CA2330187A1 - Disulfide core polypeptides - Google Patents

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.

Description

DISULFIDE CORE POLYPEPTIDES
BACKGROUND OF THE INVENTION
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.
However, when administered in vivo, aprotinin has been found to have a nephrotoxic effect in rats, rabbits and dogs after repeated injections of relativel~,~
high doses. The nephrotoxicity (appearing, i.e., in thEs form of lesions) observed for aprotinin might be ascribE~d to the accumulation of aprotinin in the proximal tubulus cells of the kidneys as a result of the high positive nest 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 o.f the inhibitor (such as cardiopulmonary bypass operations). Furthermore, aprotinin is a bovine protein, which may induce an immune response upon administration to humans.

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
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 Zdscl polypeptide). Murine Zdscl, SEQ ID NOs: 1 and 2 has a signal sequence extending from the methioni:ne at position 1 thraugh and including the alanine at position 24 of SEQ ID N0:2. The mature murine Zdscl polypeptide is also depicted by SEQ ID N0:3. SEQ ID N0:4 and 5 are examples of a mature human Zdscl polypeptide ;end polynucleotide which encodes it. A generic Zdscl polypeptide is exemplified by S:EQ ID N0:6.
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 N0:2, SEQ ID N0:3 or SEQ ID N0:5.
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 :LD N0:2, SEQ ID N0:3 or SEQ
ID N0:5.
Within an additional aspect of the invention there is provided a polynucleotide sequence which hybridizes under stringent cand:itions to either SEQ ID
NO:1 or SEQ ID N0:4 or to a complementary sequence of SF;Q
ID NO:l or to a complementary sequence of SEQ ID N0:4.

WO 99/63091 PCT/US99/12545~

Within an additional aspect of the invention there is provided a polynucleot_ide sequence which is at least 90%, 950, or 99% homologous to a polynucleotide sequence which encodes the polypeptide of SEQ ID N0:3 or SEQ ID N0:4.
Within another aspects of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a Zdscl polypeptide, containing an amino acid sequence as described above.
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 Zdscl polypeptide encoded by the DNA segment.
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 Zdscl polypeptide as described above. The invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
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 Zdscl antibody.

WO 99/63091 PCT/US99/1254:5 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
All references cited herein are incorporated in their entirety herein by reference.
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms.
The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, a:ny peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity t,ag.
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, FlagT"' peptide {Hopp et all. , Biotechnology 6:1204-10 (1988), streptavidin binding peptide, or other antigenic epitope or binding domain.
See, in general, Ford et aI, Protein Expression and Purification 2: 95-107 (1991). DNAs encoding affinity tags are available from commercial suppliers, (e. g., Pharmacia Biotech, Piscataway, NJ).

WO 99/63091 PCT/US99/12545~
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 5 phenotypic polymorphism within :populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
The 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 i:~
located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
The term "complement/anti-complement pair"
denotes non-identical moieties that form a non-covalentl_y associated, stable pair under appropriate conditions. F'or 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 dis:~ociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M-1.
The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequenc:e. For example, the sequence 5' ATGCACGGG 3~ is complementary to 5' CCCGTGC'AT
3'.
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.
The term "degenerate nucleotide sequence"
denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide).
Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.~e., GAU and GAC triplets each encode Asp).
The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segm<~nt encoding a polypeptide of interest operably linked to additional segments that provida_ 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.
The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetir_ milieu and is thus free of other extraneous or unwanted coding sequences, and i~~
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' u.ntranslated 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 316:774-7$ (1985).
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 a:nd animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It i;s preferred to provide the polypeptides in a highly purified form, i.e. greater than 95o pure, more preferably greater than 99o pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dime_rs or alternatively glycosylated or derivatized forms.
The term "operably linked", when referring to DNA segments, indicates that the=_ segments are arranged ;~o that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
The term "ortholog" denotes a polypeptide or protein obtained from one species that is the functiona7_ counterpart of a polypeptide or protein from a different.
species. Sequence differences among orthologs are the result of speciation.

"Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, (3-globin, and myoglobin are paral.ogs of each other.
A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleoti.de bases read from the 5' to the ?.' end. Polynucleot.ides 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 (abbreviate:d "bp"), nucleotides ("nt"), or k:ilobases ("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.
A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".
The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not: always, found in the 5' non-coding regions of genes.
A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-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 la are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
The term "receptor" denotes a cell-associated protein that binds to a 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 "mufti-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 molecules) in the cell. Thi;
interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are link<~d 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 inosito:l 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, WO 99/63091 PCT/US99/1254:5 IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence" denotes a 5 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 10 peptide during transit through the secretory pathway.
The term "splice variant" i_s used herein to denote alternative forms of RNA transcribed from a gene.
Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA
molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ~100.
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 al:L
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.
l0 Serine proteinase inhibitors fall into various str~actu:ral families, for example, the Kunitz family, the Kazal family, and.
the Hirudin family. The protein Zdscl 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 strand:
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 disul:Eide 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 che7_onianin, and human epididymal secretory protein E4, and trout TOFU-2, and C. Elegans C08G9. Several of these family members contain several copies of this structural motif.
Imbalances between native proteinases and a proteinase inhibitor is seen in patients where levels ofd 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-s disulfide core family of zdscl. 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 ela:Ein may prevent excessive elastase-mediated tissue proteoly:~is and damage.
Serine proteinase inhibitor activity can be measured using the method essentially described by Norris et al . , Biol. Chem. Hoppe-Seyle.r 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 x_00 mM NaCl, 50 mM Tris HC1, 0.01% TWEEN80 (Polyoxyethylenesorbitan monoleate) (pH 7.4) at 25°C.
After a 30 minute incubation, the residual enzymatic activity is measured by the degradation of a solution of:
the appropriate substrate as li:~ted 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 Ki i~;
calculated.

Table 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) Me0-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 Frei urg, 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 Fujikawa mM) University of Washington, Peptide Institute Seattle, WA Osaka, Japan human Factor Xa (3 nM) Me0-CO-CHA-Gly-Arg-pNA (0.3 mM) Dr. I. Schousboe NycoMed Copenhagen, Denmark Oslo, Norway rec. human Factor Vlla (300 H-D-lle-Pro-Arg-pNA (0.6 mM) nM) Novo Nordisk A/S Kabi Leukocyte Elastase Me0-Suc-Ala-Ala-Pro-Val-pNA
(0.6 mM) purified at Novo Nordisk (SEQ JD N0:14) A/S

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 (SEQ ID N0:15 A/S

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 kallikrein.
Inhibition assays were performed in microtiter wells in a total volume of 300 ~tl in 10 mM NaCl, 50 mM
Tris-HC1 (pH 7.4), 0.01% TWEEN80 (Polyoxyethylenesorbitan monoleate). Each reaction contained 1 ~M of the sample inhibitor and one of the proteases at the concentration listed in Table 1. The reactions were incubated at 25°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°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 1000 activity or 0%
inhibition.
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 ;end myocardial infarction. The amyloid protein precursor homologues of the present invention may be used, inter alia, to generate antibodies fo:r use in demonstrating tissue distribution of the precursor or for use in purifying such proteins.
Cysteines 3-8 in members of the four disulfide core family occur according to the motif:
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys ('ys Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys (SEQ ID N0:17) The residue Xaa can by any amino acid residue except for cysteine.
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:

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys (SEQ ID N0:18) Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys~ Xaa Xaa Xaa Xaa Xaa Xaa 5 Xaa Xaa Cys (SEQ ID N0:19) Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys (SEQ ID N0:20) 10 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys (SEQ ID N0:21) Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys (SEQ ID N0:22) The 8 cysteines in the four-disulfide core are bonded according to the pattern:
1-6, 2-7, 3-5, 4-8 Zdscl The protein Zdscl 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 t:he pattern:
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
N0:23) .
Zdscl is related by sequence homology to most of the four-disulfide core proteins, having the highest similarity t.o 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).
To further characterize the three-dimensional structure of Zdscl, including the disulfide bonding pattern and binding loop, we have constructed a homology model based on the NMR structure for porcine elafin, FLE, IO Francart, C. et al "Solution structure of R-elafin, a specific inhibitor of elastase", J. Mol. Biol. 26~: 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 Zdscl/2 can be predicted. See Grutter, M. et a.l., "The 2.5A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analyzed in its complex wii=h bovine alpha-chymotrypsin", EMBO J., 7: 345-351 (1988).
The 6 cysteines in Zdscl are bonded according to the pattern:
2-7, 3-5, 4-8 The reactive binding loop of Zdscl includes the sequence LQLLGT (SEQ ID NO: 9). Their active binding loop of human Zdsc includes the sequence DRLLGT (SEQ ID NO:
20). In Zdscl flanking residues around this binding loop are expected to interact with the target proteinase. Tree scissile bond is in the reactive binding loop between the two Leucines. Substitution at t:he P1 position (the second Leucine) is not tolerated as this residue is predicted t:o influence specificity towards the target proteinase, Bocle, W. and Huber, R. "Natural protein proteinase inhibitor;

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.
To predict the variation acceptable from positions G1n30 through Cys60 in Zdscl we have created a generalized motif which enumerates the permissible substitutions at each position.
MKLGAFLLLVSLITLSLEVQELQA (SEQ ID NO: 8) (The predicted signal sequence for Zdscl) FLE . IILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ
ID NO: 7) ZDSCl(m): AVRPLQLLGTCAELCRGDWDCGPEEQCVSIGCSHICTTN (SEQ
ID N0:3) ZDSC1(h): AGDRLLGTCVELCTGDWDCNPGDHCVSNGCGHECVAG (SEQ
ID N0:5) Multiple alignment between porcine elafin, and the predicted mature peptide for Zdscl. Cysteines 3-8 of FLE are labeled on the top of the alignment. Cysteines 1-6 of Zdscl are labeled on the bottom of the alignment, using the standard numbering for four-disulfide core proteins. Based upon the analysis of Zdscl and Zdse2 the following generic protein has been deduced as shown below in SEQ ID N0: 6.
SEQ ID N0:6 Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Gly Thr Cys Xaa Glu Leu Cys Xaa Gly Asp Trp Asp Cys Xaa Pro Xaa Xaa Xaa Cys Val Ser Xaa Gly Cys Xaa His Xaa Cys Xaa Xaa Xaa 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 i~~ Ala, Arg, Asn, Asp, Glu., Gln, Gly, His, Lys, Pro, Ser, or- Thr;
Xaa at amino acid position 25 i~~ 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;

WO 99/63091 PCT/US99/1254~i Xaa at amino acid position 35 i:~ 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 i:~ Val or Thr;
Xaa at amino acid position 38 is Ala or Thr; and Xaa at amino acid position 39 i:~ Asn or Gly.
Any resultant polypeptide based upon SEQ ID NO: 8 must be at least 80%, preferably 90 or 95o and most preferably 99o identical to SEQ ID NO: 3, SEQ ID NO: 5 or to SEQ ID N0:7.
POLYNUCLEOTIDES
The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the Zdsc polypeptide~s disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic cade, 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.
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;

WO 99/63091 PCT/US99/1254.5 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;
5 Isoleucine (Ile) is encoded by ATA, ATC or AT'.C;
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;
10 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, CC~C, 15 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;
20 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.
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 anti-sense 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 encodes 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).

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 1.3:355-64 (1981); Grosjean and Fiers, Gene 18:199-209 (1982); Holm, Nuc. Acids Res. 14:3075-87 (1986); Ikemura, J. Mol. Bio.l.
158:573-97 (1982). As used herein, the term "preferent:ial 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, yea:~t, viruses or bacteria, different 'L'hr 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 c:an be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
Within preferred embodiments of the invention the isolated polynucleotides wil.1 hybridize to similar sized regions of SEQ ID NO:1, SE:Q ID N0:4, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C
lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is WO 99Jb3091 PCT/US99/1254~5 the temperature (under defined .ionic strength and pH) at.
which 500 of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those ~_n which the salt concentration is up to about 0.03 M at pF3 7 and the temperature is at least about 60°C.
As previously noted, t=he 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 amount: of Zdsc1 RNA. Such tissues and cells are identified by Northern blotting, Thomas, Proc. Natl. Acad. Sci. LISA 77:5201 (1980), and include high expression of human Zdscl in the liver.
Total RNA can be prepared using guanidine HCl extractior.~.
followed by isolation by centrifugation in a CsCl gradient, Chirgwin et al., Biochemistry 18:52-94, 1979).
Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder, Proc . Na t1 . ficad . Sci . USA 69 : 14 0 8 -1412 (1972). Complementary DNA (cDNA) is prepared from poly(A)+ 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.
A full-length clone encoding Zdscl polypeptide can be obtained by conventianal 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.

WO 99/63091 PCT/US99/1254~5 Expression libraries can be probed with antibodies to Zdsc, receptor fragments, or other specific binding partners.
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 st=randed DNA is required f_or 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 1000. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. 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 & App.l.ications 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).
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 mRN'A
obtained from a tissue or cell type that expresses Zdsc as disclosed herein. Suitable sources of mRNA can be identified by probing Northern f>lots 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 pi°obes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR
(Mullis, U.S. Patent 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 Zdscl polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that t:he sequences disclosed in SEQ ID NO:1 and SEQ ID N0:4 represent a single alleles of murine Zdscl and human Zdscl respectively, and that allelic variation and alternative splicing are expected to occur. Allelic variants of thia sequence can be cloned by probing cDNA ar genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1, including those ~~ontaining 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 N0:2. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the Zdscl polypeptide are included within the scope of the present:
invention, as are polypeptides Encoded by such cDNAs and 5 mRNAs. Allelic variants and sp7_ice 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.
10 The present invention also provides isolated Zdscl polypeptides that are substantially identical to the polypeptides of SEQ ID N0:2, SEQ ID N0:3 and SEQ ID N0:5 and their orthologs. The term ''substantially identical"
is used herein to denote polypeptides having 50%, 15 preferably 600, more preferably at least 800, sequence identity to the sequences shown in SEQ ID N0:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID N0:2 or its orthologs.) Percent 20 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 25 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:
Total number of identical matches x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

~r ~ N M
r-~ I
L(7 N N O
I I
~ t-1 M N N
I I
I~ '-1 ~ V' M N
I i I I
lD d' N N n-I M ri I I I
tll O N ri rl rl '-1 ri I I I I I
tll ,-1 M r-I O rl M N N
I I I f I i I
a d'NNOMNr-iNri~
I I I I I I
VI N M r~ O M N '-I M rl M
N I I f 1 I I
x. CO M M r-1 N rl N rl N N N M
I I I r I I I I I I
C7 lf~ N ~ d' N M M N O N N M M
I J I I I I I I I i I
w f~ N O M M rl N M ri O ri frl N N
I I I i 1 f I I I I
l!1 N N O M N ~-1 O M rl O r-i N '-1 N
I I I 1 I i I I I
01MVIMMr-1r-1Mr-iNMw-I~-INNe-I
I I i I I I I 1 I I I I I i I
Ga l0 M O N e-~I ri M dl rl M M r-I O r-1 ~ M M
I I I I i I I I I r I I I
z lD '-1 M O O O rl M M O N M N ~-1 O d~ N M
i I I I I I t I I
A-i Lll O N M r-~ O N O M N N r-I M N rl '-i M N M
I I I I I I I I I I I I I
~. d' I-I N N O r-W -1 O N r-I ri r1 rl N ,-r ri O M N O
I i I 1 I I 1 1 I 1 I I I
I~ rx z c~ v a w c~ x H a x ~ ~, ~, ~n ~I 3 ~, ~
N
v .t7 u1 O In O
'"i f-i N

WO 99/63091 PCT/US99/1254:5 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 ident=ity shared by an amino acid sequence and the amino acid sequence of a putative variant. The FASTA algorithm is described by Pearson anc~
Lipman, Proc. Nat'1 Acad. Sci. C~SA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA
first characterizes sequence sirnilarity by identifying regions shared by the query sequence (e. g., SEQ ID N0:2) and a test sequence that have either the highest densit~~
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 t:en 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 "cvstoff"
value (calculated by a predetermined formula based upon the length of the sequence and t:he 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 she two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM ~7.
Appl. Math. 26:787 (1974), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=1, 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:Ei3 (1990) .
FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the letup value can range between one to six, preferably from four to six.
The present invention includes nucleic acid molecules that encode a polypept:ide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID N0:3 or with the amino acid sequence of SEQ ID N0:5. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 1_ocal multiple alignments of protein ~~equence segments, representing highly conserved regions of more than 500 groups of related proteins [Heni.koff and Henikoff, Proc.
Nat'1 Acad. Sci. USA 89:10915 (1.992)]. 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 95o and most preferably 99~

identical to SEQ ID N0:3 and which are able to stimulatE~
antibody production in a mammal, and said antibodies arf~
able to bind the native sequence of SEQ ID N0:3.
Variant Zdscl polypeptides or substantially identical Zdscl polypeptides arf=_ 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 (:gee Table 3) and other substitution: 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.
Table 3 Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid Table 3 cont.

aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine 10 Different species can exhibit "preferential colon usage." In general, see, Grantham et al., Nuc.
Acids Res. 8:1893 (1980), Haas er_ a1. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids Res.
15 19:3075 (1986), Ikemura, ~l. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin. Genet. Dev. Q:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995), and Makr.ides, Microbiol. Rev. 60:512 (1996). As used herein, the term "preferential colon usage" or "preferential colons" is a 20 term of art referring to protein translation colons that are most frequently used in cells of a certain species, thus favoring one or a few repre~;entatives of the possible colons encoding each amino acid. For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or 25 ACT, but in mammalian cells, ACC is the most commonly used colon; in other species, for example, insect cells, yeast, viruses or bacteria, different Th,r colons may be preferential. Preferential colons for a particular species can be introduced into the polynucleotides of the 30 present invention by a variety of methods known in the art. Introduction of preferential colon 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 colons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

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 Zdscl polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zdscl can be cloned using information and compositions providE~d 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 Zdscl as disclosed herein. Suitable sources c>f 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.
An Zdsc1-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete cr 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 Zdscl 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 Zdscl polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that the sequence disclosed in SEQ ID N0:1 represents a single allele of human Zdscl, 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 t=o standard procedures. Allelic variants of the nucleotide sequences shown in SEQ ID NO:l or SEQ ID N0:4, including those containing silent mutatio:zs and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are prote~_ns which are allelic variants of SEQ ID N0:2, SEQ ID N0:3 or SEQ ID N0:5. cDNA molecules generated from alternative7_y spliced mRNAs, which retain the properties of the Zdscl 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 i.n the art.
In general, stringent conditions are selected to be about 5°C lower than the thermal melting point ('~'my for the specific sequence at a defined ionic strength and pI-I.
The Tm is the temperature (under defined ionic strength and pH~ at which 50% of the target sequence hybridizes t:o a perfectly matched probe.
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 c:an tolerate mismatched base pairs ~n the double helix, but the stability of the hybrid is influenced by the degree of mismatch. The Tm of the mismatched hybrid decreases by 1°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°C below the Tm of the hybrid and a hybridization WO 99/63091 PCT/CJS99/1254~

buffer having up to 1 M Na+. Higher degrees of str:ingen.cy at lower temperatures can be achieved with the addition of formamide which reduces the Tm c.>f the hybrid about 1°C for each to formamide in the buffer solution. Generally, such stringent conditions include ternperatures of 20-70°'' anc~ a hybridization buffer containing up to 6x SSC and 0-500 formamide. A higher degree of stringency can be achieved at temperatures of_ from 40-70°C with a hybridization buffer having up to 4x SSC and from 0-50% formamide.
Highly stringent conditions typically encompass temperatures of 42-70°C with a hybridization buffer having up to lx SSC and 0-50o 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 pol~ynucleotide probes from hybridized complexes.
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 Tm 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 Tm 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 Tm are known in the art, and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 19$9);
Ausubel et al., (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and WO 99/63091 PCT/US99/1254!>

Kimmel (eds.), Guide to Molecul~3r Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.
Biochem. Mol. Bial. 26:227 (199C)}). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Prirr!er Premier 9.0 (Premier Biosoft International; Palo Alto, CA}, as well as sites on the Internet, are available tools for analyzing a given sequence and calculating Tm based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25°C below the calculated Tm. For smaller probes, <50 base pairs, hybridization is typically carried out at the Tm or 5-10°C
below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
The length of the polynucleotide sequence influences the rate and stability of hybrid formation.
Smaller probe sequences, <SO 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, t:he time it takes for the polynucleotide sequences to reassociate, can be calculated for a particular sequence by methods known in the art.
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 5 addition, the base pair composition can be manipulated t:o alter the Tm of a given sequence. For example, 5-methyldeoxycytidine can be substituted for deoxycytidine and 5-bromodeoxuridine can be substituted for thymidine to increase the Tm, whereas 7-deazz--2'-deoxyguanosine can be 10 substituted for guanosine to reduce dependence on Tm .
The ionic concentration of the hybridization buffer also affects the stability of the hybrid.
Hybridization buffers generally contain blocking agents 15 such as Denhardt's solution (Sigma Chemical Co., St.
Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO), heparin or SD~i, and a Na+ source, such as SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M NaCl, 1C1 mM NaH2P04, 1 mM EDTA, pH
20 7.7). By decreasing the ionic concentration of the buffer, the stability of the hybrid is increased.
Typically, hybridization buffers contain from between 1C
mM - 1 M Na+. The addition of destabilizing or denaturing agents such as formamide, tetralkylammonium salts, 25 guanidinium cations or thiocyanate cations to the hybridization solution will alter the Tm of a hybrid.
Typically, formamide is used at a concentration of up to 50o to allow incubations to be carried out at more convenient and lower temperatures. Formamide also acts to 30 reduce non-specific background when using RNA probes.
As an illustration, a nucleic acid molecule encoding a variant Zdscl polypeptide can be hybridized with a nucleic acid molecule having the nucleotide 35 sequence of SEQ ID NO:l (or its complement) at 42°C
overnight in a solution comprising 50$ formamide, SxSSC
(lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution (100x Denhardt's solution: 20 (w/v) Ficoll 400, 20 (w/v) polyvinylpyrrolidone, and 20 (w/v) bovine serum albumin), loo dextran sulfate, and 20 ~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°C, in a solution that does not contain formamide. Moreover, premixed hybridization solutions are available (e. g., EXPRESSHYB
Hybridization Solution from CLONTECH Laboratories, lnc.), and hybridization can be performed according to the manufacturer's instructions.
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 C.5x - 2x SSC
with 0.1o sodium dodecyl sulfate (SDS) at 55 - 65°C. That.
is, nucleic acid molecules encoding a variant Zdscl 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 0.5x - 2x SSC with 0.1o SDS at 55 - 65°C, including 0.5x SSC with O.lo SDS at 55°C, or 2xSSC with O.la SDS at 65°C. One of skill in the art can readily devise equivalent conditions, for example, by substituting SSPE for SSC in the wash solution.
Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0._Lo sodium dodecyl sulfate (SDS) at 50 - 65°C. In other words, nucleic acid molecules encoding a variant Zdscl 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 O.lx - 0.2x SSC with 0"l0 SDS at 50 - 65°C, including O.l:~c SSC with 0.1~ SDS at 50°C, or 0.2xSSC with C.lo SDS <~t 65°C.
The present invention also provides isolated Zdscl polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID N0:2, ox-their orthologs. The term "substantially similar sequence identity" is used herein to denote polypeptides having at least 700, at least 80°, at lea:~t 90%, at least 950 or greater than 95o sequence identity to the sequences shown in SEQ ID N0:2, or their orthologs. The present invention also includes polypeptides that comprise an amino acid sequence having at least 700, ar. least 800, at least 90%, at least 950 or greater than 95~> sequence identity to th.e sequence of amino acid residues of SEQ ID N0:3. The present invention further includes nucleic acid molecules that encode such polypeptides. Methods for determining percent identity are described below.
The present invention also contemplates Zdscl 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 N0:3, and a hybridization assay, as described above. Such Zdscl variants include nucleic acid molecules (1) that hybridize with a nucleic acid mo~.ecule having the nucleotide sequence of SEQ ID N0:1 (or it:s complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with O.lo SDS at 55 - 65°C, and (2) that encode a polypeptide having at least 70~, at least 80%, at least 900, at least 950 or greater than 95o sequence identity to the amino acid sequence of SEQ ID N0:3. Alternatively, Zdscl variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having t:he nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1x. - 0.2x SSC with 0.1%
SDS,at 50 - 65°C, and (2) that encode a polypeptide having at least 700, at least 800, at least 90°, at least 950 or S greater than 95o sequence identity to the amino acid sequence of SEQ ID N0:2.
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. Patents Nos.
5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains. Immunoglobulin-Zdscl polypeptide fusions can be expressed in genetically engineered cells [to produce a variety of multimeric Zdscl analogs]. Auxiliary domains can be fused to Zdscl polypeptides to target them to specific cells, tissues, or macromolecules (e. g., collagen). For example, a Zdsc~L polypeptide or protein could be targeted to a predetermined cell type by fusing a Zdscl 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 Zdsc_1 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 c'~.eavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9 (1996).
The proteins of the present invention can alsc>
comprise non-naturally occurring amino acid residues.
Non-naturally occurring amino acids include, without limitation, traps-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-dimethy:Lprol:ine, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluoroplzenylalanine. 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.
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 s~~stem 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 acids) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification.
5 Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci. 2:395-403 (1993}.
A limited number of non-conservative amino 10 acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zdscl amino acid residues. Essential amino acids in the polypeptides of the present invention can be identified according to 15 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 introducE:d 20 at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid re:~idues that are critical to the activity of the molecule. See also, Hilton et al., ,T.
Biol. Chem. 271:4699-4708 (1996). Sites of ligand-25 receptor or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques a:~ nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of 30 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 Let:t.
309:59-64, 1992.
PROTEIN PRODUCTION

The Zdscl 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, NY, 1989), and Ausubel et al., eds., Current Protocols in Molecular Biology, (John Wiley and Sons, Inc., NY, 1987).
In general, a DNA sequence encoding a Zdscl 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 marker:
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 matt=er of routine design within the level of ordinary skill in the art. Many such elements arE=_ described in the literature and are available through commercial suppliers.
To direct a Zdscl polypeptide into the secretary pathway of a host cell, a secret~ory signal sequence (also WO 99!63091 PCT/US99l1254:5 known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of Zdscl, 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 Zdscl 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., Welsh et al., U.S. Patent No. 5,037,743; Holland et al.,, U.S. Patent No. 5,143,830).
Alternatively, the secretory signal sequence contained in the polypeptides o.f the present invention .Ls 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 vitxo to direct peptides through the secretory pathway.
Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al., Cell WO 99/63091 PCT/US99/1254:5 14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981): Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumar~n 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. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC
No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No.
CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL
1573; Graham et al., J. Gen. Virol. 36:59-72 (1977) and Chinese hamster ovary (e. g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U. S. Patent Nos.
4,579,821 and 4,601,978) and the adenovirus major late promoter.
Drug selection is generally used to select fo:r 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 th~~
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 dihydrofolat~e reductase, which confers resistance to methotrexate.
Other drug resistance genes (e. g. hygromycin resistance, mufti-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, o:r 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 mean:
as FRCS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacteriurn rhizogenes as a vector for expressing genes in plant cells has been reviewed b~r Sinkar et al., J. Biosci. (Bangalore) 11:47-58 (1987).
Transformation of insect cells and production of foreign polypeptides therein is disclose=d by Guarino et al., U.~~.
Patent No. 5,162,222 and WIPO publication WO 94/06463.
Insect cells can be infected wit=h recombinant baculovirus, commonly derived from Autographs californica nuclear polyhedrosis virus (AcNPV). DNA encoding the Zdscl 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 Zdscl flanked by AcNPV
sequences. Suitable insect cells, e.g. SF9 cells, are infected with wild-type AcNPV and transfected with a 5 transfer vector comprising a Zdscl polynucleotide opera:bly linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences. See, King, L.A. and Possee, R.~~., The Baculovirus Expression System: A Laboratory Guide, Chapman & Hall, (London); O'Reilly, D.R. et al., 10 Baculovirus Expression Vectors: A Laboratory Manual, (Oxford University Press, New York, 1994); and, Richardson, C. D., Ed., Baculovi.rus Expression Protocols.
Methods in Molecular Biology, (Humans Press, Totowa, NJ,, 1995). Natural recombination within an insect cell wil:L
15 result in a recombinant baculov:irus which contains Zdscl driven by the polyhedrin promoter. Recombinant viral stocks are made by methods commonly used in the art.
The second method of making recombinant 20 baculovirus utilizes a transposon-based system described by Luckow, V.A, et al., J Viro1 67:4566-79 (1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBaclT"" (Life Technologies) containing a Tn7 transpoaon 25 to move the DNA encoding the Zdscl polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBaclT"" transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case Zdscl.
30 However, pFastBaclT"" can be modified to a considerable degree. The polyhedrin promoter- can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has 35 been shown to be advantageous far expressing secreted WO 99/63091 PCT/US99/1254:5 proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen Viro1 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.
Moreover, transfer vectors can be constructed which replace the native Zdscl secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native 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 Zdscl polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc Nat1 Acad Sci.
82:7952-4, 1985). Using a technique known in the art, a transfer vector containing Zdscl 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.q.
Sf9 cells. Recombinant virus that expresses Zdscl is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.
The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington,, D.C., 1994). Another suitable cell line is the High FiveOT"" cell line (Invitrogen) derived from Trichoplusia. ni (U. S. Patent #5,300,435). Commercially available serum-free media are used to grow and maintain the cells.
Suitable media are Sf900 IIr"" (Life Technologies) or ESF
921T"" (Expression Systems) for the Sf9 cells; and Ex-ce110405T"" (JRH Biosciences, Lenexa, KS) or Express FiveOT""
(Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 105 cells to a density of 1-2 x 106 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. The recombinant virus-infected cells typically produce the recombinant Zdscl 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 contain_Lng the Zdscl polypeptide is filtered through micropore filters, usually 0.45 ~m pore size. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the Zdscl polypeptide from the supernatant can be achieved using methods described herein.
Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyce~~
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.
Patent No. 4,599,311; Kawasaki et al., U.S. Patent No.
4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., WO 99/63091 PCT/US99/1254:5 U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or 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. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See al:~o U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces f.ragilis, 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. Patent No. 4,882,279.
Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No. ri,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S.
Patent No. 4,486,533.
The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO
Publications WO 97/17450, WO 97f17451, WO 98/02536, and WO
98/02565. DNA molecules for usE~ in transforming P.

WO 99/63091 PCT/US99i12545 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 o:E
the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA
sequences.
A preferred selectable marker for use in Pich:ia methanolica is a P. methanolica ADE2 gene, which encoder phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 ho:~t cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, ~.t is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG?) are deleted. For production of secreted proteins, host cells deficient in vacuolar proteinase genes (PEP4 and PR81) 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, pulsE:d 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 (T) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

WO 99/63091 PCT/US99/12545~
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 5 foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a Zdscl 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 periplasm_Lc 10 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 15 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 20 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.
25 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, 30 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 exogenous;ly 35 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-Sl transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of-carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanol.ica is YEPD (2$~ D-glucose, 2a BactoTM Peptone (Difco Laboratories, Detroit, MII, 1%
BactoTM yeast extract (Difco Laboratories), 0.0040 adenine and 0.0060 L-leucine).
Protein Isolation It is preferred to purify the polypeptides of the present invention to >_80o purity, more preferably to >_90% purity, even more preferably >_95% purity, and particularly preferred is a pharmaceutically pure state,.
that is greater than 99.9°s pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
Expressed recombinant Zdscl 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 WO 99/63091 PCT/US99/1254:5 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, cellulosi.c resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins a:nd 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 pari~ by the properties of tree chosen support. See, for example, Affinity Chromatography: Principles & Methods, (Pharmacia LKB
Biotechnology, Uppsala, Sweden, 1988).
The polypeptides of the present invention can be isolated by exploitation of their 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, WO 99/b3091 PCTNS99/12545 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 Enzyrnol., V~~l.
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 th~~
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 polypeptid~=_ 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.
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 domains) conferring a biological function may be swapped between Zdscl of the present invention with the functionally equivalent domain(:) from another family member. Such domains include, but are not limited to, the secretory signal sequence, cons~:rved motifs [provide lieu 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 th.e fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein. Zdscl 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.
Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction.
Inhibitors of Zdscl activity (Zdscl antagonists) include' anti-Zdsc1 antibodies and soluble Zdsc1 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
A Zdscl polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U.S. Patents Nos.
5,155,027 and 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 Fc region and used in an ELISA format.
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 245:8:21 (1991) .
Zdscl polypeptides can also be used to prepare antibodies that specifically bind to Zdscl epitopes, peptides or polypeptides. The Zdscl polypeptide or a fragment thereof serves as an anr_igen (immunogen) to inoculate an animal and elicit an immune response. A
suitable antigen would be the Zdscl polypeptide encoded by 5 from amino acid number 36 to amino acid number 5l, also defined by SEQ ID N0:18, or a contiguous 9 amino acid 5 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 t=he art. See, for example,_Current Protocols in Immunology,.
10 Cooligan, et al. (eds.), National Institutes of Health (John Wiley and Sons, Inc., 1995); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, NY, 1989); and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and 15 Applications (CRC Press, Inc., E3oca Raton, FL, 1982).
Polyclonal antibodies can be generated from inoculating a variety of warm-b7.ooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, 20 and rats with a Zdscl polypeptide or a fragment thereof.
The immunogenicity of a Zdscl 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 25 polypeptides, such as fusions of Zdscl 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 30 advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
As used herein, the term "antibodies" includes 35 polyclonal antibodies, affinity-purified polyclonal WO 99/63091 PCT/US99/125d5 antibodies, monoclonal antibod_~es, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments.
Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chair, antibodies and the like, as we~1 as synthetic antigen-binding peptides and polypeptides, are also included.
Non-human antibodies may be humanized by grafting IlOn-human CDRs onto human framework. and constant regions, cr by incorporating the entire norn-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residue::, 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 tc> humans is reduced.
Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to Zdscl protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zdscl protein or peptide). Genes encoding polypeptides having potential Zdscl 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 biologica_L or synthetic WO 99/63091 PCT/US99/1254a macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US
Patent NO. 5,223,409; Ladner et al., US Patent NO.
4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB
Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the Zdscl sequences disclosed herein to identify proteins which bind to Zdscl. These "binding proteins" which interact with Zdscl 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 under:Lying pathology or diseaae.
These binding proteins can also act as Zdscl "antagonist: s"
to block Zdscl binding and sign<~l transduction in vitro and in vivo.
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 herE~in specifically bind if they bind t;o a Zdscl polypeptide, peptide or epitope with a binding affinity (Ka) of 106 Nf 1 or greater, preferably 10~ M 1 or greater, more preferably 108 M 1 or greater, and most preferably 109 M 1 or greater.
The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, fo.r example, by Scatchard analysis, Scatchard, G., Ann. NY
Acad. Sci. 51: 660-672 (1949).
Second, antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly crass-react with related polypeptide molecules, for example, if they detect Zdscl 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), Zdscl polypeptides, and non-human Zd~~cl. 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 Zdscl will flow through the matrix under the proper buffer conditions.
Such screening allows isolatiot: of polyclonal and monoclonal antibodies non-crossreacti.ve 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); C~etzoff 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).
A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Zdscl 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 immunoelectrophoresi:~, radioimmunoassay, radioimmuno-precipitation, enzyme-lin)ted 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 Zdscl protein or polypeptide.
Antibodies to Zdscl may be used for tagging cells that express Zdscl; for isolating Zdscl by affinity purification; for diagnostic assays for determining circulating levels of Zdsclpolypeptides; for detecting or quantitating soluble Zdscl as marker of underlying pathology or disease; in analytical methods employing FRCS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block Zdsc~. 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 Zdscl or fragments thereof may be used in vitro to detect denatured Zdscl or fragments thereof in assays, for example, Western Blots or other assays known in the art.

BIOACTIVE CONJUGATES:
Antibodies or po.lypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for 10 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 15 instance). More specifically, Zdscl polypeptides or ant~i-Zdscl 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.
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, arid include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the 1-ike}, as well as therapeutic radionuclides, such as iodine-131, rhenium-1.88 or yttrium-90 (either directly attached to the polypepti.de 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.
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 o:r a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein includes a camplementary molecule, the anti-complementary molecule can be canjugated 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.
The bioactive polypeptide or antibody conjugates described herein can be delivera_d intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.
USES OF POLYNUCLEOTIDE/POLYPEPTIDE:
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 a~:., eds., pp.195-202 (Academic Pres:~, San Diego, CA, 1992,).

Proteins and peptides can also be radiolabeled (Methods in Enzymol., vol. 162, "Guide to Protein Purification", M.
Deutscher, ed., 721-37 (Acad. Press, San Diego, 1990,) ~~r photoaffinity labeled, Brunner et al., Ann. Rev. Bi:oche;m.
62:483-514 (1993) and Fedan et al., Biochem. Pharmacol.
33:1167-80 (1984) and specific cell-surface proteins can be identified.
GENE THERAPY:
Polynucleotides encoding Zdscl polypeptides a:re useful within gene therapy applications where it is desired to increase or inhibit Zdscl activity. If a mammal has a mutated or absent Zdscl 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 almo:~t 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 (HSVl) vector, Kaplitt et al., Molec. Cell. Neurosci.
2:320-30 (1991); an attenuated adenovirus vector, such as the vector described by Stratfoz-d-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).

In another embodiment, a Zdscl gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et~ al.
Cell 33:153 (1983); Temin et al., U.S. Patent No.
4,650,764; Temin et al., U.S. Patent No. 4,980,289;
Markowitz et al., J. Virol. 62:1120 (1988); Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al.; and Kuo et al., Blood 82:845 (1993).
Alternatively, the vector can be introduced by lipofect:ion 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. Aca~~.
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 transfecti021 to particular cell typE~s 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), protein;
such as antibodies, or non-peptide molecules can be coupled to liposomes chemically,.
It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid;
and then to re-implant the tran~~formed 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, DEAF dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., J.
Biol. Ch em. 267:963-7 (1992) ; Wu et a1. , J. Biol. C.hem.
263:14621-14624 (1988).
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 Zdscl-encoding polynucleotide (e.g., a polynucleotide as set froth in 3EQ
ID NO:1) are designed to bind to Zdscl-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.
The present invention also provides reagents which will find use in diagnostic applications. For example, the Zdscl gene, a probe comprising Zdsc1 DNA or RNA or a subsequence thereof can be used to determine if-_ the Zdse 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) analysi:~, short tandem repeat (STR) analysis employing PCR
techniques, and other genetic linkage analysis techniques known in the art (Sambrook et a:L., ibid.; Ausubel et. a~:, ibid.; Marian, Chest 108:255-65 (1995).

Transgenic mice, engineered to express the Zd:~c gene, and mice that exhibit a complete absence of Zdsc gene function, referred to as "knockout mice", Snouwaert=
5 et al., Science 257:1083 {1992), may also be generated, Lowell et al., Nature 366:740-42 (1993). These mice ma;r be employed to study the Zdsc gene and the protein encoded thereby in an in vivo system.
10 CHROMOSOMAL LOCALIZATION:
Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes, Cox 15 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 20 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, sequenc:e-tagged sites {STSs), and other nonpolymorphic and 25 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, 30 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 35 chromosomal region; and 3) cross-referencing model WO 99/63091 PCT/US99/1254~

organisms, such as mouse, which may aid in determining what function a particular gene might have.
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 o:r 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.
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 Zdscl 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 area disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed. (Mack Publishing Co., Easton, PA, 19th ed., 1995). Therapeutic doses will WO 99/63091 PCTlUS99/12545 generally be in the range of 0.1 to 100 ~g/kg of patient.
weight per day, preferably 0.5-:?0 ~tg/kg per day, with tree 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.
The invention is further illustrated by the following non-limiting examples..
Example 1 Cloning of the Murine Zdscl Gene 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 SEQ ID N0:12, 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, and sequenced to give the DNA sequence of SEQ ID N0:4. The mature protein is shown in SEQ ID NO: 5.
Example 3 Northern Blot Analysis of Zdscl WO 99/63091 PCT/US99i12545 Northern blot analysis was performed using mouse multiple tissue blot and dot blot from Clontech (Palo Alto, CA) and Mouse Multiple Tissue Blot from Origene (Rockville, Maryland) using a 400 by DNA probe containing the entire coding region of the Zdscl gene. The probe was radioactively labeled using 32P using the MULTIPRIME° DNA
labeling system (Amersham, United Kingdom) according to manufacturer s specifications. EXPRESSHYP~ 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° C, and the blot=s were than washed four times in 2X standard sodium citrate (SCC) and O.lo sodium dodecyl sulfate (SDS) at room temperature, followed by vwo washes in O.1X SSC and 0.1% SDS at 50° 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. col.i DNA was also seen.
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.

WO 99/b3091 PCT/US99/12545 SEQUENCE LIS~fING
<110> ZymoGenetics. Inc.
1201 Eastlake Avenue East Seattle, Washington 98102 United States of America <120> Disulfide Core Polypeptides <130> 98-13PC
<150> 09/090.895 <151> 1998-06-04 <160> 23 <170> FastSEQ for Windows Version 3.0 <210>1 <211>365 <212>DNA

<213>Mus musculus <220>
<221> CDS
<222> (18)...(206) <400> 1 catccttcag cagcagc atg aag cta gga gcc ttc ctt ctg ttg gtg tcc ~0 Met Lys Leu Gly Ala Phe Leu Leu Leu Val Ser ctc ate acc etc agc cta gag gta cag gag ctg cag get gca gtg aga 98 Leu Ile Thr Leu Ser Leu Glu Ual Gln Glu Leu Gln Ala Ala Val Arg cct ctg cag ctt tta ggc acc tgt get gag etc tgc cgt ggt gac tgg 1~~6 Pro Leu Gln Leu Leu Gly Thr Cys Ala Glu Leu Cys Arg Gly Asp Trp gac tgt ggg cca gag gaa caa tgt gtc agt att gga tgc agt cac atc 1!34 Asp Cys Gly Pro Glu Glu Gln Cys Val Ser Iie Gly Cys Ser His Ile tgt act aca aac taaaaacagc ttctacctgg aaaaaaaaat gtgtctgttt 246 Cys Thr Thr Asn ggagctctgt gaccaagaaa acagttgaaa atggaggcca tgtatggaga ttacaagcag ?.06 cacagtggag tgggacaagg agttgtttct tttaataaat cattaatgta aaagtctca ?65 <210>2 <211>63 <212>PRT

<213>Mus musculus <400> 2 Met Lys Leu Gly Ala Phe Leu Leu Leu Ual Ser Leu Ile Thr Leu Ser Leu Glu Val Gln Glu Leu Gln Ala Ala Ual Arg Pro Leu Gln Leu Leu Gly Thr Cys Ala Glu Leu Cys Arg Gly Asp Trp Asp Cys Gly Pro Glu Glu Gln Cys Ual Ser Ile Gly Cys Ser His Ile Cys Thr Thr Asn <210>3 <211>39 <212>PRT

<213>Mus musculus <400> 3 AlaUal ProLeu Gln Leu Leu Gly Thr Cys Ala Glu Leu Arg Cys Arg GlyAsp AspCys Gly Pro Glu Glu Gln Cys Ual Ser Ile Trp Gly Cys SerHis CysThr Thr Asn Ile <210>4 <211>501 <212>DNA

<213>Homo sapiens <220>
<221> CDS
<222> (94)...(204) <400> 4 WO 99/b3091 PCT/US99/12545 gaattcggca cgaggcagca acatgaagtt ggcagccttc ctcctcctgt gatcctcatc 60 atcttcagcc tagaggtaca agagcttcag get gca gga gac cgg ctt ttg ggt 114 Ala Gly Asp Arg Leu Leu Gly 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 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 C,ys Val Ala Gly taaggacaggtaaaaacaccaggccctccctgctttctgaaacgttgttcagtctagatg 264 aagagttatcttaaggatcatctttccctaagatcgtcatcccttcctggagttcctatc 324 ttccaagatgtgactgtctggagttccttgactaggaagatggatgaaaacagcaagcct 384 gtggatggagactacaggggatatgggaggcagggaagaggggttgtttcttttaataaa 444 tcatcattgttaaaagcaaaaaaaaaaaaaaaaaaaaaaaaaaatggttgcggccgc 501 <210>5 <211>37 <212>PRT

<213>Homo Sapiens <400> 5 AlaGly ArgLeu Leu Gly Thr Cys Val Glu Leu Cys Thr Asp Gly Asp TrpAsp AsnPro Gly Asp His Cys Val SE~r Asn Gly Cys Cys Gly His GluCys AlaGly Val <210>6 <211>39 <212>PRT

<213>Homo Sapiens <220>
<221> VARIANT
<222> (0)...(0>
<223> Xaa at amino acid position 1 is Ala or is absent:
Xaa at amino acid position 2 is Val or is absent:

WO 99/63091 PCT/US99/1254:5 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, Iie, 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;

WO 99/63091 PCT/US99/1254:5 <400> 6 Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Gly Thr Cys Xaa Glu Leu Cys Xaa Gly Asp Trp Asp Cys Xaa Pro Xaa Xaa Xaa Cys Val Ser Xaa Gly Cys Xaa His Xaa Cys Xaa Xaa Xaa <210>7 <211>40 <212>PRT

<213>Homo Sapiens <400> 7 Ile Ile Leu Ile Arg Cys Ala Met Leu Asn Pro Pro Asn Arg Cys Leu Lys Asp Thr Asp Cys Pro Gly Ile Lys Lys C,ys Cys Glu Gly Ser Cys Gly Met Ala Cys Phe Val Pro Gln <210>8 <211>24 <212>PRT

<213>Homo sapiens <400> 8 Met Lys Leu Gly Ala Phe Leu Leu Leu Val Ser Leu Ile Thr Leu Ser Leu Glu Val Gln Glu Leu Gln Ala <210>9 <211>6 <212>PRT

<213>Homo Sapiens <400> 9 Leu Gln Leu Leu Gly Thr <210>10 <211>6 <212>PRT

<213>Homo sapiens WO 99/63091 PCTNS99/125d5 <400> 10 Asp Arg Leu Leu Gly Thr <210>11 <211>371 <212>DNA

<213>Mus musculus <400> 11 gcagcatgcaagctaggagccttccttctgttggtgtccctcatcaccctcagcctagag60 gtacaggagctgcaggctgcagtgagacctctgcagctattaggcacctgtgctgagctc120 tgccgtggtgactgggactgtgggccagaggaacaatgtgtcagtattggatgcagtcac180 atctgtactacaaactaaaaacagcttctacctggaaaaaaaaatgtgtctgtttggagc240 tctgtgaccaagaaaacagttgaaaatggaggccatgt:atggagattacaagcagcacag300 tggagtgggacaaggagttgtttcttttaataaatcat;taatgtaaaagtcaaaaaaaaa360 aaaaaaaattg 371 <210>12 <211>448 <212>DNA

<2I3>Homo Sapiens <400>

cagcaacatgaagttggcagccttcctcctcctgtgatcctcatcatcttcagcctagag 60 gtacaagagcttcaggctgcaggagaccggcttttgggtacctgcgtcgagctctgcaca 120 ggtgactgggactgcaaccccggagaccactgtgtcagcaatgggtgtggccatgagtgt 180 gttgcagggtaaggacaggtaaaaacaccaggccctccctgctttctgaaacgttgttca 240 gtctagatgaagagttatcttaaggatcatctttccctaagatcgtcatcccttcctgga 300 gttcctatcttccaagatgtgactgtctggagttccttgactaggaagatggatgaaaac 360 agcaagcctgtggatggagactacaggggatatgggaggcagggaagaggggttgtttct 420 tttaataaatcatcattgttaaaaagca 448 <210>13 <211>569 <212>DNA

<213>Homo Sapiens <400>

gaggacccagggtacacagggtgggtggctattctccagaaatgtcagtttctgggcagg 60 gcttaggtgtctgcagtccctagtcccacccctggccttgcattccagctcagcgagtgg 120 aaggtataaatttcagctgctctcagccctgctgtgtttttccaaagccttccaacagca 180 acatgaagttggcagccttcctcctcctgtgatcctcatcatcttcagcctagaggtaca 240 agagcttcaggctgcaggaagaccggcttttgggtacctgcgtcgagctctgcacaggtg 300 actgggactgcaaccccggagaccactgtgtcagcaatgggtgtggccatgagtgtgttg 360 cagggtaagg acagatgaag agttatctta aggatcatct ttccctaaga tcgtcatccc 420 ttcctggagt tcctatcttc caagatgtga ctgtctggag ttccttgact aggaagatgg 480 atgaaaacag caagcctgtg gatggagact acagggggat attggaagca aggaagaggg :~40 gttgttcttt taataaatca tcattgtta X69 <210>14 <211>4 <212>PRT

<213>Homo Sapiens <400> 14 Ala Ala Pro Val <210>15 <211>4 <212>PRT

<213>Homo sapiens <400> 15 Ala Ala Pro Phe <210>16 <211>18 <212>PRT

<213>Homo Sapiens <400> 16 Thr Cys Ala Glu Leu Cys Arg Gly Asp Trp Asp Cys Gly Pro Glu Glu Gln Cys <210>17 <211>24 <212>PRT

<213>Homo Sapiens <220>
<221> VARIANT
<222> (0)...(0) <223> Xaa can be any amino acid residue except for cysteine <400> 17 Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Xaa Cys <210>18 <211>16 <212>PRT

<213>Homo Sapiens <220>
<221> VARIANT
<222> (0)...(0) <223> Xaa is any amino acid residue except for cysteine <400> 18 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210>19 <211>17 <212>PRT

<213>Homo Sapiens <220>
<221> VARIANT
<222> (0)...(0) <223> Xaa is any amino acid residue except for cysteine.
<400> 19 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys <210>20 <211>18 <212>PRT

<213>Homo Sapiens <220>
<221> VARIANT
<222> (0)...(0) <223> Xaa is any amino acid residue except for cysteine.
<400> 20 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210>21 <211>14 <212>PRT

<213>Homo Sapiens <220>
<221> VARIANT
<222> (0)...(0) <223> Xaa is any amino acid residue except for cysteine.
<400> 21 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210>22 <211>15 <212>PRT

<213>Homo sapiens <220>
<221> VARIANT
<222> (0)...(0) <223> Xaa is any amino acid residue except for cysteine.
<400> 22 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys <210>23 <211>26 <212>PRT

<213>Homo sapiens <220>
<221> VARIANT
<222> (0)...(0) <223> Xaa is any amino acid residue except for cysteine.
<400> 23 Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa WO 99/63091 PCT/US99/12545~
io Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys

Claims (8)

69 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 containing 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.

8. An isolated polypeptide comprised of SEQ ID
NO:6 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; anal Xaa at amino acid position 39 is Asn or Gly.