyzing a
given sequence and calculating T.sub.m based on user defined
criteria. Such programs can also analyze a given sequence under
defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated Tm. For smaller probes, <50 base pairs,
hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids.
[0103] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer. Generally,
incubations are carried out for a period equal to three times the
calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0104] The base pair composition of polynucleotide sequence will
effect the thermal stability of the hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contribute positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the T.sub.m, whereas
7-deazz-2'-deoxyguanosine can be substituted for guanosine to
reduce dependence on T.sub.m.
[0105] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). By decreasing the ionic
concentration of the buffer, the stability of the hybrid is
increased. Typically, hybridization buffers contain from between 10
mM-1 M Na.sup.+. The addition of destabilizing or denaturing agents
such as formamide, tetralkylammonium salts, guanidinium cations or
thiocyanate cations to the hybridization solution will alter the
T.sub.m of a hybrid. Typically, formamide is used at a
concentration of up to 50% to allow incubations to be carried out
at more convenient and lower temperatures. Formamide also acts to
reduce non-specific background when using RNA probes.
[0106] As an illustration, a nucleic acid molecule encoding a
variant Zdsc1 polypeptide can be hybridized with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1 (or its
complement) at 42.degree. C. overnight in a solution comprising 50%
formamide, 5.times.SSC (1.times.SSC: 0.15 M sodium chloride and 15
mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution (100.times. Denhardt's solution: 2% (w/v)
Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine
serum albumin), 10% dextran sulfate, and 20 .mu.g/ml denatured,
sheared salmon sperm DNA. One of skill in the art can devise
variations of these hybridization conditions. For example, the
hybridization mixture can be incubated at a higher temperature,
such as about 65.degree. C., in a solution that does not contain
formamide. Moreover, premixed hybridization solutions are available
(e.g., EXPRESSHYB Hybridization Solution from CLONTECH
Laboratories, Inc.), and hybridization can be performed according
to the manufacturer's instructions.
[0107] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. That is, nucleic acid molecules encoding a variant
Zdsc1 polypeptide hybridize with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1 (or its complement) under
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., including 0.5.times.SSC with 0.1% SDS at 55.degree. C., or
2.times.SSC with 0.1% SDS at 65.degree. C. One of skill in the art
can readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
[0108] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. In other words, nucleic acid
molecules encoding a variant Zdsc1 polypeptide hybridize with a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1
(or its complement) under highly stringent washing conditions, in
which the wash stringency is equivalent to 0.1.times.-0.2.times.SSC
with 0.1% SDS at 50-65.degree. C., including 0.1.times.SSC with
0.1% SDS at 50.degree. C., or 0.2.times.SSC with 0.1% SDS at
65.degree. C.
[0109] The present invention also provides isolated Zdsc1
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having at least 70%, at least 80%, at least 90%, at
least 95% or greater than 95% sequence identity to the sequences
shown in SEQ ID NO:2, or their orthologs. The present invention
also includes polypeptides that comprise an amino acid sequence
having at least 70%, at least 80%, at least 90%, at least 95% or
greater than 95% sequence identity to the sequence of amino acid
residues of SEQ ID NO:3. The present invention further includes
nucleic acid molecules that encode such polypeptides. Methods for
determining percent identity are described below.
[0110] The present invention also contemplates Zdsc1 variant
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:3, and a hybridization
assay, as described above. Such Zdsc1 variants include nucleic acid
molecules (1) that hybridize with a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO:1 (or its complement) under
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., and (2) that encode a polypeptide having at least 70%, at least
80%, at least 90%, at least 95% or greater than 95% sequence
identity to the amino acid sequence of SEQ ID NO:3. Alternatively,
Zdsc1 variants can be characterized as nucleic acid molecules (1)
that hybridize with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:1 (or its complement) under highly stringent
washing conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C., and (2)
that encode a polypeptide having at least 70%, at least 80%, at
least 90%, at least 95% or greater than 95% sequence identity to
the amino acid sequence of SEQ ID NO:2.
[0111] The present invention further provides a variety of other
polypeptide fusions [and related multimeric proteins comprising one
or more polypeptide fusions]. For example, a Zdsc polypeptide can
be prepared as a fusion to a dimerizing protein as disclosed in
U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing
proteins in this regard include immunoglobulin constant region
domains. Immunoglobulin-Zdsc1 polypeptide fusions can be expressed
in genetically engineered cells [to produce a variety of multimeric
Zdsc1 analogs]. Auxiliary domains can be fused to Zdsc1
polypeptides to target them to specific cells, tissues, or
macromolecules (e.g., collagen). For example, a Zdsc1 polypeptide
or protein could be targeted to a predetermined cell type by fusing
a Zdsc1 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 Zdsc1 polypeptide can be fused to two or
more moieties, such as an affinity tag for purification and a
targeting 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).
[0112] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, 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-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 aminoacylated suppressor
tRNAs.
[0113] 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 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 259:806-809 (1993); and Chung et al.,
Proc. Natl. Acad. Sci. USA 90:10145-10149 (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. 271:19991-19998
(1996). Within a third method, E. coli cells are cultured in the
absence of a natural amino acid that is to be replaced (e.g.,
phenylalanine) and in the presence of the desired non-naturally
occurring amino acid(s) (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. 33:7470-7476 (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).
[0114] 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 Zdsc1 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:
1081-1085 (1989); Bass et al., Proc. Natl. Acad. Sci. USA
88:4498-4502 (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. 271:4699-4708 (1996). Sites of ligand-receptor 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); Wlodaver et
al., FEBS Lett. 309:59-64, 1992.
[0115] Protein P
[0116] The Zdsc1 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 are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells, and
cultured higher eukaryotic cells. Eukaryotic cells, particularly
cultured cells of multicellular organisms, are preferred.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989), and Ausubel et al., eds., Current Protocols in Molecular
Biology, (John Wiley and Sons, Inc., NY, 1987).
[0117] In general, a DNA sequence encoding a Zdsc1 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0118] To direct a Zdsc1 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
Zdsc1, or may be derived from another secreted protein (e.g., t-PA)
or synthesized de novo. The secretory signal sequence is operably
linked to the Zdsc1 DNA sequence, i.e., the two sequences are
joined in the correct reading frame and positioned to direct the
newly synthesized polypeptide 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., Welch et al., U.S. Pat.
No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
[0119] Alternatively, the secretory signal sequence contained in
the polypeptides of the present invention is used to direct other
polypeptides into the secretory pathway. The present invention
provides for such fusion polypeptides. 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 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 as a receptor. Such fusions may
be used in vivo or in vitro to direct peptides through the
secretory pathway.
[0120] 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 14:725 (1978); Corsaro and
Pearson, Somatic Cell Genetics 7:603 (1981): Graham and Van der Eb,
Virology 52:456 (1973), electroporation, Neumann et al., EMBO J.
1:841-845 (1982), DEAE-dextran mediated transfection, Ausubel et
al., 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 (1989); Wang
and Finer, Nature Med. 2:714 (1996). The production of recombinant
polypeptides in cultured mammalian cells is disclosed, for example,
by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.
Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and
Ringold, U.S. Pat. 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 (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
Additional suitable cell lines are known in the art and available
from public depositories such as the American Type Culture
Collection, Rockville, Maryland. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable
promoters include those from metallothionein genes (U.S. Pat. Nos.
4,579,821 and 4,601,978) and the adenovirus major late
promoter.
[0121] 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 like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
Alternative markers that introduce an altered phenotype, such as
green fluorescent protein, or cell surface proteins such as CD4,
CD8, Class I MHC, placental alkaline phosphatase may be used to
sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0122] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58 (1987). Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). DNA
encoding the Zdsc1 polypeptide is inserted into the baculoviral
genome in place of the AcNPV polyhedrin gene coding sequence by one
of two methods. The first is the traditional method of homologous
DNA recombination between wild-type AcNPV and a transfer vector
containing the Zdsc1 flanked by AcNPV sequences. Suitable insect
cells, e.g. SF9 cells, are infected with wild-type AcNPV and
transfected with a transfer vector comprising a Zdsc1
polynucleotide operably linked to an AcNPV polyhedrin gene
promoter, terminator, and flanking sequences. See, King, L. A. and
Possee, R. D., The Baculovirus Expression System: A Laboratory
Guide, Chapman & Hall, (London); O'Reilly, D. R. et al.,
Baculovirus Expression Vectors: A Laboratory Manual, (Oxford
University Press, New York, 1994); and, Richardson, C. D., Ed.,
Baculovirus Expression Protocols. Methods in Molecular Biology,
(Humana Press, Totowa, N.J., 1995). Natural recombination within an
insect cell will result in a recombinant baculovirus which contains
Zdsc1 driven by the polyhedrin promoter. Recombinant viral stocks
are made by methods commonly used in the art.
[0123] The second method of making recombinant baculovirus utilizes
a transposon-based system described by Luckow, V. A, et al., J
Virol 67:4566-79 (1993). This system is sold in the Bac-to-Bac kit
(Life Technologies, Rockville, Md.). This system utilizes a
transfer vector, pFastBacl.TM. (Life Technologies) containing a Tn7
transposon to move the DNA encoding the Zdsc1 polypeptide into a
baculovirus genome maintained in E. coli as a large plasmid called
a "bacmid." The pFastBac1.TM. transfer vector utilizes the AcNPV
polyhedrin promoter to drive the expression of the gene of
interest, in this case Zdsc1. However, pFastBac1.TM. 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 (1990); Bonning, B. C.
et al., J Gen Virol 75:1551 (1994); and, Chazenbalk, G. D., and
Rapoport, B., J Biol Chem 270:1543 (1995). In such transfer vector
constructs, a short or long version of the basic protein promoter
can be used.
[0124] Moreover, transfer vectors can be constructed which replace
the native Zdsc1 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, Calif.), or baculovirus gp67
(PharMingen, San Diego, Calif.) can be used in constructs to
replace the native Zdsc1 secretory signal sequence. In addition,
transfer vectors can include an in-frame fusion with DNA encoding
an epitope tag at the C- or N-terminus of the expressed Zdsc1
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 Zdsc1 is transformed
into E. coli, and screened for bacmids which contain an interrupted
lacZ gene indicative of recombinant baculovirus. The bacmid DNA
containing the recombinant baculovirus genome is isolated, using
common techniques, and used to transfect Spodoptera frugiperda
cells, e.g. Sf9 cells. Recombinant virus that expresses Zdsc1 is
subsequently produced. Recombinant viral stocks are made by methods
commonly used the art.
[0125] 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 FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
are used to grow and maintain the cells. Suitable media are Sf900
II.TM. (Life Technologies) or ESF 921.TM. (Expression Systems) for
the Sf9 cells; and ExcellO405.TM. (JRH Biosciences, Lenexa, Kans.)
or Express FiveO.TM. (Life Technologies) for the T. ni cells. The
cells are grown up from an inoculation density of approximately 2-5
.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells at
which time a recombinant viral stock is added at a multiplicity of
infection (MOI) of 0.1 to 10, more typically near 3. The
recombinant virus-infected cells typically produce the recombinant
Zdsc1 polypeptide at 12-72 hours post-infection and secrete it with
varying efficiency into the medium. The culture is usually
harvested 48 hours post-infection. Centrifugation is used to
separate the cells from the medium (supernatant). The supernatant
containing the Zdsc1 polypeptide is filtered through micropore
filters, usually 0.45 .mu.m pore size. Procedures used are
generally described in available laboratory manuals (King, L. A.
and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;
Richardson, C. D., ibid.). Subsequent purification of the Zdsc1
polypeptide from the supernatant can be achieved using methods
described herein.
[0126] 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 therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. 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 POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
[0127] Suitable promoters and terminators for use in yeast include
those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat.
No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and
Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes.
See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and
4,661,454. Transformation systems for other yeasts, including
Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces
lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris,
Pichia methanolica, Pichia guillermondii and Candida maltosa are
known in the art. See, for example, Gleeson et al., J. Gen.
Microbiol. 132:3459 (1986) and Cregg, U.S. Pat. No. 4,882,279.
Aspergillus cells may be utilized according to the methods of
McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming
Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat.
No. 5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0128] 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 98/02565. DNA molecules for use in
transforming P. 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.
methanolica alcohol utilization gene (AUG1 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.
[0129] 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
proteinase genes (PEP4 and PRB1) 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 (.tau.) of from 1 to 40 milliseconds,
most preferably about 20 milliseconds.
[0130] 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 foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a Zdsc1 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.
[0131] 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. methanolica cells
are cultured in a medium comprising adequate sources of carbon,
nitrogen and trace nutrients at a temperature of about 25.degree.
C. to 35.degree. C. Liquid cultures are provided with sufficient
aeration by conventional means, such as shaking of small flasks or
sparging of fermentors. A preferred culture medium for P.
methanolica is YEPD (2% D-glucose, 2% Bacto.TM. Peptone (Difco
Laboratories, Detroit, Mich.), 1% Bacto.TM. yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0132] Protein Isolation
[0133] It is preferred to purify the polypeptides of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.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.
[0134] Expressed recombinant Zdsc1 polypeptides (or chimeric Zdsc1
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, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive 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 is a matter of routine design and is
determined in part by the properties of the chosen support. See,
for example, Affinity Chromatography: Principles & Methods,
(Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).
[0135] The polypeptides of the present invention can be isolated by
exploitation of their properties. For example, immobilized metal
ion adsorption (IMAC) chromatography can be used to purify
histidine-rich proteins, including those comprising polyhistidine
tags. Briefly, a gel 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.),pp.529-539 (Acad. Press, San Diego, 1990). 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. To direct the export of a receptor polypeptide from
the host cell, the receptor DNA is linked to a second DNA segment
encoding a secretory peptide, such as a t-PA secretory peptide.
[0136] 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 generated using known techniques
and expressed by the methods described herein. For example, part or
all of a domain(s) conferring a biological function may be swapped
between Zdsc1 of the present invention with the functionally
equivalent domain(s) from another family member. Such domains
include, but are not limited to, the secretory signal sequence,
conserved motifs [provide list if possible], and [significant
domains or regions in this family]. Such fusion proteins would be
expected to have a biological functional profile that is the same
or similar to polypeptides of the present invention depending on
the fusion constructed. Moreover, such fusion proteins may exhibit
other properties as disclosed herein. Zdsc1 polypeptides or
fragments thereof may also be prepared through chemical synthesis.
Zdsc1 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.
[0137] Antagonists are also useful as research reagents for
characterizing sites of ligand-receptor interaction. Inhibitors of
Zdsc1 activity (Zdsc1 antagonists) include anti-Zdsc1 antibodies
and soluble Zdsc1 receptors, as well as other peptidic and
non-peptidic agents (including ribozymes).
[0138] A Zdsc1 polypeptide can be expressed as a fusion with an
immunoglobulin heavy chain constant region, typically an F.sub.C
fragment, which contains two constant region domains and lacks the
variable region. Methods for preparing such fusions are disclosed
in U.S. Pat. Nos. 5,155,027 and 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 vitro assay tool,
antagonist). For use in assays, the chimeras are bound to a support
via the F.sub.C region and used in an ELISA format.
[0139] 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 (1949) and calorimetric
assays (Cunningham et al., Science 253:545 (1991); Cunningham et
al., Science 245:821 (1991).
[0140] Zdsc1 polypeptides can also be used to prepare antibodies
that specifically bind to Zdsc1 epitopes, peptides or polypeptides.
The Zdsc1 polypeptide or a fragment thereof serves as an antigen
(immunogen) to inoculate an animal and elicit an immune response. A
suitable antigen would be the Zdsc1 polypeptide encoded by from
amino acid number 36 to amino acid number 51,also defined by SEQ ID
NO:18, or a contiguous 9 amino acid residues or a fragment thereof.
Antibodies generated from this immune response 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,.sub.--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, N.Y., 1989); and
Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques
and Applications (CRC Press, Inc., Boca Raton, Fla., 1982).
[0141] Polyclonal antibodies can be generated from inoculating a
variety of warm-blooded animals such as horses, cows, goats, sheep,
dogs, chickens, rabbits, mice, and rats with a Zdsc1 polypeptide or
a fragment thereof. The immunogenicity of a Zdsc1 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 Zdsc1 or a portion thereof with an
immunoglobulin polypeptide or with maltose binding protein. The
polypeptide 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.
[0142] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.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.
[0143] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to Zdsc1 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled Zdsc1 protein or peptide). Genes encoding
polypeptides having potential Zdsc1 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., U.S.
Pat. NO. 5,223,409; Ladner et al., U.S. Pat. NO. 4,946,778; Ladner
et al., U.S. Pat. NO. 5,403,484 and Ladner et al., U.S. Pat. NO.
5,571,698) and random peptide display libraries and kits for
screening such libraries are available commercially, for instance
from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the Zdsc1 sequences disclosed
herein to identify proteins which bind to Zdsc1. These "binding
proteins" which interact with Zdsc1 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 proteins
can also be used in analytical methods such as for screening
expression libraries and neutralizing activity. The binding
proteins can also be used for diagnostic assays for determining
circulating levels of polypeptides; for detecting or quantitating
soluble polypeptides as marker of underlying pathology or disease.
These binding proteins can also act as Zdsc1 "antagonists" to block
Zdsc1 binding and signal transduction in vitro and in vivo.
[0144] Antibodies are determined to be specifically binding if: 1)
they exhibit a threshold level of binding activity, and 2) they do
not cross-react with related prior art polypeptide molecules.
First, antibodies herein specifically bind if they bind to a Zdsc1
polypeptide, peptide or epitope with a binding affinity (K.sub.a)
of 10.sup.6 M.sup.-1 or greater, preferably 10.sup.7 M.sup.-1 or
greater, more preferably 10.sup.8 M.sup.-1 or greater, and most
preferably 10.sup.9 M.sup.-1 or greater. The binding affinity of an
antibody can be readily determined by one of ordinary skill in the
art, for example, by Scatchard analysis, Scatchard, G., Ann. NY
Acad. Sci. 51: 660-672 (1949).
[0145] Second, antibodies are determined to specifically bind if
they do not significantly cross-react with related polypeptides.
Antibodies do not significantly cross-react with related
polypeptide molecules, for example, if they detect Zdsc1 but not
known related polypeptides using a standard Western blot analysis
(Ausubel et al., ibid.). Examples of known related polypeptides are
orthologs, proteins from the same species that are members of a
protein family (e.g. IL-16), Zdsc1 polypeptides, and non-human
Zdsc1. Moreover, antibodies may be "screened against" known related
polypeptides to isolate a population that specifically binds to the
inventive polypeptides. For example, antibodies raised to Zdsc1 are
adsorbed to related polypeptides adhered to insoluble matrix;
antibodies specific to Zdsc1 will flow through the matrix under the
proper buffer conditions. Such screening allows isolation of
polyclonal and monoclonal antibodies non-crossreactive to 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).
[0146] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which specifically bind to Zdsc1
proteins or peptides. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold
Spring Harbor Laboratory Press, 1988). Representative examples of
such assays include: concurrent immunoelectrophoresis,
radioimmunoassay, radioimmuno-precipitation, enzyme-linked
immunosorbent assay (ELISA), dot blot or Western blot assay,
inhibition or competition assay, and sandwich assay. In addition,
antibodies can be screened for binding to wild-type versus mutant
Zdsc1 protein or polypeptide.
[0147] Antibodies to Zdsc1 may be used for tagging cells that
express Zdsc1; for isolating Zdsc1 by affinity purification; for
diagnostic assays for determining circulating levels of Zdsc1
polypeptides; for detecting or quantitating soluble Zdsc1 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 Zdsc1 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 Zdsc1 or fragments thereof may be used in vitro to
detect denatured Zdsc1 or fragments thereof in assays, for example,
Western Blots or other assays known in the art.
[0148] Bioactive Conjugates:
[0149] 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 of the
present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule
(receptor or antigen, respectively, for instance). More
specifically, Zdsc1 polypeptides or anti-Zdsc1 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 anti-complementary
molecule.
[0150] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide or antibody, and 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). 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 polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0151] In another embodiment, 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 polypeptide has multiple
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 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 fusion
proteins thus represent a generic targeting vehicle for
cell/tissue-specific delivery of generic
anti-complementary-detectable/cytotoxic molecule conjugates.
[0152] The bioactive polypeptide or antibody conjugates described
herein can be delivered intravenously, intraarterially or
intraductally, or may be introduced locally at the intended site of
action.
[0153] Uses of Polynucleotide/Polypeptide:
[0154] Molecules of the present invention can be used to identify
and isolate receptors. For example, proteins and peptides of the
present invention can be immobilized on a column and membrane
preparations run over the column (Immobilized Affinity Ligand
Techniques, Hermanson et al., eds., pp.195-202 (Academic Press, San
Diego, Calfi., 1992,). Proteins and peptides can also be
radiolabeled (Methods in Enzymol., vol. 182, "Guide to Protein
Purification", M. Deutscher, ed., 721-37 (Acad. Press, San Diego,
1990,) 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 cell-surface proteins can be identified.
[0155] Gene Therapy:
[0156] Polynucleotides encoding Zdsc1 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit Zdsc1 activity. If a mammal has a mutated or absent Zdsc1
gene, the Zdsc1 gene can be introduced into the cells of the mammal
In one embodiment, a gene encoding a Zdsc 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 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 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. Clin. Invest. 90:626-30, 1992; and
a defective adeno-associated virus vector (Samulski et al., J.
Virol. 61:3096-101 (1987); Samulski et al., J. Virol. 63:3822-3828
(1989).
[0157] In another embodiment, a Zdsc1 gene can be introduced in a
retroviral vector, e.g., as described in Anderson et al., U.S. Pat.
No. 5,399,346; Mann et al. Cell 33:153 (1983); Temin et al., U.S.
Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., J. Virol. 62:1120 (1988); Temin et al., U.S. Pat.
No. 5,124,263; International Patent Publication No. WO 95/07358,
published Mar. 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 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 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 cellular
heterogeneity, such as the pancreas, liver, kidney, and brain.
Lipids may be chemically coupled to 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.
[0158] 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. 263:14621-14624 (1988).
[0159] Antisense methodology can be used to inhibit Zdsc gene
transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
Zdsc1-encoding polynucleotide (e.g., a polynucleotide as set froth
in SEQ ID NO:1) are designed to bind to Zdsc1-encoding mRNA and to
inhibit translation of such mRNA. Such antisense polynucleotides
are used to inhibit expression of Zdsc polypeptide-encoding genes
in cell culture or in a subject.
[0160] The present invention also provides reagents which will find
use in diagnostic applications. For example, the Zdsc1 gene, a
probe comprising Zdsc1 DNA or RNA or a subsequence thereof can be
used to determine if the Zdsc gene is present or if a mutation has
occurred. Detectable chromosomal aberrations at the Zdsc1 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).
[0161] Transgenic mice, engineered to express the Zdsc gene, and
mice that exhibit a complete absence of Zdsc gene function,
referred to as "knockout mice", Snouwaert et al., Science 257:1083
(1992), may also be generated, Lowell et al., Nature 366:740-42
(1993). These mice may be employed to study the Zdsc gene and the
protein encoded thereby in an in vivo system.
[0162] Chromosomal Localization:
[0163] Radiation hybrid mapping is a somatic cell genetic technique
developed for constructing high-resolution, contiguous maps of
mammalian chromosomes, Cox et al., Science 250:245-250 (1990).
Partial or full knowledge of a gene's sequence allows one to design
PCR primers suitable for use with chromosomal radiation hybrid
mapping panels. Radiation hybrid mapping panels are commercially
available which cover the entire human genome, such as the Stanford
G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc.,
Huntsville, AL). These panels enable rapid, PCR-based chromosomal
localizations and ordering of genes, sequence-tagged sites (STSs),
and other nonpolymorphic and polymorphic markers within a region of
interest. This includes establishing directly proportional physical
distances between newly discovered genes of interest and previously
mapped markers. The precise knowledge of a gene's position can be
useful for a number of purposes, including: 1) 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.
[0164] Sequence tagged sites (STSs) can also be used independently
for chromosomal localization. An STS is a DNA sequence that is
unique in the human genome and can be used as a reference point for
a particular chromosome or region of a chromosome. An STS is
defined by a pair of oligonucleotide primers that are used in a
polymerase chain reaction to specifically detect this site in the
presence of all other genomic sequences. Since STSs are based
solely on DNA sequence they can be completely described within an
electronic database, for example, Database of Sequence Tagged Sites
(dbSTS), GenBank, (National Center for Biological Information,
National Institutes of Health, Bethesda, Md.
http://www.ncbi.nlm.nih.gov), and can be searched with a gene
sequence of interest for the mapping data contained within these
short genomic landmark STS sequences.
[0165] For pharmaceutical use, the proteins of the present
invention are formulated for parenteral, particularly intravenous
or subcutaneous, delivery according to conventional methods.
Intravenous administration will be by bolus injection or infusion
over a typical period of one to several hours. In general,
pharmaceutical formulations will include a Zdsc1 protein in
combination with a pharmaceutically acceptable vehicle, such as
saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent
protein loss on vial surfaces, etc. Methods of formulation are well
known in the art and are disclosed, for example, in Remington: The
Science and Practice of Pharmacy, Gennaro, ed. (Mack Publishing
Co., Easton, Pa., 19th ed., 1995). Therapeutic doses will generally
be in the range of 0.1 to 100 .mu.g/kg of patient weight per day,
preferably 0.5-20 .mu.g/kg per day, with the exact dose determined
by the clinician according to accepted standards, taking into
account the nature and severity of the condition to be treated,
patient traits, etc. Determination of dose is within the level of
ordinary skill in the art. The proteins may be administered for
acute treatment, over one week or less, often over a period of one
to three days or may be used in chronic treatment, over several
months or years.
[0166] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Cloning of the Murine Zdsc1 Gene
[0167] SEQ ID NO:11, an Expressed Sequence Tag (EST) was discovered
in an EST data bank of an eosinophil cDNA library. The cDNA clone
corresponding to the EST was discovered and sequenced to give the
DNA sequence of SEQ ID NO:1. The mature protein is shown in SEQ ID
NO: 3.
EXAMPLE 2
Cloning of the Human Zdsc1 Gene
[0168] SEQ ID NO:12, an EST was discovered in an EST data bank of a
senescent human fibroblast cDNA library. The cDNA clone
corresponding to the EST was discovered, the clone ordered from the
IMAGE Consortium, Washington University School of Medicine and
sequenced to give the DNA sequence of SEQ ID NO:4. The mature
protein is shown in SEQ ID NO: 5.
EXAMPLE 3
Northern Blot Analysis of Zdsc1
[0169] Northern blot analysis was performed using mouse multiple
tissue blot and dot blot from Clontech (Palo Alto, Calif.) and
Mouse Multiple Tissue Blot from Origene (Rockville, Md.) using a
400 bp DNA probe containing the entire coding region of the Zdsc1
gene. The probe was radioactively labeled using .sup.32P using the
MULTIPRIME.RTM. DNA labeling system (Amersham, United Kingdom)
according to manufacturer's specifications. EXPRESSHYP.RTM.
solution (Clontech) was used for prehybridization and as a
hybridizing solution for the Northern analysis. Hybridization of
the probe on the blots took place overnight at 65.degree. C., and
the blots were than washed four times in 2.times. standard sodium
citrate (SCC) and 0.1% sodium dodecyl sulfate (SDS) at room
temperature, followed by two washes in 0.1.times.SSC and 0.1% SDS
at 50.degree. C. The blots were then exposed. only one strong
transcript was seen in liver for both multiple tissue blots. The
dot blot showed a strong dot for liver. A faint dot for spleen and
E. coli DNA was also seen.
[0170] 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.
Sequence CWU 1
1
23 1 365 DNA Mus musculus CDS (18)...(206) 1 catccttcag cagcagc atg
aag cta gga gcc ttc ctt ctg ttg gtg tcc 50 Met Lys Leu Gly Ala Phe
Leu Leu Leu Val Ser 1 5 10 ctc atc acc ctc agc cta gag gta cag gag
ctg cag gct gca gtg aga 98 Leu Ile Thr Leu Ser Leu Glu Val Gln Glu
Leu Gln Ala Ala Val Arg 15 20 25 cct ctg cag ctt tta ggc acc tgt
gct gag ctc tgc cgt ggt gac tgg 146 Pro Leu Gln Leu Leu Gly Thr Cys
Ala Glu Leu Cys Arg Gly Asp Trp 30 35 40 gac tgt ggg cca gag gaa
caa tgt gtc agt att gga tgc agt cac atc 194 Asp Cys Gly Pro Glu Glu
Gln Cys Val Ser Ile Gly Cys Ser His Ile 45 50 55 tgt act aca aac
taaaaacagc ttctacctgg aaaaaaaaat gtgtctgttt 246 Cys Thr Thr Asn 60
ggagctctgt gaccaagaaa acagttgaaa atggaggcca tgtatggaga ttacaagcag
306 cacagtggag tgggacaagg agttgtttct tttaataaat cattaatgta
aaagtctca 365 2 63 PRT Mus musculus 2 Met Lys Leu Gly Ala Phe Leu
Leu Leu Val Ser Leu Ile Thr Leu Ser 1 5 10 15 Leu Glu Val Gln Glu
Leu Gln Ala Ala Val Arg Pro Leu Gln Leu Leu 20 25 30 Gly Thr Cys
Ala Glu Leu Cys Arg Gly Asp Trp Asp Cys Gly Pro Glu 35 40 45 Glu
Gln Cys Val Ser Ile Gly Cys Ser His Ile Cys Thr Thr Asn 50 55 60 3
39 PRT Mus musculus 3 Ala Val Arg Pro Leu Gln Leu Leu Gly Thr Cys
Ala Glu Leu Cys Arg 1 5 10 15 Gly Asp Trp Asp Cys Gly Pro Glu Glu
Gln Cys Val Ser Ile Gly Cys 20 25 30 Ser His Ile Cys Thr Thr Asn 35
4 501 DNA Homo sapiens CDS (94)...(204) 4 gaattcggca cgaggcagca
acatgaagtt ggcagccttc ctcctcctgt gatcctcatc 60 atcttcagcc
tagaggtaca agagcttcag gct gca gga gac cgg ctt ttg ggt 114 Ala Gly
Asp Arg Leu Leu Gly 1 5 acc tgc gtc gag ctc tgc aca ggt gac tgg gac
tgc aac ccc gga gac 162 Thr Cys Val Glu Leu Cys Thr Gly Asp Trp Asp
Cys Asn Pro Gly Asp 10 15 20 cac tgt gtc agc aat ggg tgt ggc cat
gag tgt gtt gca ggg 204 His Cys Val Ser Asn Gly Cys Gly His Glu Cys
Val Ala Gly 25 30 35 taaggacagg taaaaacacc aggccctccc tgctttctga
aacgttgttc agtctagatg 264 aagagttatc ttaaggatca tctttcccta
agatcgtcat cccttcctgg agttcctatc 324 ttccaagatg tgactgtctg
gagttccttg actaggaaga tggatgaaaa cagcaagcct 384 gtggatggag
actacagggg atatgggagg cagggaagag gggttgtttc ttttaataaa 444
tcatcattgt taaaagcaaa aaaaaaaaaa aaaaaaaaaa aaaatggttg cggccgc 501
5 37 PRT Homo sapiens 5 Ala Gly Asp Arg Leu Leu Gly Thr Cys Val Glu
Leu Cys Thr Gly Asp 1 5 10 15 Trp Asp Cys Asn Pro Gly Asp His Cys
Val Ser Asn Gly Cys Gly His 20 25 30 Glu Cys Val Ala Gly 35 6 39
PRT Homo sapiens VARIANT (0)...(0) Xaa at amino acid position 1 is
Ala or is absent; 6 Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Gly Thr Cys Xaa
Glu Leu Cys Xaa 1 5 10 15 Gly Asp Trp Asp Cys Xaa Pro Xaa Xaa Xaa
Cys Val Ser Xaa Gly Cys 20 25 30 Xaa His Xaa Cys Xaa Xaa Xaa 35 7
40 PRT Homo sapiens 7 Ile Ile Leu Ile Arg Cys Ala Met Leu Asn Pro
Pro Asn Arg Cys Leu 1 5 10 15 Lys Asp Thr Asp Cys Pro Gly Ile Lys
Lys Cys Cys Glu Gly Ser Cys 20 25 30 Gly Met Ala Cys Phe Val Pro
Gln 35 40 8 24 PRT Homo sapiens 8 Met Lys Leu Gly Ala Phe Leu Leu
Leu Val Ser Leu Ile Thr Leu Ser 1 5 10 15 Leu Glu Val Gln Glu Leu
Gln Ala 20 9 6 PRT Homo sapiens 9 Leu Gln Leu Leu Gly Thr 1 5 10 6
PRT Homo sapiens 10 Asp Arg Leu Leu Gly Thr 1 5 11 371 DNA Mus
musculus 11 gcagcatgca agctaggagc cttccttctg ttggtgtccc tcatcaccct
cagcctagag 60 gtacaggagc tgcaggctgc agtgagacct ctgcagcttt
taggcacctg tgctgagctc 120 tgccgtggtg actgggactg tgggccagag
gaacaatgtg tcagtattgg atgcagtcac 180 atctgtacta caaactaaaa
acagcttcta cctggaaaaa aaaatgtgtc tgtttggagc 240 tctgtgacca
agaaaacagt tgaaaatgga ggccatgtat ggagattaca agcagcacag 300
tggagtggga caaggagttg tttcttttaa taaatcatta atgtaaaagt caaaaaaaaa
360 aaaaaaaatt g 371 12 448 DNA Homo sapiens 12 cagcaacatg
aagttggcag ccttcctcct cctgtgatcc tcatcatctt cagcctagag 60
gtacaagagc ttcaggctgc aggagaccgg cttttgggta cctgcgtcga gctctgcaca
120 ggtgactggg actgcaaccc cggagaccac tgtgtcagca atgggtgtgg
ccatgagtgt 180 gttgcagggt aaggacaggt aaaaacacca ggccctccct
gctttctgaa acgttgttca 240 gtctagatga agagttatct taaggatcat
ctttccctaa gatcgtcatc ccttcctgga 300 gttcctatct tccaagatgt
gactgtctgg agttccttga ctaggaagat ggatgaaaac 360 agcaagcctg
tggatggaga ctacagggga tatgggaggc agggaagagg ggttgtttct 420
tttaataaat catcattgtt aaaaagca 448 13 569 DNA Homo sapiens 13
gaggacccag ggtacacagg gtgggtggct attctccaga aatgtcagtt tctgggcagg
60 gcttaggtgt ctgcagtccc tagtcccacc cctggccttg cattccagct
cagcgagtgg 120 aaggtataaa tttcagctgc tctcagccct gctgtgtttt
tccaaagcct tccaacagca 180 acatgaagtt ggcagccttc ctcctcctgt
gatcctcatc atcttcagcc tagaggtaca 240 agagcttcag gctgcaggaa
gaccggcttt tgggtacctg cgtcgagctc tgcacaggtg 300 actgggactg
caaccccgga gaccactgtg tcagcaatgg gtgtggccat gagtgtgttg 360
cagggtaagg acagatgaag agttatctta aggatcatct ttccctaaga tcgtcatccc
420 ttcctggagt tcctatcttc caagatgtga ctgtctggag ttccttgact
aggaagatgg 480 atgaaaacag caagcctgtg gatggagact acagggggat
attggaagca aggaagaggg 540 gttgttcttt taataaatca tcattgtta 569 14 4
PRT Homo sapiens 14 Ala Ala Pro Val 1 15 4 PRT Homo sapiens 15 Ala
Ala Pro Phe 1 16 18 PRT Homo sapiens 16 Thr Cys Ala Glu Leu Cys Arg
Gly Asp Trp Asp Cys Gly Pro Glu Glu 1 5 10 15 Gln Cys 17 24 PRT
Homo sapiens VARIANT (0)...(0) Xaa can be any amino acid residue
except for cysteine 17 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa
Xaa Cys Cys Xaa Xaa 1 5 10 15 Xaa Cys Xaa Cys Xaa Xaa Xaa Cys 20 18
16 PRT Homo sapiens VARIANT (0)...(0) Xaa is any amino acid residue
except for cysteine 18 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Xaa Xaa Xaa Cys 1 5 10 15 19 17 PRT Homo sapiens VARIANT
(0)...(0) Xaa is any amino acid residue except for cysteine 19 Cys
Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Cys 20 18 PRT Homo sapiens VARIANT (0)...(0) Xaa is any amino
acid residue except for cysteine 20 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 1 5 10 15 Xaa Cys 21 14 PRT Homo
sapiens VARIANT (0)...(0) Xaa is any amino acid residue except for
cysteine 21 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys
1 5 10 22 15 PRT Homo sapiens VARIANT (0)...(0) Xaa is any amino
acid residue except for cysteine 22 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Cys Xaa Xaa Xaa Cys 1 5 10 15 23 26 PRT Homo sapiens
VARIANT (0)...(0) Xaa is any amino acid residue except for cysteine
23 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
1 5 10 15 Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys 20 25
* * * * *
References