MXPA01000349A - Disintegrin homologs - Google Patents
Disintegrin homologsInfo
- Publication number
- MXPA01000349A MXPA01000349A MXPA/A/2001/000349A MXPA01000349A MXPA01000349A MX PA01000349 A MXPA01000349 A MX PA01000349A MX PA01000349 A MXPA01000349 A MX PA01000349A MX PA01000349 A MXPA01000349 A MX PA01000349A
- Authority
- MX
- Mexico
- Prior art keywords
- polypeptide
- seq
- molecule
- residues
- zdintl
- Prior art date
Links
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Abstract
The present invention relates to polynucleotide and polypeptide molecules for zdint1, a novel member of the Disintegrin Proteases. The polypeptides, and polynucleotides encoding them, are believed to be cell-cell interaction modulating and may be used for delivery and therapeutics. The present invention also includes antibodies to the zdint1 polypeptides.
Description
HOMOLOGOS DE DISINTEGRI AS
Field of the Invention
Disintegrin homologues are described from zdintl polypeptide molecules.
Background of the Invention
It has been shown that the integrins bind to surface cell molecules, such as platelet cells, fibroblasts, tumors, endothelial, muscles, neurons, bones and sperm. Disintegrin or disintegrins are 15 unique and potentially useful tools for the investigation of cell-matrix and cell-cell interactions. Additionally, they have been useful in the development of anti-thyroid and antimicrobial agents due to their anti-adhesive and anti-tumor properties of certain tumor cells and antigenegenic activities.
Protein families that have disintegrin domains include ADAMs (A 5 metalloprotease and integrin), MDCs
Ref. 126298
* '' 'S43W * ?? JS ** *. "" t i (Metalloprotease / Disintegrin / Cystein'a-rich) and SVMPs (Metalloprotease of Snake Venom).
For a review of ADAMs, see Olfsberg and White, Developmental Biology, 180: 389-401, 1996. ADAMs have been shown to exist as independent functional units or in conjunction with other members of this family in heterodimeric complexes. Some members of the family exist in multiple isoforms which may have resulted from an alternative splice. The ADAMs proteins have been shown to have adhesive as well as anti-adhesive functions. Some members of the ADAM family have a very specific tissue distribution while others are widely distributed. Not all members of this family are able to manifest all the potential functions represented by the domains common to their genetic structure.
ADAMs are characterized by having a propeptide domain, a domain similar to metalloprotease and a domain similar to disintegrin, a domain rich in cysteine, a domain similar to EGF and an opliotic cytotic domain.
aafeJMifcJfa An example of a prototype of this family is ADAM 12. ADAM 12, also known as meltrin a, has a truncated isoform, as well as a full-length isoform, and is involved in the fusion and differentiation of muscle cells (Gilpin et al. ., J. Biol. Chem. 273: 157-166, 1998).
Another prototypical example of this family is ADAM 1, which forms a heterodimer with ADAM 2 and is involved in sperm / egg fusion (Wolfsberg and White, supra).
The SVMP family is represented by three classes (P-I, P-II, P-III). All three classes contain domains of propeptides and met aloprot easas. Classes P-II and P-III also contain a domain of des int igor, and class P-III also contains a domain rich in cysteine. These domains are similar in sequence to those found in ADAMs. Some members of the SVMP family have a conserved amino acid sequence "RGD". This tripeptide has been shown to form a hairpin circuit whose conformation can break the fibrinogen bond with activated platelets. This sequence of RGD can be substituted for RSE, MVD, MSE and KGD in SVMPs P-II and by MSEC, RSEC, IDDC and RDDC (a tripeptide together with a carboxy-terminal cysteine residue) in the SVMPs of P-III. Thus, these sequences may be responsible for the binding of the mtegrin in the SVMPs of P-II and P-III.
A prototypical example of an SVMP is jararagine, which mediates platelet aggregation by binding to platelet subunit a2 (GPIa) through the disintegrin domain followed by proteolysis of the βx subunit (GPIIA) (Huang and Liu, J. Tox col-Toxin Reviews 16: 135-161, 1997).
Proteins from the family of metalloproteases / desint egrinas / cysteine rich, are involved in various tasks, ranging from roles in fertilization and muscle fusion, release of TNFa from plasma membranes, partition of mt cell proteins and essential functions in neuronal development (Blobel, Cell 90_: 589-592, 1997). This family is also characterized by the domains of
G. "^^ rtm ^^? metalloprotease, disintegrin and rich cysteine as described above.
The present invention provides a novel disintegrin homologue and related compositions whose uses should be apparent to those skilled in the art of these teachings.
Brief Description of the Invention
Within one aspect, the present invention provides an isolated polypeptide molecule comprising a contiguous sequence of 14 amino acids of SEQ ID NO: 2. Within one embodiment, the polypeptide molecule comprises residues 437 to 450 of SEQ ID NO: 2. Within another embodiment, the polypeptide molecule is between 82 and 232 amino acids in length. Within additional embodiments, the polypeptide molecule is from residues 164 to 382 of SEQ ID NO: 2; residues 383 to 464 of SEQ ID NO: 2; and / or residues 465 to 696 of SEQ ID NO: 2.
Within another aspect, the invention provides a polypeptide-isolated molecule selected from the group consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO: 2; b) a polypeptide molecule comprising residues 383 to 464 of SEQ ID NO: 2; c) a polypeptide molecule comprising residues 465 to 696 of SEQ ID NO: 2; d) a polypeptide molecule comprising residues 438 to 449 of SEQ ID NO: 2; e) a polypeptide molecule comprising residues 164 to 464 of SEQ ID NO: 2; f) a polypeptide molecule comprising
residues 164 to 696 of SEQ ID NO: 2; g) a polypeptide molecule comprising residues 383 to 696 of SEQ ID NO: 2; h) a polypeptide molecule comprising residues 164 to 449 of SEQ ID NO: 2; i) a polypeptide molecule
comprising the residues 438 to 696 of SEQ ID NO: 2; and j) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO: 2.
Within another aspect there is provided an isolated polynucleotide molecule encoding a polypeptide molecule, wherein the polypeptide molecule comprises a contiguous sequence of 14 amino acids of SEQ ID NO: 2. Within one embodiment, the polypeptide molecule comprises residues 437 to 450 of SEQ ID NO: 2. Within a further embodiment, the polypeptide molecule is between 82 and 232 amino acids
length. Within additional embodiments, the polypeptide molecule is residues 164 through 383 of SEQ ID NO: 2; residues 383 to 464 of SEQ ID NO: 2; and / or residues 465 to 696 of SEQ ID NO: 2.
Within another aspect, the invention provides an isolated polynucleotide molecule that encodes a polypeptide molecule, wherein
The polypeptide molecule is selected from the group consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO: 2; b) a polypeptide molecule comprising residues 383 to 464 of SEQ ID NO: 2; C) a polypeptide molecule comprising residues 465 to 696 of SEQ ID NO: 2; d) a polypeptide molecule comprising residues 438 to 449 of SEQ ID NO: 2; e) a polypeptide molecule comprising residues 164 to 464
of SEQ ID NO: 2; f) a polypeptide molecule comprising residues 164 to 696 of SEQ ID NO: 2; g) a polypeptide molecule comprising residues 383 to 696 of SEQ ID NO: 2; h) a polypeptide molecule comprising the residues
164 to 449 of SEQ ID NO: 2; i) a polypeptide molecule comprising residues 438 to 696 of SEQ ID NO: 2; and j) a molecule of
polypeptide comprising residues 1 to 696 of SEQ ID NO: 2.
Within another aspect, there is provided an isolated polynucleotide encoding a fusion protein having a first segment and a second segment, wherein the first segment comprises a first polypeptide encoding a polypeptide having a protease domain and the second segment comprises a second polynucleotide encoding a polypeptide having a contiguous sequence of 14 amino acids between residues 383 and 464 of SEQ ID NO: 2, and wherein the first segment is positioned at the amino terminus of the second segment. Within one embodiment, the protease domain is selected from the group consisting of; a) a protease domain that is a member of the disintegrin proteases; and b) a protease domain that is at least 80% identical to the amino acid residues 164 to 382 of SEQ ID NO: 2.
Within another aspect, the invention provides an isolated polynucleotide molecule that encodes a polypeptide molecule wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80 % identical to residues 383 to 464 of SEQ ID NO: 2; and b) a polynucleotide molecule that is complementary to a). Within one embodiment, the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80% identical to residues 383 to 696 of SEQ ID NO: 2; and b) a polynucleotide molecule that is complementary to a). Within a further embodiment, the polynucleotide molecule is selected from the group consisting of; a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80% identical to residues 1 to 696 of SEQ ID NO: 2; and b) a polynucleotide molecule that is complementary to a).
Within another aspect, an expression vector is provided comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment encoding the polypeptide of claim 1; and c) a transcription terminator. Within one embodiment, the DNA segment also encodes an affinity tag.
In another aspect, the invention provides a cultured cell into which said expression vector has been introduced, wherein the cell expresses the polypeptide encoded by the DNA segment.
Within another aspect, the invention provides a method for producing a polypeptide comprising the culture of the cell expressing the polypeptide encoded by the DNA segment; and the recovery of the polypeptide.
Within another aspect, a method for the modulation of cell-cell interactions is provided by combining the polypeptide comprising the sequence of 14 contiguous amino acids, with cells i n vi v e and n vi t ro. Within one embodiment, the cells are derived from tissues selected from the group consisting of: a) heart tissues; b) brain tissues; c) tissues of the spine; and d) skeletal muscle tissues.
Within another aspect, the invention provides an asylated polypeptide molecule comprising a contiguous amino acid sequence, wherein the contiguous sequence of
Y. ? ß? zjémik5íi? 6Í? & amp; B * amino acids is selected from the group consisting of: a) SEQ ID NO: 7; b) SEQ ID NO: 8; c) SEQ ID NO: 9; d) SEQ ID NO: 10; and e) SEQ ID NO: 11.
Within another aspect there is provided an isolated polynucleotide molecule encoding a polypeptide-isolated molecule, wherein the polypeptide comprises a contiguous amino acid sequence and is selected from the group consisting of: a) SEQ ID NO: 7; b) SEQ ID NO: 8; c) SEQ ID NO: 9; d) SEQ ID NO: 10; and e) SEQ ID NO: 11.
These and other aspects of the invention will become apparent with reference to the following detailed description of the invention and the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a Hopp / Woods hydrophilic property profile of the zdintl protein sequence shown in SEQ ID NO: 2. The profile is based on a sliding window of six residues. The buried waste G, S and T and the exposed waste H, Y and W were ignored. These residues are indicated in the figure by lowercase letters.
Figure 2 shows schematically an alignment of the domain level of members of ADAMs, MDCs and SVMPs. DISA_TRIGA is an SVMP. MS2_HUMAN is an ADAM. HSUTSP1 (TACE) is an MDC. And HSU52370_1 is a fertilin-ß, ADAM2. "sig" denotes the secretory signal peptide; "propep" denotes the propeptide domain; the "metal protease" denotes the metalloprotease domain; "disint" denotes the disintegrin domain; "cys" denotes the domain rich in cysteine; "RGD" denotes a tripeptide, arginine-glycine-asparagine; and "TMD" denotes a transmembrane domain.
Detailed description of the invention
Before establishing the invention in detail, it may be useful for the understanding of it to define the following terms:
The term "affinity tag" is used herein to denote a segment of polypeptide that can be placed on a second polypeptide to provide purification or detection of the second polypeptide or provide sites for the placement of the second polypeptide on a substrate. In the main, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a tract of pol i-hist idine, 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. Nati, Acad. Sci. USA 82: 7952-4, 1985), Flag peptide Hopp et al.
Biotechnology 6: 1204-10, 1988), streptavidin binding peptide or other antigenic epitope or binding domain. See generally, Ford, et al., Protein Expression and Purification 2: 95-107, 1991. Affinity labels encoding DNAs are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).
The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene that occupies the same chromosomal location. Allelic variation appears naturally through mutation, and can result in phenotypic polymorphism within populations. The gene mutations can be silent (without change in the encoded polypeptide) or they can encode polypeptides that
-4 # «» gifeM | S¿g have altered an 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 the polypeptides. Wherever the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a carboxyl terminal positioned in a certain sequence within a polypeptide is located near the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
The term "complement pair / / ant i-complement" denotes non-identical portions that form a stable pair associated in non-covalent form under suitable conditions. For example, biotin and avidin (or streptavidin) are prototypic members of a complement pair / ant i-complement. Other exemplary complement or / ant i-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs and the like. Where subsequent dissociation of the complement / ant i-complement pair is desirable, the complement / ant i-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 compared to a reference sequence. For example, the 5 'sequence ATGCACGGG 3' is complementary to the 5 'CCCGTGCAT 3'. The term "contig" denotes a polynucleotide having a contiguous extension of sequence identical or complementary to another polynucleotide. The contiguous sequences are said to "overlap"
a given extension of polynucleotide sequence either in its entirety or along a partial extension of the polynucleotide. For example, contigs representative of the 5'-ATGGAGCTT-3 'polynucleotide sequence are 5'-AGCTTgagt-25 3' and 3 '-tcgacTACC-5 *.
«JftSghlfart-to LMi ^ fafchwfe ^ wSBMfcftftJMw-.
The term "corresponding to", when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are aligned optimally.
The term "degenerate nucleotide sequence" denotes a nucleotide sequence 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 (that is, GAU tppletes and GAC each encode Asp).
The term "expression vector" is used to denote a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences and can also include one or more origins of replication, one or more selectable markers, an enrichment signal, a polyadenylation signal, etc. Expression vectors are generally derived from a plasmid or viral DNA, or may contain elements of both.
The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been separated from its genetic environment and is thus released from other undesirable or foreign coding sequences, and is in a form suitable for use within the protein production systems prepared by genetic engineering. Said isolated molecules are those that are separated from their natural environment and include genomic and cDNA clones. The isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. The identification of the associated regions will be apparent to someone of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985).
An "isolated" polypeptide or protein is a polypeptide or protein that is in a condition different from its natural environment, such as separated from animal tissue and blood. In a
In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, ie, more than 95% pure, more preferably more than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derived forms.
The term "operably linked", when referring to the DNA segments, indicates that the segments are placed so that they function in concert for the intended purposes, for example, the transcription is initiated at the promoter and proceeds through the encoder segment to the terminator.
The term "ortholog" denotes a polypeptide or protein obtained from a species that is a functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of species formation.
"Paralogos" are different but structurally related proteins made by an organism. Paralogos are thought to appear through gene duplication. For example, a-globin, b-globin and myoglobin are paralogos of each other.
A "polynucleotide" is a single or double filamentous polymer of soxir ibonucleotide or r ibonucleotide base read from the 5 'end to the 3' end. The polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vi, or prepared from the combination of natural and synthetic molecules. The sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the last two terms can describe polynucleotides that are double or single filament. When the term is applied to double-filament molecules, it is used to denote the global length and it will be understood that it is 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
IÜJES- Í r. .at £ r: ~ & 'result of the enzymatic partition; Thus, all nucleotides within a double stranded polynucleotide molecule may not be even. Such unpaired extremes will generally not exceed 20 nt in length.
A "polypeptide" is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or synthetically. The
polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".
The term "promoter" is used herein for its meaning recognized by the art, to denote a portion of DNA sequences containing genes that provide the binding of the RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes.
A "protein" is a macromolecule comprising one or more polypeptide chains. A
The protein may also comprise non-peptide components, such as the carbohydrate groups. Carbohydrates and other non-peptide substituents
they can be added to a protein by the cell in which the protein is produced, and will vary with the cell type. Proteins are defined here in terms of their core amino acid structures; Substituents such as carbohydrate groups are not generally specified but may nevertheless be present.
The term "receptor" denotes a protein associated with a cell that binds to a bioactive molecule (ie, a ligand) and mediates the effect of the ligand on the cell. Membrane-linked receptors are characterized by a muI t -domain or muI ti -peptide structure comprising an extracellular ligand-binding domain and an intracellular determinant domain that is typically involved in signal transduction. The binding of the ligand to the receptor results in a change in the conformation in the receptor, which causes an interaction between the domain of the determinant and another molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, calcium mobilization
ñ ^ m-fff > - Cell turnover, mobilization of membrane lipids, adhesion of cells, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be linked by membranes, cytosolic or nuclear; monomeric (eg, thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (eg, PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence" denotes a DNA sequence encoding 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 synthesize. The major polypeptide is commonly split to separate the secretory peptide during transit through the secretory pathway.
The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splicing variation occurs naturally through the use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and can result in several mRNAs transcribed from the same gene. The splice variants can encode polypeptides having an 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.
The molecular weights and lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) will be understood as approximate values. When such value is expressed as "around" X or "approximately" X, the set value of X will be understood to be accurate by + 10%.
All the references cited here are incorporated as a reference in their entirety.
The present invention is based on the discovery of a novel sequence of cDNA (SEQ ID NO: 1) and the corresponding polypeptide (SEQ ID NO: 2) having homology with family members similar to the des intg ers inas (ADAMs). , SVMPs and MDCs, referred to herein as Proteases of
Disintegrins, or "DPs"). See, for example, Blobel, Cell 9_0_: 589-592, 1997, and Wolfsberg and White, Developmental Biology 180: 389-401, 1996. Disintegrins can be involved in for example, 5 anticoagulation, fertilization, muscle fusion, connective tissues, chondrogenesis, arthritis, metastasis and neurogenesis.
The domain of the secretory signal (also
known as the leader sequence, pre-sequence or pre-sequence) of these polypeptides, directs the polypeptide through a secretory pathway of a cell in which it is synthesized. The secretory signal and the propeptide domain are
unfold from the full-length molecule, resulting in the mature form of the zdmtl polypeptide. The protease domain can be active or inactive. Some members of the disintegrin family have catalytic zinc sites
"assets" that can be regulated by a "cysteine switch" in the cysteine-rich domain. Examples of family members that have "active" protease domains are ADAM 1 and ADAM 10, which are involved in the fusion
sperm / egg and in the degradation of the myelin basic coating protein, respectively. Other members of this family do not
they have such a catalytic site and are "inactive." An example of a family member that contains an inactive protease domain is ADAM 11, which may be involved in the suppression of tumors. Other protein families that are known to have inactive protease domains are serine proteases.
The adhesion domain (disintegrin) of this protein binds to the integrin domains on the surface of a multitude of cells, depending on the specificity of the disintegrin. The predicted binding site within this disintegrin domain is often an amino acid circuit comprising about 13 amino acids. The conformation of this sequence when multiplied, results in a hairpin circuit that has an amino acid sequence at its tip. This sequence is often "RGP", but it can be replaced by a variety of other amino acid residues
(Wolfsberg and White, supra; and Ji a, J. Biol. Chem.
272: 13094-13102 1997). The diversity of these sequences may reflect that: 1) not all disintegrin domains serve as ligands for integrins (or other surface cell receptors); 2) the disintegrin domains with different sequences are linked to different types of surface cell receptors, or 3) the important part of the disintegrin structure circuit is its structure, not its sequence and so, that the receptors of the Specific classes of disintegrin domains can recognize a multitude of disintegrin link circuit sequences. Disintegrin domains have been shown to be responsible for cell-cell interactions, including the inhibition of platelet aggregation by binding GPIIb / IIIa (fibronectin receptor) and / or GPIa / IIa (collagen receptor) as well as the fusion of cells
Many members of the disintegrin family have a fusion domain, a relatively hydrophobic domain of about 23 amino acids. This domain is present within some of the members of the ADAM family, and has been shown to be involved in cell-cell fusion, and particularly in sperm / egg fusion and muscle fusion.
The cysteine-rich domain varies among members of the DP family and is thought to be
- > . * - & ^ ¿A ^ ^ ^ »vJ ^ .-, involved in structurally presenting the region int igr ina-link to the integrins.
Many members of the DP family have a transmembrane domain, which acts to anchor the polypeptide to the cell membrane.
The signaling domain of disintegrine family members tends to
preserved in length and sites for phosphorylation. However, beyond that, they tend to be unique in the composition of amino acids. Some members of the disintegrine family can point to the domain link
SH3 of Abl, Src and / or SH3 domains related to Src.
The zdmtl polypeptides of the present invention are a novel member of the family
DP. The presence of zdintl isoforms which also comprises a transmembrane domain, suggesting that the zdintl will have an alternatively spliced variant with a signaling domain. The novel zdintl polynucleotides encoding polypeptides of the present invention,
£., .., ..- ¿j < a ».: > j were initially identified by performing a Blast similarity search. An expressed sequence tag corresponding to nucleotides 1097 to 1415 of SEQ ID NO: 1 was used to obtain a clone that had been isolated from a collection of plasmids from infant brains.
The examination of the deduced amino acid zdintl sequence (SEQ ID NO: 2) allowed the
Identification of the following domains: a sequence of propeptides, ending at residue 163 of SEQ ID NO: 2; a protease sequence, residues 164 to 382 of SEQ ID NO: 2; a disintegrin sequence, residues 383 through 464
of SEQ ID NO: 2, and a cysteine-rich sequence, residues 465 to 696 of SEQ ID NO: 2. Within the disintegrin domain, there is a sequence of "disintegrin circuit", residues 438 to 449 of SEQ ID NO: 2. The
The amino acid sequence, ECD, which corresponds to residues 443 to 445 of SEQ ID NO: 2, is analogous to the "RGD link circuit" of some other members of the DPs.
The analysis of zdintl tissue distribution was carried out by the Northern spotting technique using Multiple Human Tissue,
Master and stained human vascular. Strong signals of three transcribed sizes, approximately 3.0 kb, 4.4 kb, and 7.5 kb, were observed in the heart over the Northern blotches of multiple tissues. Weak signals were observed on the same transcript sizes in the brain and spinal cord. Weaker signals of the three transcribed sizes were observed in the skeletal muscle. Spot Spotting showed stronger signals in the brain, heart, fetal brain and fetal heart. Human vascular staining showed a stronger signal at 3-3.5kb in human aortic endothelial cells and weaker signals in uniform aortic muscle cells and normal human lung fibroblast cells.
The zdintl protease domain had 49.5% identity in the protease domain of the closest family neighbor, ADAM 11 at the polypeptide level, and 58% identity at the polynucleotide level. The disintegrin domain of zdintl had 66.7% identity in the disintegrin domain of the closest family neighbor, ADAM 11, at the polypeptide level, and 64.3% identity at the polynucleotide level. The expression of ADAM 11 has been shown to decrease in breast cancer tissues and thus, it is suggested that it acts as a tumor suppressor in breast cancer (Emi et al., Nature Gen. 5_: 151-157, 1993) . Additionally, it has been shown that ADAM 11 has multiple isoforms as a result of an alternative splice.
Another protein that is an example of alternative splicing in DPs is ADAM 12, meltrin a. The truncated form of this molecule, which lacks the propeptide and metalloprotease domains, is associated with the formation of ectopic muscles in vi, but not in vitro, indicating that the cells that produce this gene produce a growth factor that it acts on vicinal progenitor cells.
Other ADAMs have been considered for the treatment of angioplasty, acute coronary syndrome, prevention of restenosis in implants, and prevention of excess adhesion following surgical procedures, prevention of metastasis, as well as for the degradation of specific proteins such as, for example, the precursor protein of ami loides.
POLYUCLEOTIDES
The highly conserved amino acids in the zdintl disintegrin domain can be used as a tool to identify new members of the family. For example, the reverse transcription polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved disintegrin domain of RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the zdintl sequences are useful for this purpose.
The present invention also provides polynucleotide molecules, including DNA and RNA molecules, which encode the zdintl polypeptides described herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable variation between these polynucleotide molecules is possible. SEQ ID NO: 3 is a degenerate DNA sequence encompassing all of the DNAs encoding the zdintl polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 3 also provides all of the RNA sequences encoding SEQ ID NO: 2 by replacing U with T. Thus, polynucleotides encoding zdintl polypeptides comprise from nucleotide 1 to nucleotide 2088 of SEQ ID NO: 3 and its RNA equivalents are contemplated by the present invention. Table 1 establishes the one-letter codes used within SEQ ID NO: 3 to denote the positions of degenerate nucleotides. The
"resolutions" are the nucleotides denoted by a code letter. The "complement" indicates the code for the nucleotide (s) complement anus (s). For example, the code Y denotes C Ó T, and its complement R denotes A or G, A is
complementary to T and G is complementary to C.
25 Í! «. ± í ím ^ sssigí i & i TABLE 1
Nucleotide Resolution Nucleotide Complement
AATTCCGGGGCCTTAARA / GYC / TYC / TRA / GMA / CKG / TKG / TMA / CSC / GSC / GWA / TWA / THA / C / TDA / G / TBC / G / TVA / C / GVA / C / GBC / G / TDA / G / THA / C / TNA / C / G / TNA / C / G / T
The degenerate codons used in SEQ ID NO: 3 which encompass all possible codons for a given amino acid are set forth in Table 2.
i * ^ ¿r ¿* ¡¡TABLE 2
Amino Acid Code Codon A Degenerate Letter Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG WSN Thr T TCT ACN Pro P ACÁ ACC ACG ACT CCN Wing A CCA CCC CCG CCT GCN Gly G GCA GCC GCG GCT GGN Asn N GCA GGC GGG GGT AAY Asp D AAC AAT GAY Glu E GAC GAT GAR Gln Q GAA GAG CAR His H CAA CAG CAY Arg R CAC CAT MGN Lys K AGA AGG CGA CGC CGG AAR Met M CGT ATG He I AAA AAG ATH Leu L ATG YTN Val V ATA ATC ATT GTN Phe F CTA CTC CTG CTT TTA TTY Tyr AND TTG TAY Trp W GTA GTTC GTG TGT TGT Ter TTC TTT TRR
Someone with ordinary skill in the art will appreciate that some ambiguity is introduced in determining the degenerate codon, representative of all possible codons that encode each amino acid. For example, the degenerate codon for serine (WSN) can under certain circumstances encode arginine (AGR), and the degenerate codon for arginine (MGN) can in certain circumstances encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence can encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences with reference to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be easily tested for functionality as described herein.
Someone with ordinary skill in the art will also realize that different species can exhibit a "preferential use of the codon". In general, see, Grantham, et al., Nuc. Acids Res. 8_: 1893-912, 1980; Haas, et al., Curr. Biol. _6: 315-24, 1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 18: 199-209, 1982; Holm, Nuc. Acids Res. 1 4_: 3075-87, 1986; Ikemura, J. Mol. Biol. 158: 573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of the art that refers to protein translation codons that are most frequently used in cells of certain species, thus favoring one or a few representatives of the possible codons that encode each amino acid (See Table 2). For example, the amino acid Treomna (Thr) can be encoded by ACA, ACC, ACG or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, may be preferential codons other than Thr. Preferred codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferential codon sequences within recombinant DNA can, for example, increase the production of protein by making protein translation more efficient within a particular type of cell or species. Therefore, the sequence of the degenerate codon described in SEQ ID NO: 3, serves as a template to optimize the expression of polynucleotides in various cell types and species commonly used in the art and described herein. The sequences containing preferential codons can be tested and optimized for expression in various species and tested for functionality as described herein.
Within the preferred embodiments of the invention, isolated polynucleotides are hybridized to similar sized regions of SEQ ID NO: 1, or a sequence complementary thereto, under severe conditions. In general, severe conditions are selected to be around 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence is hybridized to a perfectly coupled probe. Typical conditions
s, < to «Bfc» ?? t? ? «> The most severe are those in which the salt concentration is up to about 0.03 M at a pH of 7 and the temperature is at least about 60 ° C.
The isolated polynucleotides of the present invention include DNA and RNA. Methods for the preparation of DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zdintl RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Nati, Acad. Sci. USA 77: 5201, 1980), and include heart, brain, skeletal muscle, spine, fetal heart, and fetal brain. Total RNA can be prepared using guanidine extraction with HCl 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. Nati, Acad. Sci. USA 6J9: 1 08-12, 1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. In the alternative, genomic DNA can be isolated. The nucleic acids encoding zdintl polypeptides are then identified and isolated by, for example, hybridization or PCR.
A full-length clone encoding zdintl can be obtained by conventional cloning procedures. Complementary clones of DNA (cDNA) are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or modify a cDNA clone to include at least one genomic intron. The methods for the preparation of the cDNA and the genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence broken down here, or parts thereof, to probe or detonate a collection. Expression collections can be probed with antibodies to zdintl or other specific binding partners.
The nucleic acid sequences of zdintl described herein can also be used as probes or primers to clone the 5 'non-coding regions of a zdintl gene. In view of the tissue-specific expression observed for zdintl by Northern blotting, this region of genes is expected to supply the specific expression for heart, brain, spine and skeletal muscle. The promoter elements of
Thus, a zdintl gene can thus be used to direct the specific expression of tissues of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of boundary sequences 51 also facilitates the production of zdintl proteins by "gene activation" as described in U.S. Pat. No. 5,641,670. Briefly, the expression of an endogenous zdintl gene in a cell is altered by introducing into the zdintl site a DNA construct comprising at least one target sequence, a regulatory sequence, an exon and an unpaired splice donor site. The target sequence is a 5 'non-coding sequence of zdintl that allows for homologous recombination of the construct with the endogenous zdintl site, whereby the sequences within the construct become operably linked to the endogenous zdintl coding sequence. In this manner, an endogenous zdintl promoter can be substituted or supplemented with other regulatory sequences to provide enriched, tissue-specific or otherwise regulated expression.
The polynucleotides of the present invention can also be synthesized using DNA synthesizers. Currently the method of choice is the phosphoramidite method. If a chemically synthesized double-strand DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically direct and can be achieved by synthesizing the complementary filaments and then softening them. For the production of longer genes (> 300 bp) however, special strategies must be applied since the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic (double-stranded) genes are assembled in modular form, from single filament fragments having from 20 to 100 nucleotides in length. See Glick and Pasternak, Mol ecul a r Bi o techno lgy, Prin cipi es and Appl i ca t i ons of Recom bman t DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-356 (1984) and Cumie et al., Proc. Nati Acad. Sci. USA 87: 633-637 (1990).
The present invention also provides counterpart polypeptides and polypeptides from other species (orthologs). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fish and insects and other vertebrate and invertebrate species. Of particular interest are the zdintl polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine or other primate polypeptides. Human zdintl orthologs can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using the mRNA obtained from a type of tissue or cell that expresses zdintl as described herein. Such type of tissue or cell include it, for example, heart, brain, spine and skeletal muscle. The appropriate sources of mRNA can be identified by Northern spotting probes with probes designed from the sequences described herein. A collection is then prepared from mRNA of a positive tissue or cell line. A cDNA encoding zdintl can then be isolated by a variety of methods, such as probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the described 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 a representative human zdintl sequence described herein. Within a further 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 the zdintl polypeptide. Similar techniques can be applied for the isolation of genomic clones.
Those skilled in the art will recognize that the sequence described in SEQ ID NO: 1 represents a single allele of human zdintl and that allelic variation and alternative splicing is expected to occur. Allelic variants of this sequence can be cloned by probing the cDNA or genomic collections from different individuals according to normal procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, including those containing silent mutations and those in which mutations result in changes in the amino acid sequence, are within the scope of the present invention, as are the proteins that are allelic variants of SEQ ID NO: 2. The cDNAs generated from alternately spliced mRNAs, which retain the properties of the zdintl polypeptide, are included within the scope of the present invention, as are the polypeptides encoded by such cDNAs and mRNAs. The allelic variants and splice variants of these sequences can be cloned by probing the cDNA or the genomic collections of different individuals or tissues according to the normal procedures known in the art. The present invention also provides isolated zdintl polypeptides that are substantially homologous to the polypeptides of SEQ ID NO: 2 and their orthologs. The term
"Substantially homologous" is used herein to denote polypeptides having about 50%, preferably 60% and more preferably at least 70% and even more preferably 80% sequence identity to the sequences shown in SEQ ID.
NO: 2 or your orthologs. Such polypeptides will more preferably be at least 90% identical and most preferably 95% or more identical to SEQ ID NO: 2 or their orthologs. The percentage of sequence identity is determined by methods
conventional. See for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 89: 10915-9,
£ "a -» - ^ - ^^^^ 1 ^^ - »'1992. Briefly, two amino acid sequences are aligned to optimize the alignment records using a space opening penalty of 10, an extension penalty of space of 1, and the "blosum 62" recording matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by normal one-letter codes). The identity percentage is then calculated as:
Total number of identical matches x 100 (length of the longest sequence plus the number of spaces entered within the longest sequence in order to align the two sequences)
1? N CM CM or 1 1 > "3" rH O CM CM 1 1 1 a. r-l-H PO CM 1 1 1 1 1. vo CM CM rH ro 1 1 1
-C co O ro rH CM CM CM CM ro 1 1 1 1 1 1 1 ü V £ > CM CM ro ro CM CM CM CM ro ro 1 1 1 1 1 1 1 1 1 1? LO CM o ro PO rH O rH o rH PO CM CM 1 1 1 1 1 1 1 1 1
O m CN) CM or O CM rH O r or r H CM r-t CM 1 1 1 1 1 1 1 1 u s > O PO rH! -H M O CM CM rH 1 1 1 I 1 1 1 1 1 1 Q 1JD or CM rH • -I O ro PO o r-l ro ro 1 1 1 1 1 1 1 1 1 1 1
2 VD O or OO iH O PO o ro CN lH O CM PO 1 1 1 1 1 1 1 1 cr CM O n • -H o CM o PO CM CM ro CM rH ro CM PO 1 1 1 1 1 1 1 1 1 1 CM or O CM l? CM rH rH or PO CM 1 or 1 - 1 1 1 1 1 1 1 < 0- z Q? CU? or I 1-1 • J X s bμ Cu C? -E > >
The sequence identity of polynucleotide molecules is determined by similar methods using a ratio as described above.
Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lip an is an appropriate alignment method of proteins, to examine the level of identity shared by an amino acid sequence described herein and the amino acid sequence of a putative variant zdintl. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat A Acad. Sci. USA 85: 2444 (1988), and by Pearson, Meth. Enzymol. 183: 63 (1990).
Briefly, FASTA first characterizes the sequence similarity by identifying the regions shared by the interrogation sequence (e.g., SEQ ID NO: 2), and a test sequence that has either the highest density of identities (if the variable ktup is 1) or pairs of identities (if ktup = 2) without considering substitutions, insertions or conservative deletions of amino acids. The ten regions with the highest density of identities are further qualified by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "cut" to include only those residues that contribute to the highest rating. If there are several regions with ratings higher than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the initial cut regions are examined to determine if the regions can be joined to form a approximate alignment with the spaces. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 4_8_: 444 (1970); Sellers, SIAM J. Appl. Math 2_6: 787 (1974)), which allows the insertions and deletions of amino acids. The preferred parameters for the FASTA analysis are: ktup = l, penalty for space opening = 10, penalty for space extension = l, and substitution matrix = BLOSUM62. These parameters can be entered into a FASTA program by modifying the matrix rating file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 119 ^ 63 (1990).
FASTA can also be used to determine the identity of the sequence of nucleic acid molecules using a ratio as described above. For comparisons of the nucleotide sequences, the ktup value may be between one and six, preferably from three to six, more preferably three, with other parameters set by default.
The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, as compared to the amino acid sequence of SEQ ID NO: 2. Table BLOSUM62 is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 related protein groups (Henikoff and Henikoff, Proc. Nat '1 Acad. Sci USA 8_9: 10915 (1992)). In this manner, substitution frequencies BLOSUM62 can be used to define conservative amino acid substitutions that can 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 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. The preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2). or 3), whereas the most preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (eg, 2 or 3).
Zdintl variant polypeptides or polypeptides substantially homologous to zdintl are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is, they are conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the multiplication or activity of the polypeptide; minor eliminations, typically from one to about 30 amino acids; and the small amino- or carboxy-terminal extensions, such as an amino-terminal methionine residue, a small peptide linker of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides from 383 to 464 amino acid residues comprising a sequence that is at least 50%, preferably at least 60% and more preferably 80% or more identical to the corresponding region of SEQ ID NO: 2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zdintl polypeptide and the affinity tag. Such preferred sites include thrombin cleavage sites and factor Xa cleavage sites.
8! á & amp; & K & S & Table 4
Conservative amino acid substitutions
The present invention also provides a variety of polypeptide and related multimeric protein fusions comprising one or more polypeptide fusions. For example, a disintegrin polypeptide domain can be prepared as a fusion to a dimerizing protein, as described in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this respect include other disintegrin polypeptide domains or fragments of disintegrin polypeptide domains. Disintegrin polypeptide domain fusions, or mergers of disintegrin polypeptide domain fragments, can be expressed in cells prepared by genetic engineering, to produce a variety of multimeric analogues such as disintegrin. Auxiliary domain polypeptides can be fused to the desmtegrin domain polypeptides to direct them to specific cells, tissues or macromolecules (e.g., heart, brain, spine, skeletal muscle, platelets). For example, a protease polypeptide domain, or a protease polypeptide fragment or protein, can be targeted to a predetermined cell type by fusing it to a disintegrin polypeptide domain or fragment that specifically binds to an integrin or polypeptide. a integrin-like polypeptide on the surface of the target cell. In this way, the polypeptides, fragments of
In addition, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. Such domains or fragments of protease or disintegrin polypeptides can be fused to two or more portions, such as an affinity tag for purification and a disintegrin target domain. Polypeptide fusions may also comprise one or more cleavage sites, particularly between domains. See Tuan et al., Connective Tissue Research 34: 1-9, 1996.
The polypeptide fusions of the present invention will generally contain no more than about 1,500 amino acid residues, preferably no more than about 1,200 residues, more preferably no more than about 1,000 residues and will be considerably smaller in many cases. For example, residues of the zdintl polypeptide can be fused to the β-galactosidase of E. col i (1021 residues, see Casadaban et al., J. Bacteriol., 143: 971-980, 1980, a spacer of 10 residues, and a factor Xa cleavage site of residue 4. In a second example, residues of zdintl polypeptide can be fused to the maltose-binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhist idine label.
The proteins of the present invention may also comprise amino acid residues that occur non-naturally. Amino acids that occur naturally include, without limitation, t -rans-3-methyproline, 2,4-me tanoproline, cis-4-hydroxyproline, t -rans-4-hydroxyproline, N-methylic, alo-thonin, Met ilt reonin, hydroxyethylcysteine, hydroxyethyl lhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and -methylproline, 3, 3-dimethoprol ina, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4 -a za feni lalanina, and 4-f luorofeni lalanina. Various methods are known in the art to incorporate amino acid residues that occur not naturally within proteins. For example, an i n vi t ro system may be employed where nonsense mutations are suppressed using chemically aminoacylated suppressors tRNAs. Methods for synthesizing amino acids and amino acid tRNAs are known in the art. The transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an S30 extract of E. col i and commercially available enzymes and other reagents. The proteins are purified by chromatography. See for example, Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-9, 1993; and Chung et al., Proc. Nati Acad. Sci. USA 9_0: 10145-9, 1993). In a second method, the
The translation is carried out in Xen opus oocytes by microinjection of mutated mRNA and chemically ammoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 1999-1-8, 1996). Within a third method, cells are cultured
E. co li in the absence of a natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the naturally occurring amino acid (s) (e.g., 2-azaphenylalanine, 3-azaphene lalanine, 4) - 20 azaphenylalanine or 4-fluorophenylalanine). The amino acid that occurs is not naturally incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem 3_3_: 7470-6, 1994. The amino acid residues that
occur naturally, can be converted to species that occur not naturally by a chemical modification i n vi t ro. The modification
a »a ^ Ajah» a > Thus, chemistry can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).
A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids and unnatural amino acids, can be substituted for zdintl amino acid residues.
The essential amino acids in the polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Nati, Acad. Sci. USA 88: 4498-502, 1991). In the latter technique, simple alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested for biological activity as described below, to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-708, 1996. Disintegrin-integrin or protease interaction sites can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photophase tagging, in conjunction with putative amino acid mutation of contact site. See, for example, de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Biol. 224: 899-904,
1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identities of the essential amino acids can also be inferred from the analysis of homologies with related molecules similar to the integrin. 15 Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those described by Reidhaar-Olson and Sauer (Science
241: 53-7, 1988) or Bowie and Sauer (Proc. Nati, Acad. Sci. USA 8_6: 2152-6, 1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide,
and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Others
&J, methods that can be used include phage display (e.g., Lowman et al., Biochem, 3_0: 10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 4_6: 145, 1986, Ner et al., DNA 1_: 12 1, 1988).
Variations of the described zdintl DNA and polypeptide sequences can be generated through DNA redistribution, as described by Stemmer, Nature 370: 389-91, 1994, Stemmer, Proc. Nati Acad. Sci. USA jU: 10747-51, 1994 and WIPO publication WO 97/20078. Briefly, variant DNAs are generated by homologous recombination in vi t ro by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assays provide a rapid "evolution" of sequences when selecting mutations
Jb?? SH desirable while simultaneously selecting against damaging changes.
Mutagenesis methods such as those described herein can be combined with high throughput automatic screening methods to detect the activity of mutagenized polypeptides, cloned into host cells. The mutagenized DNA molecules that encode
Active polypeptides (e.g., cell-disintegrin surface binding or protease activity) can be recovered from host cells and rapidly sequenced using modern equipment. These methods allow the
rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
Using the methods described herein, one of ordinary skill in the art can identify and / or prepare a variety of fragments of polypeptides or variants of SEQ ID NO: 2 or that which retains disintegrin and / or activity of the
protein protease zdintl of irregular type. Such polypeptides may include additional amino acids of, for example, a secretory domain,
a propeptide domain, a protease domain, part or all of intracellular and transmembrane domains, including amino acids responsible for intracellular signaling; fusion domains; Affinity tags; and similar.
For any zdintl polypeptide, including variants and fusion proteins, one of ordinary skill in the art can easily generate a completely degenerate polynucleotide sequence encoding that variant using the information set forth in the above Tables 1 and 2.
PROTEIN PRODUCTION
The zdintl polypeptides of the present invention, including full length polypeptides, biologically active fragments and fusion polypeptides, can be produced in host cells prepared by genetic engineering in accordance with conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells and cultured higher eukaryotic cells. Preferred
eukaryotic cells, particularly cultured cells of multicellular organisms. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2a. 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 zdintl polypeptide is operably linked to other genetic elements required for its expression, which generally include a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers can be provided on separate vectors, and replication of the exogenous DNA can be provided by the integration within the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial providers.
To direct a zdintl polypeptide into a secretory path of a host cell, a secretory signal sequence (also known as leader sequence, sequence) is provided.
prepro or pre sequence) in the expression vector. The secretory signal sequence can be derived from another protein secreted (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the
The sequence of zdintl DNA, that is, the two sequences are linked in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory path of the host cell. The
secretory signal sequences are commonly positioned 5 'to the DNA sequence encoding the polypeptide of interest, although certain sequences of secretory signals can be positioned anywhere in the sequence of
DNA of interest (see, for example, Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). The
? SSS, fragments of peptides and polypeptides of the present invention are considered biologically active in the absence of the native signal sequence.
The protease domain of zdintl can be replaced by a heterologous sequence that provides a different protease domain. In this case, the fusion product can be segregated and the disintegrin domain of zdintl can direct the protease domain to the specific tissue described above. This substituted protease domain can be chosen from the protease domains represented by the DP protein families, or domains of other known proteases.
Cultured cells of mammals are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (er et al., Cell 1_4_: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981: Graham and Van der Eb, Virology 5_2: 456, 1973), electroporation (Neumann et al., EMBO J. A841-5, 1982), transfection mediated by DEAE-dext rano (Ausubel et al., ibid.), "and transfection mediated by liposomes (Hawley-Nel son et al., Focus 15:73, 1993, Ciccarone et al., Focus _15_: 80, 1993, and viral vectors (Miller and Rosman, BioTechniques 5: 980"90 '1989; Finer, Nature Med. 2: 714-6, 1996) The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., U.S. Patent No. 4,713,339, Hagen et al.
American U.S. No. 4,784,950; Palmiter et al., U.S. No. 4,579,821; and Ringold, U.S. No. 4,656,134. Appropriate mammalian cultured cells include 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. 3_6: 59-72, 1977) and Chinese hamster ovarian cell lines (e.g. CHO-K1; ATCC No. CCL 61). Cell lines are known
Additional appropriate in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters, such as SV-40 promoters, are preferred.
cytomegalovirus. See, for example, U.S. No. 4,956,288. Other appropriate promoters include those of genes from the
ionein metalot (US Patent Nos. 4,579,821 and 4,601,978) and the major late promoter of the adenovirus.
The drug selection is generally used for cultured cells of mammals into which the outer 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 capable of passing the gene of interest to their progeny are referred to as "trans fect ant are stable". A preferred selectable marker is a gene that encodes resistance to antibiotic neomycin. The selection is carried out in the presence of a neomycin-type medicament, such as G-418 or the like. Selection systems can also be used to increase the level of expression of the gene of interest, a process referred to as "amplification". The amplification is carried out by the culture transfectants in the presence of a low level of a selective agent and then increasing the amount of selective agent to select the cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is the reductase of
rr_r ^ rÁn ^ dihydrofolate, which confers resistance to methotrexate. Other drug resistance genes can also be used (for example, hygromycin resistance, immunomodulatory resistance, puromycin acetyl transferase). Alternate markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins, such as CD4, CD8, MHC class I or placental alkaline phosphatase, can be used to order transfected cells from non-transfected cells by means such as FACS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and bird cells. The use of Agroba c t eri um rh i zogen is as a vector for expressing genes in plant cells, it has been reviewed by Sinkar et al., J. Biosci. (Banqalore) 11: 47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is described by Guarino et al., U.S. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with
8 &fett-.Mj • »-; *. -? Att < * *: * áfas aiáfc ».. -.a.:y and ....! r? . r ^ baculovirus commonly derived from the polynephrine virus of the Au tographa cation (AcNPV). See King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994 and, Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. A second method for making zdintl recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, VA et al., J Virol 67: 4566-79, 1993) . This system, which uses transfer vectors, is sold in the Bac-to-Bac ™ case
(Life Technologies, Rockville, MD). This system uses a transfer vector, pFastBacl ™
(Life Technologies) which contains a Tn7 transposon to move the DNA encoding the zdintl polypeptide into a baculovirus genome conserved in E. col i as a large plasmid called "bacmido". See, Hi 1 I-Per kins, M.S. and Possee, R.D., J Gen Virol 71: 971-6, 1990; Bonning, B.C. et al., J Gen Virol 75: 1551-6, 1994; and Chazenbalk, G.D., and Rapoport, B., J Biol Chem 270: 1543-9, 1995. In addition, transfer vectors can include a fusion in the
~ z ~ i? »G? A, structure with DNA encoding an epitope tag at the C- or N- terminus of the expressed zdintl polypeptide, eg, a Glu-Glu epitope tag (Grus senmeyer, T. et al., Proc. Nati. Acad. Sci. 8_2: 7952-4, 1985). Using a technique known in the art, a transfer vector containing zdintl is transformed into E. col i and sifted for bacmidos which contain an interrupted lacZ gene indicative of recombinant baculovirus. Bacmid DNA containing the baculovirus recombinant genome is isolated, using common techniques, and used to transfect cells of Spodop t was giperda fru, for example Sf9 cells. The recombinant virus that expresses zdintl is produced later. Recombinant viral batches are made by methods commonly used in the art.
The recombinant virus is used to infect host cells, typically a cell line derived from maggot larvae, Spodop t was fru giperda. See in general, Glick and Pasternak,
Molecular Biot echnology: Principies and
Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another appropriate cell line is the High FiveO ™ cell line.
(Invitrogen) derived from Tri ch opl u s i a n i (U.S. Patent 5,300,435). The commercially available serum free media is used to grow and maintain the cells. The appropriate media are Sf900 II ™ (Life Technologies) or
ESF 921 TM Expression Systems) for cells
Sf9; and Ex-cellO405 ™ (JRH Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for T cells. n i. The cells grow from an inoculation density of about 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral batch is added at a multiplicity of infection (MOI) of 0.1 to 10. , more typically close to 3. The procedures used are generally described in available laboratory manuals (King, LA and Possee, RD, ibid., O'Rielly, DR et al., ibid., Richardson, CD, ibid.). The subsequent purification of the zdintl polypeptide from the supernatant can be achieved using the methods described herein.
Fungal cells, including yeast cells, can also be used within the present invention. The yeast species of particular interest in this regard include Sa ccha romyces cerevi s i a e, Pi ch i a pa s t or i s and Pi ch i a m e th a n e i n ca l. Methods for the transformation of S. cerevi s cells to exogenous DNA and producing recombinant polypeptides thereof are described, for example, in Kawasaki, U.S. Pat. 4,599,311; Kawasaki et al., U.S. No. 4,931,373; Brake, U.S. No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. No. 4,845,075. Transformed cells are selected by the phenotype determined by the selectable marker, commonly resistant to drugs or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Sa cch a romyce s cerevi s a e is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373) which allows the transformed cells to be selected by growth in glucose-containing media. Promoters and terminators suitable for use in yeast include those of glycolytic enzyme genes (see for example, Kawasaki, US Patent No. 4,599,311, Kingsman et al., US Patent No. 4,615,974, and Bitter, US Pat. 4, 977, 092) and alcohol dehydrogenase genes. See also U.S. Us.
4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts are known in the art, including Hansenula polymorpha, Schi zosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustigalo maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose. See, for example, Gleeson et al., J. Gen Microbiol. 132: 3459-65, 1986 and Cregg, U.S. No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for the transformation of Acremonium chrysogenum are described by Sumino et al., U.S. No. 5,162,228. Methods for the transformation of Neurospora are described by Lambowitz, U.S. No. 4,486,533.
The use of Pichia methanolica as a host for the production of recombinant proteins is described in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536 and WO 98/02565. DNA molecules for use in the transformation of P. methanolica will be commonly prepared as double circular plasmids
ta- "¿& faith é > - ~ s * r¿.y.r filament, which are preferably linearized before the transformation. For the production of polypeptides in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as the alcohol utilization gene of P. methanolica (AUG1 or AUG2). Other useful promoters include those of dihydroxyacetone synthase (DHAS), dehydrogenase
formate (FMD) and catalase genes (CAT). To facilitate integration of the DNA into the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by the host sequences of the DNA. A preferred selectable marker for use in Pichia methanolica is the ADE2 gene from P. methanolica, which codes for fos for ibos i 1 -5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows cells guest of ade2
grow in the absence of adenine. For large-scale industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes are eliminated. { AUGl and AUG2). 25 For the production of secreted proteins, host cells deficient in genes are preferred
of vacuolar protease. { PEP4 and PRB1). Electroporation is used to facilitate the introduction of a plasmid containing a DNA encoding a polypeptide of interest within the cells of P. methanolica. It is preferred to transform the P. methanolica cells by electroporation, using an exponentially decaying pulsed electric field having a field strength of 2.5 to 4.5 kV / cm, preferably around 3.75 kV / cm, and a time constant (t ) from 1 to 40 milliseconds, more preferably around 20 mi 1 i seconds.
The prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillys and other genera, are also useful host cells within the present invention. Well-known techniques in the art are
Transformation of these hosts and expression of external DNA sequences cloned therein (see for example, Sambrook et al., Ibid.). When a zdintl polypeptide is expressed in bacteria such as E. coli, the polypeptide is
can retain in the cytoplasm, typically as insoluble granules or can be directed to the periplasmic space by a sequence of secretion
,?? Yyyu ^^ »? & ^^ Z £ & * - < :, Jfc? A £ ¿SSÉ ^, bacterial. In the above case, the cells are lysed and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can be multiplied and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of oxidized and reduced glutathione, followed by dialysis against a buffered saline solution. In the last
In this case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by breaking the cells (for example, sonication or osmotic shock) to release the contents of the periplasmic space and
recover the protein, which obviates the need for denaturation and multiplication.
Transfected or transfected host cells are cultured according to the procedures
in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of appropriate means are known in the art, including defined means and means
complexes, and generally include a carbon source, a source of nitrogen, essential amino acids, vitamins and minerals. The media
^^ ^^ á & ^^^ ~. ^^ &6 ~ ^ '* y ^^ ~ yi ¿¿¿¿¿¿¿¿¿¿¿¿¿-and- "iuSiBiSlb-? á & kfc? ~ they may also contain components such as growth factors or serum as required.The growth medium will generally be selected for cells that contain the exogenously added DNA for example, drug selection or deficiency in the essential nutrients which is complemented by the selected marker carried in the expression vector or co-transfected in the host cell.
of P. m e tha n o l ca are grown in a medium comprising suitable sources of carbon, nitrogen and trace nutrients at a temperature of about 25 to 35 ° C. Liquid crops are supplied with sufficient aeration by means of
commercial, such as shaking small flasks or bubbling fermentors. A preferred culture medium for P. m e tha n ol i ca is YEPD (2% D-glucose, 2% Bacto ™ Peptone (Difco Laboratories, Detroit MI), 1% ex.
yeast Bacto ™ (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
Protein Isolation
It is preferred to purify the polypeptides of the present invention up to L.80% purity, more preferably up to > 90% pure, even more
preferably > _95% purity and particularly preferred is the pure state, which is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of pyrogenic and infectious agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. The expressed recombinant zdintl polypeptides (or zdintl chimeric polypeptides) can be purified using conventional purification methods and means and / or by
fractionation. Precipitation by ammonium sulfate and acid or chaotropic extraction can be used for the fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and
reverse phase high performance liquid chromatography. Suitable chromatographic media include dextrans derivatives, agarose, cellulose, polyacrylamide, special silicas and the like. Preferred are derivatives of PEI, DEAE, QAE and Q. Exemplary chromographic media include those media derived with phenyl, butyl or octyl groups, such as Phenyl-Sepharose FF.
(Pharmacia), Toyo butyl 650 beads (Toso Haas, Montgomeryville, PA), Oct il-Sepharose (Pharmacia) and the like; or polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica based resins, cellulosic resins, agarose beads, crosslinked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like, which are insoluble under the conditions in which they are to be used . These supports can be modified with reactive groups that allow the placement of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of coupling chemistries include activation by cyanogen bromide, activation by N-hydroxysuccinimide, activation by epoxides, activation by sulfhydryl, activation by hydrazide and amino and carboxyl derivatives for coupling chemicals by carbodiimide. 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. The selection of a particular method is a matter of routine design and is determined in
r ^ s ^ - ^ áwaa part by the properties of the chosen support.
See for example, Affinity Chromatography:
Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
The polypeptides of the present invention can be isolated by a combination of methods including, but not limited to, anion and cation exchange chromatography, size exclusion, and affinity chromatography. See example 3 for the procedure. For example, immobilized metal ion adsorption chromatography (IMAC) can be used to purify histidine rich proteins, including those comprising histidine tags. Briefly, first a gel is charged with bivalent metal ions to form a chelate
(Sulkowski, Trends in Biochem. 3_: 1-7, 1985). Histidine rich proteins will adsorb to this matrix with affinities that differ, depending on the metal ion used, and will be eluted by competitive elution, lowering the pH, or using strong chelating agents. Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purif ication", M. Deutscher, (ed.) ', Acad. Press, San Diego, 1990, pp. 529-39). Within the additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, maltose binding proteins, an immunoglobulin domain) can be constructed to facilitate purification.
Fragment os / Fusion Proteins
To direct export of a zdintl polypeptide from the host cell, the zdintl DNA is ligated to a second segment of DNA encoding a secretory peptide, such as a t-PA secretory peptide. To facilitate the purification of the secreted receptor polypeptide, a C-terminal extension, such as a poly-histidine tag, substance P, Flag peptide (Hopp et al., Bio / Technoloqy 6_: 1204-1210, 1988; available from Eastman Kodak Co. ., New Haven, CT) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the zdint 1 polypeptide.
Moreover, the use of the methods described in the art, polypeptide fusions, or hybrid zdintl proteins, are constructed using regions or domains of the inventive zdintl in combination with those of other molecules such as disintegrin (eg, ADAM, MDC and SVMP), or heterologous proteins (Sambrook et al., Ibd., Altschul et al., Ibid., Picard, Cur. Opin. Biology, 5: 511-5, 1994 and references therein). These methods allow the determination of the biological importance of domains or major regions in a polypeptide of interest. Such hybrids can alter the reaction kinetics, link, limit or expand the substrate specificity or alter the cellular or tissue location of a polypeptide, and can be applied to polypeptides of unknown structure.
The 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 it. Alternatively, a polynucleotide that encodes both components of the fusion protein in the proper reading frame can be generated using techniques known and expressed by the methods described herein. For example, a part or all of the domains conferring a biological function can be exchanged between the zdintl of the present invention with the functionally equivalent domains of another member of the family, such as ADAM, MDC and SVMP. Such domains include but are not limited to, conserved chain portions such as the secretory signal sequence, protease, RGD, cysteine and disintegrin domains. Such fusion proteins would be expected to have a functional biological profile that is the same or similar to the polypeptides of the present invention or other known proteins of the disintegrin-like family (e.g., ADAMs, MDCs and SVMPs), depending on the fusion built Moreover, such fusion proteins can show other properties as described herein.
Zdintl polypeptides or fragments thereof can also be prepared through chemical synthesis. The zdintl polypeptides can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
* & »• * ** rV. S &A Chemical Synthesis of Polypeptides
The zdintl polypeptides, peptides, variants and / or fragments thereof can be prepared through chemical synthesis. The TML polypeptides can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; amidated or non-amidated; sulphated or non-sulfated; and may or may not include an initial methionine amino acid residue. For example, TML polypeptides can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by synthesis of solid phase peptides, for example, as described by Merrifield, J. Am. Chem. Soc. 85: 2149, 1963. The synthesis is carried out with amino acids that are protected at the alpha-amino terminus. Trifunctional amino acids with labile side chains are also protected with appropriate groups to avoid undesirable chemical reactions that occur during the assembly of the polypeptides. The alpha amino protecting group is selectively removed to allow the subsequent reaction to be carried out at the amino terminus.
The conditions for the separation of the alpha-amino protecting group do not eliminate the side chain protecting groups.
The alpha-amino protecting groups are those known to be useful in the art of polypeptide synthesis in stages. Acyl-protecting groups (e.g., formyl, t-trifluoroacetyl, acetyl), aryl-type protecting groups (e.g., biotinyl, aromatic urethane-type protecting groups (e.g., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl, and 9-) are included. f luorenilmet i loxycarbonyl (Fmoc), aliphatic urethane protecting groups (e.g., t-butoxycarbonyl (tBoc), isopropyloxycarbonyl, cyclohexyloxycarbonyl, and alkyl type protecting groups (e.g., benzyl, triphenylmethyl.) Preferred protecting groups are tBoc and Fmoc .
The selected side chain protecting groups must remain intact during coupling and not be separated during deprotection of the amino terminus protecting group or during coupling conditions. The side chain protective groups must also
^ ^. & ^ ¿^^^^ & amp; & amp; £. ** 1bA &~ $. * á ~ * be separable at the end of the synthesis using reaction conditions that will not alter the finished polypeptide. In tBoc chemistry, the side chain protective groups for trifunctional amino acids are mainly benzyl based. In Fmoc chemistry, they are mainly tert-butyl or trityl based.
In tBoc chemistry, the preferred side chain protecting groups are tosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for glutamic acid, serine and threonine, benzyloxymethyl (and dinitophenyl) for histidine, 2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and 2-bromobenzyl for tyrosine. In the Fmoc chemistry, the preferred side chain protecting groups are 2, 2, 5, 7, 8 -pent amet ilchroman-6-sulfonyl (Pmc) or 2,2,4,6,7-pentametyl-dihydrobenzofuran-5- sulfonyl (Pbf) for arginine, trityl for asparagine, cysteine, glutamine and histidine, tert-butyl for aspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc for lysine and tryptophan.
For the synthesis of phosphopeptides, direct or subsequent incorporation of the phosphate group is used. In the strategy of direct incorporation, the phosphate group on serine, threonine or tyrosine, can be protected by methyl, benzyl or tert-butyl in the chemistry of Fmoc or by methyl, benzyl or phenyl in the chemistry of tBoc. The direct incorporation of phosphotyrosine without the phosphate protection can also be used in Fmoc chemistry. In the strategy of post-assembly incorporation, unprotected hydroxyl groups of serine, threonine or tyrosine are derived in solid phase with di-tert-butyl, dibenzyl or dimethyl-NN'-diisopropyl phosphoramidite and then oxidized by hydroperoxide of tert-butyl.
Solid phase synthesis is usually carried out from the carboxy terminus by coupling the protected alpha-amino acid amino acid (protected in the side chain) to an appropriate solid support. An ester ligation is formed when placing a chloromethyl, chlorotrityl or hydroxymethyl resin, and the resulting polypeptide will have a free carboxyl group at the C terminus. Alternatively, when an amide resin such as the benzhydrylamine resin is used or p-met i lbenzhydrylamine (for tBoc chemistry) and Rink amide or PA1 resin (for Fmoc chemistry), an amide bond is formed and the resulting polypeptide will have a carboxamide group on the C terminus. These resins, either polyamide based or polystyrene or grafted with polyethylene glycol, with or without a handle or linker, with or without the first amino acid placed, are commercially available, and their preparations have been described by Stewart et al., "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co., Rockford, IL, 1984) and Bayer & Rapp Chem. Pept. Prot. 3: 3 (1986); and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989.
The amino acid at the C-terminus, protected in the side chain if necessary, and in the alpha-amino group, is placed on a hydroxymethane resin using various activating agents including dicyclohexy Icarbodiimide (DCC), NN'-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). The chloromethyl or chlorotryril resin can be placed directly in its cesium tetramethylammonium salt or in the presence of triethylamine (TEA) or diisopropylethylamine
(DIEA). The first amino acid placement to an amide resin is the same as the formation of the amide bond during the coupling reactions.
ffmtwü Following the placement of the resin support, the alpha-amino protecting group is separated using several reagents depending on the protection chemistry (eg, tBoc, Fmoc). The degree of Fmoc separation can be monitored at 300-320 nm or by a conductivity cell. After separation of the alpha-amino protecting group, the protected remnant amino acids are coupled
by steps in the order required to obtain the desired sequence.
Various activating agents can be used for coupling reactions including
DCC, DIPCDI, 2-chloro- 1, 3-dimeti1 imide hexafluorophosphate (CIP), benzotria zol-1-yl-oxytris- (dimethylamino) -phosphonium hexafluorophosphate (BOP) and its pyrrolidine analog (PyBOP), bromine - tris-pyrrolidino-phosphonium hexafluorophosphate
(PyBroP), O- (benzotria zol- 1 -i 1) - 1, 1, 3, 3-tetramet-il-uronium hexafluorophosphate (HBTU) and its tetrafluoroborate analogue (TBTU) or its pyrrolidine analogue (HBPyU), 0- (7 -a zabenzot ria zol-1-yl) -1, 1, 3, 3-tetramethyl-uronium hexafluorophosphate
(HATU) and its tetrafluoroborate analogue (TATU) or its pyrrolidine analog (HAPyU). The most common catalytic additives used in reactions of
Couplings include 4-dimethylaminopyridine (DMAP), 3-hydroxy-3,4-dihydro-4-oxo-l, 2,3-benzotriazine (HODhbt), N-hydroxybenzothiazole (HOBt) and l-hydroxy-7-azabenzotriazole (HOAt). Each protected amino acid is used in excess (> 2.0 equivalents) and the couplings are usually carried out in N-methylpyrrolidone (NMP) or in DMF, CH2CL2 or mixtures thereof. The degree of termination of the coupling reaction can be observed at each stage, for example, by the ninhydrin reaction as described by Kaiser et al., Anal. Biochem. 34: 595, 1970.
After the complete assembly of the desired peptide, the resin-peptide is cleaved with a reagent with suitable sequestrants. The Fmoc peptides are usually cleaved and deprotected by TFA with sequestrants (e.g.
H20, ethanedithiol, phenol and thioanisole). The tBoc peptides are usually cleaved and deprotected with liquid HF for 1-2 hours at -5 to 0 ° C, which cleaves the polypeptide from the resin and removes most of the side chain protecting groups. Sequestrants such as anisole, dimet ilsul furo and p-thiocresol are usually used with the liquid HF to prevent cations formed during the splitting of the alkylation and acylation of the amino acid residues present in the polypeptide. The formyl group of tryptophan and the dinitrophenyl group of histidine need to be separated, respectively, by piperidine and thiophenyl in DMF before cleavage with HF. The acetyl amidomethyl cysteine group can be separated by mercury (II) acetate and alternatively by iodine, thallium trifluoroacetate (III) or silver tetrafluoroborate which simultaneously oxidizes cysteine to cysteine. Other strong acids used for cleavage and deprotection of tBOC peptides include trifluoromethansuiphonic acid (TFMSA) and trimethylsilyl trifluoroacetate (TMSOTf).
The disintegrin circuit (residues 438 to 449 of SEQ ID NO: 2) is of particular interest for use in assays and treatment of disorders of the heart, brain, spine and skeletal muscle. For these purposes, the peptide from the synthesized disintegrin circuit includes the terminal cysteine residues and thus, would be from residue 437 to residue 450 of SEQ ID NO: 2. This peptide can be synthesized as a linear peptide or a peptide linked with bisulfide. The peptides that have bisulfide bonds between the residues can be 438, 444 and 450, are of interest
7 < S & k. < particular. See Jia, L.G. ibi d for further description of the synthesis of peptides and the bisulfide bond.
One skilled in the art will recognize that it is useful to design and synthesize new binding peptides using the zdintl integrin binding peptides as a model. Methods for the synthesis of such peptides are described by PL Barker et al., J. Med. Chem. 35: 2040-2048, 1992, and L. Jia et al., J. Biol. Chem. 272: 13094- 13102, 1997. Since the structural conformation of the integrin linkage is critical, it is recognized that although some amino acid substitutions will not change the conformation of the peptides, the cyclization of the peptide is advantageously retained. The synthetic peptides are useful as agonists or antagonists for zdintl and can be tested.
ESSAYS
The activity of zdintl polypeptides can be measured using a variety of assays that measure e.g., cell-cell interactions, proteolysis, formation or remodeling of extracellular matrix. Additionally, other biological functions associated with members of the disintegrin family or with integrin / disintegrin interactions, apoptosis, proliferation or differentiation can also be measured. Of particular interest is a change with platelet aggregation. Tests that measure platelet aggregation are well known in the art. For a general reference, see Dennis, Proc. Nati Acad. Sci. 87: 2471-2475, 1989.
Another test of interest, measures or detects changes in the differentiation, development and / or electrical coupling of muscle cells or myocytes. Additionally, the effects of zdintl polypeptides on the cell-cell interactions of fibroblasts, myoblasts, nerve cells, white blood cells, endothelial cells and tumor cells, would be of interest to measure. Still other trials examine changes in protease activity and apoptosis.
The activity of the molecules of the present invention can be measured using a variety of assays that, for example, measure neogenesis or hyperplasia (ie, proliferation) of cardiac cells based on the tissue specificity in the heart of adults. The activities
additional probably associated with the polypeptides of the present invention include the proliferation of endothelial cells, cardiomyocytes, fibroblasts, skeletal myocytes directly or indirectly through other growth factors; action as a chemotactic factor for endothelial cells, fibroblasts and / or phagocytic cells; osteogenic factor; and factor for the expansion of the 10 mesenchyme stem cell and precursor populations.
Proliferation can be measured using cardiac cells cultured or in vivo, by
administration of molecules of the claimed invention to an appropriate animal model. Generally, proliferative effects are observed as an increase in the number of cells and therefore, may include
inhibition of apoptosis as well as mitogenesis. Cultured cells include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, endothelial cells of the human umbilical vein of primary cultures. The lines
of established cells include: NIH 3T3 fibroblast (ATCC No. CRL-1658), CHH-1 heart chum cells (ATCC No. CRL-1680), heart myoblasts
of rat H9c2 (ATCC No. CRL-1446), Shionogi mammary carcinoma cells (Tanaka et al., Proc. Nati, Acad. Sci. 8_9: 8928-8932, 1992) and LNCap adenocarcinoma cells. FGC (ATCC No. CRL-1740). Tests that measure cell proliferation are well known in the art. For example, assays to measure proliferation include assays such as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8_: 347-354, 1990), incorporation of radiolabeled nucleotides.
(Cook et al., Analytical Biochem., 179: 1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 8_2: 169-179, 1985), and the use of tetrazolium salts (Mosmann, J. Immunol Methods _6_5: 55-63, 1983; Alley et al., Cancer Res. _8_: 589-601, 1988; Marshall et al. , Growth Req. 5_: 69-84, 1995; and Scudiero et al., Cancer Res. 48: 4827-4833, 1988).
Differentiation is a dynamic and progressive process, beginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when a commitment is made with a cell lineage. The progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress back to the cell lineage path toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products and receptors. The stage of a differentiation of the cell population is observed by the identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, crondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchyme stem cell (Owen et al., Ciba Fdn, Symp 136: 42-46, 1988). Markers for mesenchymal stem cells have not been well identified (Owen et al., J of Cell Sci. 8_7_: 731-738, 1987), so that identification is usually done in the mature and progenitor cell stages. The existence of progenitor cells of early-stage cardiac myocytes (often referred to as cardiac myocyte stem cells) has been speculated, but not demonstrated, in adult cardiac tissue. The novel polypeptides of the present invention are useful for studies to isolate mesenchymal stem cells and cardiac myocyte progenitor cells, both in vivo and ex vivo.
H a and evidence suggesting that factors that stimulate specific cell types back on a path towards terminal differentiation or dedifferentiation, affect the entire population of cells that originate from a common precursor or stem cell. Thus, zdintl polypeptides can stimulate the inhibition or proliferation of myocytes, uniform muscle cells, theobjects, adipocytes, crondrocytes and endothelial cells. The molecules of the present invention can, while stimulating the proliferation or differentiation of cardiac myocytes, inhibit the proliferation or differentiation of adipocytes, by virtue of their effect on common precursor / stem cells. Thus, the molecules of the present invention have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
Tests to measure differentiation include, for example, editing markers of
L - '?' surface of associated cells with specific expression of tissue stages, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5_: 281-284, 1991; Francis, Differentiation 57: 63-75, 1994; Raes, Adv. Anim Cell Biol. Technol. Bioproces ses, 161-171, 1989, all of which are incorporated herein by reference).
The trials to evaluate cardiac neogenesis or hyperplasia include the treatment of mature and neonatal rats with the molecules of the present invention. The cardiac function of animals is measured as heart rate, blood pressure and cardiac output to determine left ventricular function. Post-mortem methods to assess cardiac improvement include: increased cardiac weight, toplasmic volume / nuclei, staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res. 7_5: 1050-1063, 1994 and Reiss et al., Proc. Nati Acad. Sci. 93: 8630-8635, 1996).
The effects of the measurement of in vi ve trials of synthetic zdintl agonists include a model of Left Ventricular Hypertrophy (AM Feldman et al., Circ Res. 73: 184-192, 1993), which measures remodeling and repair after of congestive heart failure and chronic pressure overload.
The proteins, including alternatively spliced peptides of the present invention, are useful for the suppression of tumors
and growth and differentiation either working alone or in conjunction with other molecules (growth factors, cytokines, etc.) in brain, heart, spine or skeletal muscle cells. The alternative splicing of
zdintl can be specific cell and confer activity to specific tissues.
The proteins of the present invention are useful for the delivery of therapeutic agents
such as, but not limited to, proteases, radionuclides, chemotherapy agents and small molecules. The effects of these therapeutic agents can be measured i n vi t ro using cultured cells or i n vi ve by administering
molecules of the claimed invention to the appropriate animal model. For example, transfected zdintl expression host cells can be
fe ^^ w- ^ a ^ j ^^ á ^ j ^^? ^^ = ^^^ i? * ^^ e * «í. - *. * & amp; & ** - * sl 'lodge in an alginate medium and inject (implant) into the recipient animals. The microencapsulation of alginate-poly-L-lysine, encapsulation of permoselective membranes and diffusion chambers, have been described as a means to trap cells from transfected mammals or primary mammalian cells. These types of non-immunogenic "encapsulations" or microenvironments allow the transfer of nutrients within a microenvironment, and also allow the diffusion of proteins and other macromolecules secreted or released by the cells captured through the environmental barrier to the recipient animal. More importantly, the capsules or microenvironments mask and protect the outer cells, housed in the immune response of the recipient animal. Such microenvironments can extend the life of the injected cells from a few hours or days (bare cells) to several weeks (housed cells).
Alginate yarns provide a simple and fast means to generate hosted cells. The materials required to generate alginate yarns are readily available and relatively inexpensive. Once done, the threads
P ^. * ^ W * g &ki &yyy * -.
of alginate are relatively strong and durable, both in vi t ro and, based on data obtained using the threads, i n vi vo. The alginate threads are easily manipulated and the methodology is scalable for the preparation of numerous threads. In an exemplary procedure, 3% alginate is prepared in sterile H20 and sterile filtered. Just before the preparation of the alginate threads, the alginate solution is filtered again. A cell suspension of about 50% (containing about 5 x 10 5 to about 5 x 10 7 cells / ml) is mixed with the 3% alginate solution. One ml of the alginate / cell suspension is extruded in a sterile 100 mM filtered CaCl 2 solution for a period of about 15 min, forming a "thread". The extruded yarn is then transferred into a 50 mM CaCl 2 solution, and then into a 25 mM CaCl 2 solution. The yarn is then rinsed with deionized water before coating the yarn upon incubation in a 0.01% poly-L-lysine solution. Finally, the thread is rinsed with a Lactated Ringer's Solution and removed from the solution in a syringe cylinder (without the needle installed). A large-bore needle is then placed in the syringe, and the wire is injected intraperitoneally into the recipient in a minimum volume of Lactated Ringer's Solution.
An alternative approach to testing 5 proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpes virus, lentivirus, vaccinia virus and adeno-associated virus (AAV): Adenovirus, a virus of
double-stranded DNA, is currently the best-studied gene transfer vector for the delivery of heterologous nucleic acids (for a review, see TC Becker et al., Meth Cell Biol. 4_3: 161-89, 1994; and JT Douglas and DT Curiel,
Science & Medicine 4_: 44-53, 1997). The adenovirus system offers several advantages: the adenovirus can (i) accommodate relatively large inserts of DNA; (ii) grow up to high-titration; (iii) infect a wide range of cell types
of mammals; and (iv) used with a large number of available vectors containing different promoters. Also, because the adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. 25 By removing portions of the adenovirus genome, larger inserts can be accommodated
k®by¿ »S! u ^ - t $ ßg ^ ¡S & (up to 7 kb) of heterologous DNA. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transgenase plasmid. In an exemplary system, the essential gene El has been removed from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (human cell line 293 is exemplary). When administered intravenously to intact animals, the adenovirus mainly attacks the liver. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, host tissue (e.g. liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. The secreted proteins will go into circulation in the highly vascularized liver, and the effects in the infected animal can be determined.
The adenovirus system can also be used for the production of proteins in vitro. By culturing non-293 adenovirus-infected cells under conditions where the cells do not divide rapidly, the cells can produce proteins for extended periods of time. By
For example, BHK cells grow to coalesce into cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells then grow under serum free conditions, which allows the infected cells to survive for several weeks without significant cell division. Alternatively, 293S cells infected with the adenovirus vector can grow in suspension culture at a relatively high density of cells to produce significant amounts of protein (see Garnier et al., Cytotechnol 15: 145-55, 1994). With any protocol, a secreted heterologous protein, expressed, can be repeatedly isolated from the supernatant of the cell culture. Within the production protocol of the infected 293S cell, the non-secreted proteins can also be obtained effectively.
Within yet another embodiment, there is provided an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 25.
. ^ -sf ^ ^^^^ 2 __ ^ ^ __ ^^ ^^^ s - ^^^^^^ ¿^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
In view of the distribution of tissues (heart, brain, spine and skeletal muscle) observed for the expression of zdintl, agonists (including the native protease and disintegrin domains) and antagonists have enormous potential in applications in vi vo ein vi t ro Compounds identified as zdintl agonists and antagonists are useful for studying cell-cell interactions, myogenesis, apoptosis, neurogenesis, connective tissue disorders, chondrogenesis, arthritis, tumor suppression and proliferation, extracellular matrix proteins, reperfusion repair and remodeling of ischemia and inflammation in vi t ro ein vi vo. For example, the zdintl and agonist compounds are useful as components of defined culture media, and can be used alone or in combination with other cytokines and hormones to replace the serum that is commonly used in cell culture. Agonists are thus useful in specifically promoting the growth and / or development of cells of myeloid lineages in cultures. Additionally, zdintl polypeptides and zdintl agonists, including small molecules, are useful as a reagent of
»: A« r¿-^ -., - research, such as for the expansion, differentiation and / or cell-cell interactions of heart, brain, spine or skeletal muscle cells. The zdintl polypeptides are added to tissue culture media for these cell types.
Antagonists
Antagonists are also useful as research reagents for characterizing complementary ary / antiecomplement interaction sites. Inhibitors of zdintl activity (zdintl antagonists) include anti-zdintl antibodies and soluble zdintl receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
Zdintl can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays described herein to identify compounds that inhibit zdintl activity. In addition to these assays described herein, samples for the inhibition of zdintl activity can be tested within a variety of assays designed to measure the disintegrin / integrin binding or the stimulation / inhibition of zdintl-dependent cellular responses. For example, the zdintl response cell lines can be transfected with a reporter gene construct that responds to a cell path stimulated by zdintl. Reporter gene constructs of this type are known in the art, and will generally comprise a response element.
of DNA operably linked to a gene encoding a test able protein, such as luciferase or a metabolite. DNA response elements may include, but are not limited to, AMP cyclic response elements
(CRE), hormone response elements (HRE), insulin response elements (IRE) (Nasrin et al., Proc. Nati. Acad. Sci. USA 87: 5273-7, 1990) and response elements to serum (SRE) (Shaw et al., Cell 56: 563-72, 1989). The elements of
AMP cyclic response are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1988 and Habene r, Molec. Endocrinol 4_ (8): 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 5_6: 335-44; 1989. The
The most likely reporter gene construct would contain a disintegrin which, when linked to an integrin, would intially designate a
through, for example, a SRE reporter. The candidate compounds, solutions, mixtures or extracts are tested for their ability to inhibit zdintl activity in target cells, as evidenced by a decrease in zdintl stimulation of reporter gene expression. Assays of this type will detect compounds that directly block the bond of zdintl to surface cell receptors, that is, integrin or the anticomplementary member of a complementary / anticomplementary pair, as well as compounds that block processes in the cellular path subsequent to the complement link / ant i-complement. In the alternative, compounds or other samples can be tested for direct blocking of the zdintl link to an integrin using zdintl labeled with a detectable label (eg, 125I, biotin, horseradish peroxidase, FITC and the like). Within assays of this type, the ability of a test sample to inhibit the binding of the integrin-labeled zdintl is indicative of the inhibitory activity, which can be confirmed by secondary assays. The integrins used with binding assays can be cellular or isolated integrins, immobilized integrins.
^^^ á ^^^ r l ^^^^^^ A ^^^^^. ~ - An amino acid sequence comprising the integrin binding component "ECD" of zdmtl, (residues 443 to 445 of SEQ ID NO: 2), which is analogous to the integrin binding circuit "RGD", can also used as an inhibitor. Such an inhibitor would bind an integrin different from its mtegrin which occurs naturally by its multiplier structure. A particular interest in such an inhibitor would be mediating platelet aggregation. Assays that measure binding and inhibition as well as platelet aggregation are known in the art.
A zdintl polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fe fragment, which contains two domains of constant region and lacks variable region. Methods for the preparation of such fusions are described in U.S. Us.
,155,027 and 5,567,584. Such fusions are typically segregated as multimeric molecules wherein the Fe and bisulfide portions linked to each other and two no_Ig polypeptides are placed in close proximity to one another. Mergers of this type can be used to evaluate the effects and dimerization potential of zdintl
** a &g5 with himself or other members of the disintegrine family. Such fusions would also be useful for isolating the corresponding integrin (s) that bind zdintl. For use in assays, the chimeras are linked to the support via the Fe region and are used in an ELISA format.
A zdintl integrin binding polypeptide can also be used for the purification of the
integrin. The polypeptide is immobilized on a solid support, such as agarose beads, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide or similar materials
which are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art and include amine chemistry, activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation
with epoxides, activation with sulfhydryl and activation with hydrazide. The resulting medium will generally be configured in the form of a column and the fluids containing the integrins are passed through the column one or more times to
Allow the integrins to bind to the integrin binding circuit polypeptide. The integrin is then eluted using
Bfa * & Changes in salt concentration, chaotropic agents (guanidine hydrochloride) or pH to break the integrin-receptor bond.
A test system using a ligand-binding receptor (or an antibody, a member of a complementary / ant i-complementary pair) or a binding fragment thereof, and a commercially available biosensing instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) can be used advantageously. Such receptor, antibody, member of a complement / anticomplement pair or fragment is immobilized on the surface of a receptor sensor. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor, antibody, member or fragment is covalently placed, using amine or sulfhydryl chemistry, to dextran fibers that are placed to the gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope or opposite member of the complementary / anticomplementary pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the index
of refraction of the medium, which is detected as a change in the surface plasmon resonance of the gold film. This system allows the determination of on and off speeds, from which the link affinity and the evaluation of the link stoichiometry can be calculated.
Another test method of cell-cell interactions caused by zdintl polypeptides, peptides or variants, is with a silicon-based biosensor microfi isometer, which measures the rates of extracellular acidification or proton excretion associated with receptor binding and the subsequent cellular physiological responses. An exemplary device is the Cytosensor ™ Microfiometer manufactured by Molecular Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, enregy production, inflammatory response, regulatory and recirculator activation and the like, can be measured by this method. See for example, McConnell, H.M. et al., Science 257: 1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228: 84-108, 1997; Arimilli S. et al., J. Immunol. Meth. 212: 49-59, 1998; Van Liefde, I. et al., Eur. J.
Pharmacol. 346: 87-95, 1998. The microphysiometer can be used to test adherent or non-adherent prokaryotic or eukaryotic cells. By measuring the changes of extracellular acidification in cell media over time, the microfiometer directly measures cellular responses to various stimuli, including zdintl polypeptides, peptides, variants, agonists or antagonists. Preferably, the
The microphysiometer is used to measure responses of a eukaryotic cell responsive to zdintl, compared to a control eukaryotic cell that does not respond to the polypeptide, peptide or zdintl variant. The eukaryotic cells of
The response to zdintl comprises cells within which a zdintl receptor has been transfected creating a cell responsive to the polypeptide, peptide or zdintl variant; or cells that respond naturally to zdintl such
for example, cells derived from the kidney or small intestine. The differences measured by a change for example, an increase or decrease in extracellular acidification, in the response of cells exposed to the polypeptide, peptide or
variant of zdintl in relation to a control not exposed to the polypeptide, peptide or variant of zdintl, are a direct measure of the responses
cell phones modulated by the zdintl. Moreover, such responses modulated by the zdintl can be tested under a variety of stimuli. Using the microfiometer, a method is provided for identifying zdintl polypeptide agonists, which comprises providing cells responsive to a zdintl polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the
cells in the presence of a test compound, and detecting a change, e.g., an increase or decrease, in a cellular response of the second portion of cells compared to the first portion of the cells. The change in
The cellular response is shown as a measurable change in the rate of cellular acidification. Moreover, by culturing a third portion of the cells in the presence of the zdintl polypeptide and the absence of a test compound, one can
used as a positive control for the zdintl response cells, and as a control to compare the activity of the agonist of a test compound with that of the zdintl polypeptide. Furthermore, when using the microf isometer, a
The method of identifying antagonists of the zdintl polypeptide, comprising supplying cells responsive to a zdintl polypeptide,
culturing a first portion of the cells in the presence of zdintl and the absence of a test compound, culturing a second portion of the cells in the presence of zdintl and the presence of a test compound, and detecting a change, e.g., an increase or decrease in the cellular response of the second portion of the cells compared to the first portion of the cells. The change in cellular response is
shows as a measurable change in the rate of extracellular acidification. Antagonists and agonists, for the zdintl polypeptide, can be quickly identified using this method.
Furthermore, the polypeptides, peptides and variants of zdintl can be used to identify cells, tissues or cell lines which respond to a path stimulated by zdintl. The microfi s meter, described above, is
can be used to rapidly identify ligand response cells, such as the polypeptide, peptide and zdintl response cells of the present invention. The cells can be cultured in the presence or absence of
polypeptides, peptides and zdintl variants. Those cells that obtain a measurable change in extracellular acidification in the presence of
* & 5t) * J! á¡ £? & amp; ^^ & ^ £ ^ ^ & £. "^^^^ tJsS ^? ¡^^.
Polypeptides, peptides or zdintl variants have a response to zdintl. Such cell lines can be used to identify antagonists and zdintl polypeptide agonists as described above.
Integrin polypeptides and other receptor polypeptides which bind disintegrin polypeptides and variants thereof, can also be used within other assay systems known in the art. Such systems include Scatchard analysis for the determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253: 545-48, 1991; Cunningham et al., Science 245: 821-25, 1991).
A "soluble receptor" is a receptor polypeptide that does not bind to a cell membrane. Soluble receptors are more commonly ligand-binding polypeptides that lack cytoplasmic and transmembrane domains. Soluble receptors may comprise additional amino acid residues, such as affinity tags that are supplied for the purification of the polypeptide or
provide sites for the placement of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have soluble counterparts that occur naturally, that are produced by proteolysis or are translated from alternatively spliced mRNAs. The receptor polypeptides are said to be substantially free of segments of transmembrane or intracellular polypeptides when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction respectively.
Soluble forms of zdintl polypeptides can act as antagonists for zdintl polypeptides, and would be useful for modulating the effects of zdintl on the heart, brain, skeletal muscle and spine. Additionally, soluble zdintl peptides and fragments can break the integrin-mediated placement of a cell to the extracellular matrix.
ANTIBODIES
Zdintl polypeptides can also be used to prepare antibodies that specifically bind epitopes, peptides or zdintl polypeptides. The zdintl polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and evoke an immune response. The appropriate antigens
would include fragments of the zdintl polypeptide encoded by SEQ ID NO: 2 which represents six or more contiguous hydrophilic amino acids. Such antigenic regions would be, for example, from amino acid residue 159 to 164 (SEQ.
N NOO :: 7 7));; amino acid residue 158 to 163 (SEQ ID NO: 8); amino acid residue 518 to 523 (SEQ ID NO: 9); amino acid residue 658 to 663 (SEQ ID NO: 10 (); and amino acid residue 190 to 195 (SEQ ID NO: 11) .The antibodies generated from this
The immune response can be isolated and purified as described herein. Methods for the preparation of monoclonal and polyclonal isolating antibodies are well known in the art. See for example, Current Protocols in Immunology,
Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual,
fe ^^ S ^ s ^^ < «F ^? ¿¿^ -ij« ^ J ^ Í ^^^ feL ^ »rt« «. -..- .-;, '-fea Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J.G.R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.
As will be apparent to one of ordinary skill in the art, monoclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats with a zdintl polypeptide or a fragment thereof. The immunogenicity of a zdintl polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as zdintl fusions or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "as hapten", said portion may be advantageously linked or ligated to a macromolecular carrier (such as limpet hemocyanin).
and "& S, " i. " a " Mab fag (KLH), bovine serum albumin (BSA), or tetanus toxoid for immunization.
As used herein, the term "antibodies" includes polyclonal antibodies, affinity purified polyclonal antibodies, monoclonal antibodies and antigen binding fragments, such as Fab and F (ab ') 2 proteolytic fragments. Antibodies are also included or
intact fragments prepared by genetic engineering such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as peptides and synthetic antigen binding polypeptides. The antibodies do not
humans can be humanized by grafting non-human CDRs onto a human structure and constant regions, or by incorporating the entire non-human variable domains (optionally by "hiding" them with a human type surface by substituting the
exposed residues, where the result is a "disguised" antibody). In some cases, humanized antibodies can retain non-human residues within the human variable region structure domains to increase the
own characteristics of link. Through humanizing antibodies, the biological half-life can be increased and the potential for
Adverse immune reactions by administration to humans.
Alternative techniques for generating or selecting antibodies useful herein include exposure to lymphocytes to the zdintl protein or peptide, and selection of antibody display libraries on phages or similar vectors (e.g., through the use of
zdintl proteins or peptides labeled or immobilized). Genes encoding polypeptides that have potential zdintl polypeptide binding domains can be obtained by screening from randomized peptide collections
displayed on the phages (phage display) or on bacteria, such as E. col i. The nucleotide sequences encoding the polypeptides can be obtained by a variety of ways, such as through mutagenesis
randomization and random synthesis of polynucleotides. These randomly displayed collections of peptides can be used to screen peptides that interact with a given target which can be a protein or polypeptide, such as a ligand or
Receptor, a biological or synthetic macromolecule, or organic or inorganic substances. The techniques for creating and sifting such collections of
-Tyy * -... 3 * g. -i & The deployment of randomized peptides is known in the art (Ladner et al., US Pat. No. 5,223,409; Ladner et al., American patent US NO. 4,946,778; Ladner et al., US Patent No. 5,540,484 and Ladner et al., US Patent No. 5,571,698) and collections and kits for the display of randomized peptides are commercially available to screen such collections, for example Clontech ( Palo Alto, CA), Invitrogen
Inc. (San Diego, CA), New England Biolabs, Inc.
(Beverly, MA) and Pharmacia LKB Biotechnology Inc.
(Piscataway, NJ). The random peptide display collections can be screened using the zdintl sequences described herein for
identify proteins that bind to zdintl. These "binding proteins" that interact with the zdintl polypeptides can be used to label cells; to isolate homologous polypeptides by affinity purification; you can
directly or indirectly conjugate drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as collections of expression by screening and neutralizing activity.
The binding proteins can also be used for diagnostic assays to determine polypeptide circulation levels; for
• tS & '-afta' lüWf y ™ ¿.S ''. t & I S? * A¡Mi: * detect or quantify soluble polypeptides as a marker of the underlying pathology or disorder. These binding proteins can also act as zdintl "antagonists" to block zdintl binding and signal transduction i n vi t ro e i n vi vo. These anti-zdintl binding proteins would be useful to inhibit for example, platelet aggregation, apoptosis, neurogenesis, myogenesis, tumor formation and cell-cell interactions in general.
The antibodies are determined to be specifically linkable if: 1) they show a threshold level of binding activity and / or 2) they do not have significant cross-reactions with the related polypeptide molecules. First, the antibodies herein specifically bind if they bind to a zdintl polypeptide, peptide or epitope with a binding affinity (Ka) of 106 M "1 or greater, preferably 107 M" 1 or greater, more preferably 108 M "1 or greater and most preferably 109 M "1 or greater. The binding affinity of an antibody can be easily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann., NY Acad. Sci. 51: 660-672, 1949).
Second, the antibodies are determined to bind specifically if they do not have significant cross-reactions with the related polypeptides. Antibodies do not have significant cross-reactions with molecules related to polypeptides for example, if they detect zdintl but not related polypeptides not known using a normal Western blot analysis (Ausubel et al., Ibid.). Examples of polypeptides
Known related are orthologs, proteins of the same species that are members of a protein family, zdintl polypeptides and non-human zdintl. In addition, antibodies can be "screened against" related polypeptides
known to isolate a population that specifically binds to the inventive polypeptides. For example, antibodies raised to zdintl are adsorbed to related polypeptides adhered to an insoluble matrix; the antibodies
specific to zdintl will flow through the matrix under the proper conditions of damping. This sieving allows the isolation of monoclonal and polyclonal antibodies that do not cross-react,
towards 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). The isolation and screening of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. In Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principie 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 used to detect antibodies which specifically bind proteins or zdintl 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: immunoelect concurrent rhoplais, radioimmunoassays, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), spot staining or Western spotting test, inhibition or competition test and sandwich assay. In addition, the antibodies can be screened to bind to a
Irregular type against zdintl mutant protein or polypeptide.
The zdintl antibodies can be used to label cells expressing zdintl; to isolate zdintl by affinity purification; for diagnostic tests to determine the circulatory levels of zdintl polypeptides; to detect or quantify soluble zdintl as a marker of the underlying pathology or disorder; in analytical methods that use FACS; to sift collections of expression; to generate anti- idotypic antibodies; and as neutralizing antibodies or as antagonists to block 5 zdintl i n vi t ro n e vi n. Suitable direct labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, luminescent labels, magnetic particles and the like; indirect labels can characterize the use of biotin-avidin or other complement / ant i-complement pairs as intermediaries. The antibodies herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, 5 and these conjugates used for diagnostic or therapeutic applications. In addition, antibodies to zdintl or fragments thereof are
^^? They can use vi ng to detect denatured zdintl or fragments thereof in tests eg Western blotting or other assays known in the art.
The present invention also provides fragments of polypeptides or peptides comprising a portion that supports an epitope of a zacrp2 polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope" which is part of a protein that evokes an antibody response when the entire protein is used as an immunogen. Immunogenic peptides that support epitopes can be identified using normal methods (see, for example, Geysen et al., Proc. Nati, Acad. Sci. USA 81: 3998, 1993),
In contrast, the polypeptide or peptide fragments may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous extension of amino acids, and the antigenicity of such an epitope is not broken by denaturing agents. It is known in the art that relatively synthetic peptides
L-L-tS? Afet ~ «- short that can mimic the epitopes of a protein, can be used to stimulate the production of antibodies against the protein (see for example, Sutcliffe et al., Science 219: 660, 1983) . In this manner, the antigenic peptides and polypeptides that support epitopes of the present invention are useful for raising antibodies that bind to the polypeptides described herein.
The antigenic peptides and polypeptides that support epitopes, preferably contain at least four to ten amino acids, at least ten to fifteen amino acids or about 15 to about 30 amino acids of the sequence SEQ ID NO.
NO: 2. such peptides and polypeptides that support epitopes can be produced by cleaving a zacrp2 polypeptide or by chemical synthesis of peptides as described herein. In addition, epitopes can be selected by display of
phages from random peptide collections (see, for example, Lane and Stephen, Curr Opin, Immunol 5: 268, 1993, and Cortese et al., Curr Opin, Biotechnol 7: 616, 1996). Standard methods to identify epitopes and produce antibodies to
Starting from small peptides comprising an epitope, they are described for example by Mole "Epitope Mapping" in Methods in Molecular Biology,
S? Ej-iy? Á ^? S? I¡s > »Vol. 10, Manson (ed.) Pages 105-16 (The Humana Press, Inc. 1992), Price," Production and Characterization of Synthetic Peptide-Derived Antibodies ", in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter and Ladyman (eds.), Pages 60-84 (Cambridge University Press 1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1 9.3.5 and pages 9.4.1 - 9.4.11 (John Wiley &Sons 1997). The polypeptides or fragments thereof of the present invention, which comprise amino acid sequences of, for example, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: ll, they support epitopes.
BIOACTIVE CONJUGATES
The antibodies or polypeptides herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates used for diagnostic or therapeutic applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anticomplementary molecule (integrin or antigen, respectively for example). Plus
specifically, the zdintl polypeptides or anti-zdintl antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to mammals having cells, tissues or organs that express the ant i-complementary molecule.
Appropriate detectable molecules can be placed directly or indirectly to the polypeptide or
antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. The appropriate cytotoxic molecules can be placed directly or
indirectly to the polypeptide or antibody, and include bacterial or plant toxins (e.g., diphtheria toxin, Ps e udomon a s exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides such as iodine 131,
Rhodium 188 or Yttrium 90 (either directly attached to the polypeptide or antibody, or indirectly placed through chelating moiety media for example). The polypeptides or antibodies can also be conjugated to
cytotoxic drugs such as adriamycin. For the indirect placement of a detectable or cytotoxic molecule, the molecule
t ^ s ^, •• b "'* tl &? ^. i¡> i® & amp; -i-cytotoxic or detectable may be conjugated with a member of a complementary / anticomplementary pair, wherein the other member is linked to the polypeptide or antibody portion.For these purposes, the biot ina / est reptavidin is an exemplary complement pair / anti- complement complement.
In another embodiment, the ido-toxin polypeptide fusion proteins or antibody-toxin fusion proteins can be used for the inhibition or ablation of target cells or tissues (eg, to treat cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains
(ie, an activation domain or a domain that binds a ligand, plus a target domain), a fusion protein may be appropriate including only the target domain to direct a detectable molecule, a cytotoxic molecule or a molecule complementary to a cell or type of tissue of interest. In cases where the domain-only fusion protein includes a complementary molecule, the anticomplementary molecule can be conjugated to a detectable or cytotoxic molecule. Such fusion proteins of molecule complementary to the domain, represent in themselves a generic target vehicle for the cell / tissue specific delivery of conjugates of complementary cytotoxic / detectable anti-i molecules.
In another embodiment, the cytokine-zdint 1 fusion proteins or cytokine-antibody fusion proteins can be used to increase the extermination of target tissues (by
example, malignant brain, heart, spine and skeletal muscle), if the zdintl polypeptide or anti-zdintl antibody attacks the hyperproliferative brain, heart, spine or skeletal muscles. (See, generally, Hornick
et al., Blood 8_9: 4437-47, 1997). They describe fusion proteins that allow the attack of a cytokine at a desired site of action, thereby providing a high local concentration of cytokine. The appropriate polypeptides of zdintl a
Anti-zdintl antibodies attack an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates enhanced target cell lysis by determining cells. Appropriate cytokines for this purpose include
interleukin 2 and macrophage-granulocyte colony stimulating factor (GM-CSF) for example.
In yet another embodiment, if the zdintl polypeptide or anti-zdintl antibody attacks vascular cells or tissues, such polypeptides or antibodies can be conjugated with a radionuclide, and particularly with a beta emission radionuclide to reduce restenosis. Such a therapeutic approach has less risk for the clinical staff administering radioactive therapy. For example, strips impregnated with iridium 192 placed in implanted patient vessels until the radiation dose has been delivered, showed decreased tissue growth in the vessel and a larger luminal diameter than the control group, which received tapes of placebo. In addition, revascularization and thrombosis of the implant were significantly lower in the treatment group. Similar results are predicted with the attack of a bioactive conjugate containing a radionuclide, as described herein.
The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intially or intially or can be introduced locally into the intended site of action.
USE OF POLYUCLEOTIDE / POLYPEPTIDE
The molecules of the present invention can be used to identify and isolate receptors and integrins involved in cell-cell interactions. For example, the proteins and peptides of the present invention can be immobilized on a column and run membrane preparations on the column (Immobilized Affinity Ligand
Techniques, Hermanson et al., Eds. Academic Press, San Diego, CA, 1992, pp. 195-202). The polypeptides and peptides which bind to the polypeptides, peptides and zdintl variants of the present invention can then be eluted and
characterize using methods known in the art. Proteins and peptides may also radioet iquetar (Methods Enzymol m., Vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or
labeled photoaffinity (Brunner et al, Ann Rev. Biochem 62_:... 483-514, 1993 and Fedan et al, Biochem Pharmacol 3_3:... 1167 -80, 1984) and can be identified cell-surface proteins specific. The molecules of the present invention will be useful in repairing and remodeling after an event
& * Mal¿Sií af > ? a tik * a-% Bs? & em &s3, y * yt j »Sa -» w - - < ? *, ischemic and / or inhibit platelet aggregation. The polypeptides, nucleic acids and / or antibodies of the present invention can be used in the treatment of diseases associated with infarction in cerebral or cardiac tissue, and / or platelet aggregation. The molecules of the present invention can be used to modulate proteolysis, apoptosis, neurogénes is, myogenesis, connective tissue disorders, arthritis, condrogénes is, cell adhesion, cell fusion and signaling or to treat or prevent the development of conditions pathological in various tissues such as heart, brain, spine and skeletal muscle. In particular, certain diseases may be responsible for such diagnosis, treatment or prevention. The molecules of the present invention can be used to modulate the inhibition and proliferation of neurons and myocytes in heart, brain, spine and skeletal muscle tissues. Disorders that may be responsible for the diagnosis, treatment or prevention polypeptides zdintl include for example, Alzheimer's disease, tumor formation, multiple sclerosis, congestive heart failure, or myocardial ischemia-reperfusion and degenerative diseases.
^^^ ¿i ^ ^^ ..- fta¿ - ".-- Ja '^ JI ^ 3TIP & you &II molecules zdintl of' the present invention may be particularly useful in the treatment of intimal hyperplasia or restenosis due to acute vascular damage. Acute vascular injuries are those that occur rapidly (that is, in days or months), in contrast to chronic vascular damage (for example, atherosclerosis), which develops over a period of time. Acute vascular injuries often result from surgical procedures such as vascular reconstruction, where the techniques of angioplasty, endarterectomy, atherectomy, vascular implant placement or the like are employed. Hyperplasia can also happen as a delayed response in response to for example, graft placement or organ transplantation. The dose of zdintl in the treatment for restenosis will vary with each patient but will generally be in the range of those suggested above.
Advances in the treatment of coronary vascular diseases include the use of mechanical interventions to remove or displace material from offensive plates in order to reestablish adequate blood flow through the coronary arteries. Regardless of the
use of multiple forms of mechanical interventions, including balloon angioplasty balloons, atherectomy reduction, placement of vascular grafts, laser therapy or rotoblador, the effectiveness of these techniques remains limited by a restenosis rate of approximately 40% within 6 months after the treatment.
Restenosis is thought to result from a complex interaction of biological processes that include platelet deposition and thrombus formation, the release of chemotactic and mitogenic factors, and the migration and proliferation of vascular uniform muscle cells within the intimacy of the Dilated arterial segment.
Inhibition of platelet accumulation at sites of mechanical damage may limit the rate of restenosis in human subjects. The therapeutic use of a monoclonal antibody to the GpIIb / IIIa platelet is capable of limiting the level of restenosis in human subjects (Califf et al., N. Engl. J. Med., 330: 956-961 (1994)). The antibody is capable of binding to the GpIIb / IIIa receptor on the surfaces of platelets and therefore inhibit the accumulation of
platelets. These data suggest that the inhibition of platelet accumulation at the site of mechanical damage in human coronary arteries is beneficial for the final healing response that occurs.
GENES THERAPY
Polynucleotides that encode polypeptides
of zdintl, are useful within gene therapy applications where it is desired to increase or inhibit the activity of zdintl. If a mammal has a mutated or absent zdintl gene, the zdintl gene can be introduced into the cells
of the mammal. In one embodiment, a gene encoding a zdintl polypeptide is introduced into 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 are preferred, which completely or almost completely lack viral genes. A defective virus is not infectious
after entering a cell. The use of defective viral vectors allows administration to cells in a localized area
specific, without the worry that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a herpes simplex 1 defective virus vector (HSV1) (Kaplitt et al., Molec., Cell, Neurosci.2: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61: 3096-101, 1987; Samulski et al., J. Virol. 63: 3822-8, 1989).
In another embodiment, a zdintl gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. No. 5,399,346; Mann et al., Cell 3_3_: 153, 1983; Temin et al., U.S. No. 4,650,764; Temin et al., U.S. No. 4,980,289; Markowitz et al., J. Virol. 6_2: 1120, 1988; Temin et al., U.S. No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al., and Kuo et al., Blood 8_2: 845, 1993. Alternatively, the vector can be introduced by lipofection in vi vo using liposomes Synthetic cationic lipids can be used to prepare liposomes for the transfection of a gene encoding a marker (Felgner et al., Proc. Nati, Acad. Sci. USA 8_4_: 7413-7, 1987; Mackey et al. , Proc. Nati, Acad. Sci. USA 8_5: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs has certain practical advantages. The molecular attack of liposomes on specific cells represents an area of benefit. More particularly, directing the transfection to particular cells represents an area of benefit. For example, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney and brain. The lipids can be chemically coupled to other molecules for the purpose of attack. Target peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be chemically coupled to the 1 iposomes.
It is possible to separate the target cells from the body to introduce the vector as a naked DNA plasmid; and then reimplant the transformed cells within the body. Bare vectors of DNA for gene therapy can be
introducing into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a transporter DNA vectors. See, for example, Wu et al., J. Biol. Chem. 267: 963-7, 1992; Wu et al., J. Biol. Chem. 263: 14621-4, 1988. The antisense methodology can be used to inhibit the transcription of the zdintl gene, such as inhibiting cell proliferation in vivo. The polynucleotides that are complementary to a
The polynucleotide segment encoding zdintl (eg, a polynucleotide as set forth in SEQ ID NO: 1) are designed to bind the zdintl coding mRNA and inhibit the translation of each mRNA. Such antisense polynucleotides are
used to inhibit the expression of genes encoding zdintl polypeptides in cell culture or in a subject.
The present invention also provides
reagents that will find use in diagnostic applications. For example, the zdintl gene, a probe comprising zdintl DNA or RNA or a
Its subsequence can be used to determine if the zdintl gene is present on chromosome 2q33 or if a mutation has occurred. Chromosomal aberrations detectable at site 5 of the zdmtl gene include but are not limited to, aneuploidy, changes in the number of gene copies, insertions, deletions, changes in restriction sites and rearrangements. Such aberrations can be detected using
polynucleotides of the present invention when employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and
other genetic binding analysis techniques known in the art (Sambrook et al., Ibid; Ausubel et al., Ibid; Marian, Chest 108: 255-65, 1995)
Transgenic mice, engineered to express the zdintl gene or fragments thereof, and mice exhibiting a complete absence of zdintl gene function, referred to as "knockout mice" (Snouwaert et al.
al., Science 257: 1083, 1992), can also be generated (Lowell et al., Nature 366: 740-42, 1993) by one skilled in the art. These mice are
* 1 * 52.
they can be used to study the zdintl gene, gene fragments and the protein encoded by it in a m vi vo system.
CROMOSOMAL LOCATION
Hybrid radiation mapping is a genetic somatic cell technique developed to construct contiguous high resolution maps for
mammalian chromosomes (Coz et al., Science 250: 245-50, 1990). The partial or complete knowledge of a gene sequence allows someone to design PCR primers suitable for use with hybrid mapping groups by chromosomal radiation. The
groups of hybrid radiation mapping are commercially available, which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These 0 panels allow for chromosomal, PCR-based, rapid localizations and gene sorting, sequence tagged sites (STSs) and other polymorphic and non-polymorphic markers within a region of interest. This includes establishing 5 directly proportional physical distances between the barely discovered genes of interest and the previously mapped markers. He
itfeMaa-.a- 'fn aatt < a.a r, - y.
Accurate knowledge of the position of a gene can be useful for a variety of purposes, including: 1) determining whether 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) provide a possible candidate gene for a hereditary disease that shows binding to the same chromosomal region; and 3) organisms of transversal reference models such as the mouse, which can help in determining the function that a particular gene may have.
Sequence labeled sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique to the human genome and can be used as a reference point for a particular chromosome or region of the 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 only on the DNA sequence, they can be fully described within an electronic database, for example, the Datábase 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 sequence of genes of interest for the mapping data contained within these genomic STS guide sequences.
For pharmaceutical use, the proteins of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, locally (as powders, ointments, drops or transdermal patch) buccally or as a lung or nasal inhaler. Intravenous administration will be by bolus injection or infusion in a typical period of one to several hours. In general, the pharmaceutical formulations will include a zdintl protein, alone or in conjunction with a dimeric partner, in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like. The formulations may also include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein on the surfaces of the vial, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19a. ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 μg / kg patient weight per day, preferably 0.5-20 mg / kg per day, with the exact dose determined by the clinical staff according to the standards accepted, taking into account the nature and severity of the condition to be treated, patient characteristics, etc. The determination of the dose is within the level of ordinary skill in the art. The proteins can be administered for acute treatment, for a week or less, often for a period of one to three days or they can be used in chronic treatment for several months or years. In general, a therapeutically effective amount of zdintl is an amount sufficient to produce a clinically important change in remodeling of the extracellular matrix, scar tissue formation, tumor suppression, platelet aggregation, apoptosis, myogenesis, neurogenesis, electrical coupling , blood flow and / or proliferation of cells in brain, heart, spine and skeletal muscle.
The invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1 Extension of the EST Sequence
Polynucleotides encoding novel zdintl polypeptides of the present invention were initially identified by consulting an EST database. This query identified an expressed sequence tag (EST) at nucleotide 1097 at nucleotide 1415 of SEQ ID NO: 1. A cDNA clone was obtained, corresponding to this EST and the deduced amino acid sequence was determined to be incomplete. The primers ZC17,991 (SEQ ID NO: 4) and ZC17,992 (SEQ ID NO: 5) were used to sift a collection of arranged fetal brain cDNA plasmid to identify the zdintl clones. The conditions of the thermocycler were as follows: one cycle at 94 ° C for 1 minute 30 seconds, followed by thirty cycles at 94 ° C for 10 seconds, 64 ° C
«£ for 20 seconds, 72 ° C for 30 seconds, followed by a cycle at 72 ° C for 5 minutes, followed by 4 ° C sustained. A sample of the contents of the reaction was analyzed by electrophoresis on a 4% agarose gel to identify positive collections. These collections were screened by the polymerase chain reaction with ZC17,992 (SEQ ID NO: 5) and the vector primer ZC13,006 (SEQ ID NO: 6). Thermocycler conditions were: one cycle at 94 ° C for 1 minute 30 seconds, followed by five cycles at 94 ° C for 10 seconds, 68 ° C for 2 minutes, followed by twenty-five cycles at 94 ° C for 10 seconds, 62 ° C for 20 seconds, 72 ° C for 2 minutes, followed by a cycle at 72 ° C for 10 minutes, followed by 4 ° C sustained. A mixture of the reaction contents was analyzed by electrophoresis on a 1% agarose gel and analyzed by elect rofores is also a band of about 1.5 kb on a 1% preparative gel and the resulting band gel was purified using reagents and commercially available gel purification protocol (QIAEX II Gel Extraction Kit); Qiagen, Inc., Santa Clarita CA): This fragment was sequenced and determined to extend the amino acid sequence of zdintl in the 5 'direction.
Example 2 Distribution of Fabrics
The tissue distribution analysis was carried out by the Northern spotting technique using multiple human tissue and spot spots
master of Clontech (Palo Alto, CA), and a stain of human vascular tissue prepared internally. Human vascular stain was prepared from the following cell lines: human umbilical vein endothelial cells (Cascade Biologics, Inc.,
Portland, OR.), Endothelial cells of human pulmonary arteries (Cascade Biologics, Inc., Portland, OR.), Human aortic endothelial cells (Cascade Biologics Inc., Portland OR :), uniform aortic muscle cells (Clonetics,
San Diego, CA), human intestinal muscle uniform cells (American Type Culture Collection, Manasas, Virginia), normal human lung fibroblast, (Clonetics, San Diego, Ca), human dermal neonatal fibroblast, (Clonetics,
San Diego, CA). The messenger RNA was extracted and stained RNAs were prepared by methods known in the art. The probe was obtained by digestion of
restriction of the original cDNA clone with a restriction endonuclease, PstI. The reaction mixture was analyzed by electrophoresis in an agarose preparative gel and two bands, corresponding to a base pair fragment 239 and a base pair fragment 233 of the cDNA clone, were purified by gel using reagents and gel purification protocols commercially. available from Qiagen, Inc. A probe was made
Collect the purified DNA from both bands and label the 32P random primer using a commercially available kit (Rediprime DNA Labeling System, Amersham Corp., Arlington 'Heights, IL). The probe
was then purified using a NUCTRAP pusher column (Stratagene Cloning Systems, La Jolla CA). An EXPRESSHYB solution (Clontec) was used for prehybridization and hybridization. The hybridization solution consisted of 8 ml of EXPRESSHYB, 80 μl
of sheared salmon sperm DNA (10 mg / ml, 5 prime-3 prime, Boulder, CO), 48 μl of human Cot-1 DNA (1 mg / ml, Gibco BRL) and 57 μl of labeled probe (2.3 x 10-5 CPM / μl). Hybridization took place overnight at 50 ° C and the
stains were then washed in 2X SSC and 0.1% SDS at room temperature, then 2X SSC and SDS
0.1% at 60 ° C, followed by 0.1X SSC and 0.1% SDS at 60 ° C. Stains were exposed and developed overnight. Strong signals were observed from the three transcribed sizes, approximately 3.0 kb, 4.4 kb and 7.5 kb in the heart of the Northern blotches of multiple tissues. Weak signals of the same sizes of transcripts were observed in brain and spine. A weaker signal of the three was observed
sizes of transcripts in skeletal muscle. Spot spotting showed strong signals in brain, heart, fetal cerebellar and fetal heart. For human vascular staining, a strong signal at 3-3.5 kb was observed in
human aortic endothelial cells and weaker signals in uniform aortic muscle cells and human lung fibroblast cells.
Example 3 Purification of Proteins
Purification conditions for zdintl with EE labels of N and C terminals: 25 E cells. col l, Pi ch i a, CHO and BHK were transfected with expression vectors that
l ^ y ^ »^? ¿^ S ^ $ £ ^^ ~ y.4m yyyy yy * ^ - ^^ ^^^^ ^^ S ^^ Jyi yy yy ^^ ^^ A ^ g?. »_ _ ~ _ .. & and ^ _ gj &X and * m * -. YS ^ Jyy containing the DNA sequence of SEQ ID NO: l, or a portion of the misna, linked in operable manner to a polinucléot gone encoding a Glu-Glu tag. The zdintl protein is expressed in conditioned media of E. col i, Pi ch i a m e tha n ol i ca and / or Chinese hamster ovary cells (CHO) and newborn hamster kidney. For the zdintl expressed in E. col i and Pi ch i a, the means were not concentrated before the purification. Unless otherwise be observed, all operations were carried out at 4 ° C. A total of 25 liters of conditioned BHK sterile capsule 4 inches filtered sequentially through a filter, Millipore Opticap 0.2 mM (Bedford, MA) and a Supercap 50 Gelman 0.2 mM (Ann Arbor, MI). The material is then concentrated to about 1.3 liters using a Millipore ProFlux A30 tangential flow concentrator connected with an Amicon S10Y3 membrane of 3000 kDa (Bedford, MA). The concentrated material is sterile filtered again with the Gelman filter as described above. A mixture of protease inhibitors is added to the conditioned conditioned medium to final concentrations of 2.5 mM ethylenediamine tetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0.0001 mM leupeptin (Boehringer-Mannheim,
Indianapolis, IN), 0.001 mM pepsatatin (Boehringer-Mannheim) and 0.4 M Pefabloc (Boehpnger-Mannheim). A 50.0 ml sample of anti-SE Sepharose, prepared as described below is added and the mixture is gently shaken in a Wheaton roller culture apparatus (Millville, NJ) for 18.0 h at 4 ° C.
The mixture was then emptied into an Econo-10 Column 5.0 x 20.0 cm (Bio-Rad, Laboratories, Hercules, CA) and the gel was washed with volumes of 30 columns of phosphate buffered saline (PBS). The non-retained outflow fraction was discharged. Once the absorbance
of the effluent at 280 nM is less than 0.005, the flow through the column is reduced to zero, and the Sepharose anti-EE gel is washed with 2.0 columns of PBS containing 0.2 mg / ml of EE peptide ( AnaSpec, San José, CA). The peptide that is
usa has the sequence GluTyrMet ProValAsp. After 1.0 h at 4 ° C, the flow is restored and the eluted protein is collected. This fraction is referred to as peptide elution. The anti-EE Sepharose gel is then washed with 2.0 volumes
columns of 0.1 M glycine, pH 2.5 and the glycine wash is collected separately. The pH of the fraction eluted by glycine is adjusted to 7.0 by the
J «¿^ ^ ^^^? A- & Jj ^ & ^^ aA ^^^^^^ - j ^^^ J ^ & .M -, ^« - ^ - la - ^^^ lttK & £ A ^^ ^ ^ & ^ S ^ "^^^ &? FeA ^ ... ~ w;
add a small volume of 'PBS 10X and store at 4 ° C for future analysis if necessary.
Peptide elution is concentrated to 5.0 ml using a 15,000 molecular weight cut-off membrane concentrator (Millipore, Bedford, MA), according to the manufacturer's instructions. The concentrated peptide elution is then separated from
of the free peptide by chromatography on a Sephadex G-50 column of 1.5 x 50 cm (Pharmacia, Piscataway, NJ) equilibrated in PBS at a flow rate of 1.0 ml / rnin using Sprint BioCad HPLC (PerSpetive BioSystems Framingham, MA). They were collected
fractions of two milliliters and the absorbance was observed at 280 nM and collected by eluting near the empty volume of the column. This fraction is NEE zdintl puro or CEE zdintl. The pure material is concentrated as described above, analyzed by
SDS-PAGE and Western staining with anti-EE antibodies, in aliquots, and stored at -80 ° C according to normal procedures.
Preparation of Sepharose anti-EE
A 100 ml bed volume of G-Sepharose protein (Pharmacia, Piscataway, NJ) was washed 3 times with 100 ml of PBS containing 0.02% sodium azide using a 0.45 micron filter unit of 500 ml Nalgene. The gel was washed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, MO) and an equal volume of EE antibody solution containing 900 mg of antibody was added. After an overnight incubation at 4 ° C, the unbound antibody is removed by washing the resin with 5 volumes of 200 mM TEA as described above. The resin was resuspended in 2 volumes of TEA, transferred to an appropriate container, and 2-dimethyl-pyrimidimidate hydrochloride (Pierce, Rockford, IL) dissolved in TEA was added to a final concentration of 36 mg / ml gel. The gel is maintained at room temperature for 45 min and the liquid was removed using the filter unit as described above. The non-specific sites on the gel are then blocked by incubating for 10 minutes at room temperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gel is then washed with 5 volumes of PBS
Containing 0.02% sodium azide and stored in this solution at 4 C.
Purification of zdint 1 not labeled.
E. coli, Pichia, CHO and BHK cells were transfected with expression vectors containing the DNA sequence for SEQ ID NO: 1 or a portion thereof. The procedure below
described is used for the protein expressed in a conditioned medium of E. coli, Pichia methanolica, and Chinese hamster ovary cells (CHO) and newborn hamster kidney (BHK). For the zdintl expressed in E. coll and Pichia without
However, the medium is not concentrated before purification. Unless otherwise stated, all operations are carried out at 4 ° C. They are sequentially filtered sterile, a total of 25 liters of conditioned medium from
BHK cells through an OptiCap Millipore capsule filter (Bedford, MA) 0.2 mM 4 inches and a Supercap50 Gelman (Ann Arbor, MI). The material is then concentrated to about 1.3 liters using a tangential flow concentrator
Millipore ProFlux A30 coupled with an Amicon S10Y3 membrane of 3000 kDa (Bedfor, MA). The concentrated material is sterile filtered again
with the Gelman filter as described above. A mixture of protease inhibitors is added to the conditioned conditioned medium to final concentrations of 2.5 mM ethylenediamine tetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO) 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, IN), 0.001 mM pepstatin. (Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). The procedures described below are adaptations of those used to purify disintegrins / venom metalloprotease from Cro ta lus vi ri dus and Cró ta l os a trox (Liu et al.,
Toxicol. 33: 1289-1298, 1995; Shimokawa et al., Arch Biochem Biophys 343: 35-43, 1997). A combination of procedures that include, but are not limited to, anion cation exchange chromatography, size exclusion and
affinity chromatography to purify unlabeled zdintl.
Concentrated conditioned medium is diluted 1/10 in line with borate buffer
mM, pH 9.0, 0.1 M NaCl and 2.0 mM CaC12, using Sprint BioCad HPLC (PerSeptive BioSystems, Framingham, MA). The material is pumped on a
HQ Poros column 3.5 x 20 cm (PerSeptive BioSystems, Framingham, MA) at 5 ml / min. The column is washed with charge buffer and when the absorbance of the effluent is less than 5 0.05, the column develops with a linear gradient of NaCl from 0.1 M to 1.0 M NaCl. Fractions containing zdintl are identified by SDS-PAGE and Western blotting with anti-zdintl peptide antibodies. Fractions that contain
zdintl are pooled together and concentrated using an Amicon stirred cell concentrator coupled with a YM-10 membrane. The collected Poros HQ is then chromatographed on a Sephadex G-75 column equilibrated in 10 mM sodium phosphate, pH
7.0. Fractions containing zdintl are identified and pooled together as described above, and applied to a Poros HA hydroxyapatite column 1.0 x 5 cm at 1.0 ml / min using the Sprint BioCad HPLC. The column is washed
with a charge buffer and develops with a linear gradient of sodium phosphate from 10 mM to 500 mM. The fractions containing the pure zdintl are identified by SDS-PAGE and Western spotting as described above.
The purified material is placed in aliquots and stored as described above.
Jlí & j ^ & ?? t ^ ^ Example 4 Assignment of Chromosomes and Placement of Zdintl
The zdintl was mapped to chromosome 2 using the commercially available version of the "Stanford G3 Hybrid Radiation Mapping Group" (Research Genetics, Inc., Huntsville, AL). The "RH Stanford G3 panel" contains DNAs that can be used in PCR of each of the hybrid radiation clones 83 of the complete human genome, plus two control DNAs (the RM donor and the A3 receptor). A publicly available WWW server (http://shgc-www.stanford.edu) allows the chromosomal localization of the markers.
For the zdintl mapping with the "Stanford G3 RH Group", 20 μl reactions were established in a 96-well microtiter plate using PCR (Stratagene, La Jolla, CA) and used in a thermal cycler "RoboCycler Gradiente 96"
(Stratagene). Each of the 85 PCR reactions consisted of a 2 μl KlenTaq PCR 10X reaction buffer (CLONTECH
Laboratories, Inc., Palo Alto, CA), 1.6 ul dNTP mix (2.5 mM each, Pekin Elmer, Foster City, CA), 1 ul sense primer, ZC20,843 (SEQ ID
"A and * ^ to l ^ t ^ & 3 '&' fi 'J ^» TTI * »J-NO: 12), primer anti-sense 1 ul, ZC20,844 (SEQ ID NO: 13), Redfiload 2 μl (Research Genetics, Inc., Huntsville, AL), mixture of KlenTaq Advantage 50X polymerase 0.4 μl (Clontech Laboratories, Inc.), 25 ng of DNA from a single hybrid or control clone and distilled water for a total volume of 20 μl The reactions were overlapped with an equal amount of mineral oil and sealed.The conditions of the PCR cycler were as follows: an initial cycle 1 denaturation of 5 min at 94 ° C, 35 cycles of denaturation from 45 seconds to 94 ° C, 45 seconds of cooking at 66 ° C and 1 minute and 15 seconds extension at 72 ° C, followed by a final extension of cycle 1 of 7 minutes at 72 ° C. the reactions were separated by electrophoresis in a gel 2% agarose (Li feTechnologies, Gaithersburg, MD).
The results showed a zdintl coupling to structure marker SHGC-56733 with an LOD record of > 12 and at a distance of 0 cR_10000 from the marker. The use of zdintl surrounding marker positions in the 2q33 region on map 2 of the integrated LDB chromosome (The Genetic Location Database, University of Sothhampton, WWW server http: // cedar.genetics.soton.ac.uk/public_html/).
Example 5 Synthesis of Peptides
Zdintl-1, a peptide corresponding to amino acid residue 437 (Cys) to amino acid residue 450 (Cys) of SEQ ID NO: 2, is
synthesized by solid phase peptide synthesis using a Model 431A Peptide Synthesizer (Applied Biosystems / Per kin Elmer, Foster City, CA). The Fmoc-Glutamine resin (0.63 mmol / g; Advanced Chemtech, Lousville, KY) is used as the
initial support resin. The 1 mmol amino acid cartridges (Anaspec, Inc. San Jose, CA) are used for synthesis. A mixture of 2- (1-H-benzothiazol-11,1,3,3-tetramethylururonium hexafluorophosphate (HBTU), 1- 20 hydroxybenzotriazole (HOBt), N, N-diisopropylethylamine 2m, N-methylpyrrolidone, dichloromethane ( all of Applied Biosys t ems / Per kin Elmer) and piperidine (Aldrich Chemical Co., St. Louis, MO) are used as synthesis reagents.25 The Peptide Companion computation package (Peptides International, Louisville, KY) is used
to predict the aggregation potential and the level of difficulty for the synthesis of the zdint-1 peptide. The synthesis is carried out using simple coupling programs, in accordance with the manufacturer's specifications.
The peptide is split from the solid phase following a normal TFA split procedure (according to the manual of
peptide splitting, Applied Biosystems / Per kin Elmer). The purification of the peptide is done by RP-HPLC using a 10 μm semipreparative column, C18 (Vydac, Hesperial, CA). The fractions eluted from the
column and analyzed for their correct mass and purity by elect roatomization mass spectrometry. Collections of eluted material are collected. If they are pure, the collections are combined, frozen and lyophilized. Example 6 Anticoagulant activity of zdintl
The capacity of the zdintl protein for
inhibit coagulation is measured in a one-stage coagulation assay using zdintl in a state
irregular as a control. Recombinant proteins are prepared essentially as described above from cells grown in media containing 5 mg / ml vitamin K. Variable amounts of zdintl or zdmt of recombinant irregular type are diluted in 50 mM Tris, pH 7.5, BSA at 0.1% up to 100 ml. The mixtures are incubated with 100 ml of zdintl-deficient plasma and 200 ml of thromboplastin C (Dade, Miami, FL;
contains rabbit brain thromboplasty and Ca ++ 11.8 mM).
The coagulation test is carried out on an automatic coagulation timer (MLA Electra
800, Medical Laboratory Automation Inc., Pleasantville, NY) and coagulation times are converted to units of zdintl activity using a normal curve constructed with 1: 5 to 1: 640 dilutions of normal human plasma collected
(assuming it contains one unit per ml of zdintl activity, prepared by collecting coded serum from healthy donors).
The zdintl activity is observed as a reduction in the coagulation time in the control samples.
E emplo7 Inhibition of platelet accumulation with zdint 1
Zdintl is tested for its ability to inhibit platelet accumulation at sites of arterial thrombosis due to mechanical damage in non-human primates. A model of aortic endarterectomy is used in mandrels, essentially
as described by Lumsden et al. (Blood 81: 1762-1770 (1993)). A mandrel aorta section of 1-2 cm in length was removed, inverted and scraped to separate intimacy from the artery and approximately 50% of the media. The artery
changed back to its correct orientation, cannulated at both ends and placed on an extracorporeal implant in a mandrel, whereby the mechanically damaged artery was exposed to the mandrel blood by means of the implant. Just before the
After opening the implant to the circulating blood, autologous platelets labeled with n? In were injected intravenously into the animal. The level of platelet accumulation at the site of the damaged artery was determined by gamma camera imaging
in real time.
The evaluation of zdintl for the inhibition of platelet accumulation is done using zdintl bolus injections or saline control and is provided just before the opening of the implant. Damaged arteries are measured continuously for 60 minutes.
The activity of zdintl is observed as an inhibition in platelet accumulation. From the foregoing, it will be appreciated that although specific embodiments of the invention have been described herein for purposes of illustration, various embodiments may be made.
modifications without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except that by the appended claims.
It is noted that with respect to this date, the best known method for carrying out the invention is that which is clear from the present description of the invention.
Having described the invention as above, property is claimed as contained in the following
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LIST OF SEQUENCES
< 110 > ZymoGenetics, Inc. < 120 > DISINTEGRINE HOMOLOGOUS
< 130 > 98-29PC
< 160 > 13 < 170 > Version 3.0 for Windows FastSEQ
< 210 > 1 < 211 > 2268 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (3) ... (2090) < 221 > rrnsc_feature < 222 > (D. J2268) < 223 > n - A.T.C or G < 400 > 1 ce act gtg ttg gaa ttc ggc acg agg ctt gac here aag gca aga cae 47
Thr Val Leu Glu Phe Gly Thr Arg Leu Asp Thr Lys Wing Arg His 1 5 10 15 cag caaaa cat aat aag gct gtc cat ctg gcc cag gca age ttc cag 95 Gln Gln Lys His Asn Lys Ala Val His Leu Ala Gln Ala Ser Phe Gln 20 25 30 att gaa gcc ttc gcc tcc aaa ttc att ctt gac etc ata ctg aac aat 143 He Glu Wing Phe Gly Ser Lys Phe He Leu Asp Leu He Leu Asn Asn 35 40 45 ggt ttg ttg tct tct gat tat gtg gag att falls tac gaa aat ggg aaa 191 Gly Leu Leu Being Ser Asp Tyr Val Glu He His Tyr Glu Asn Gly Lys 50 55 60 cea cag tac tct aag ggt gga gag falls tgt tac tac cat gga age ate 239 Pro Gln Tyr Ser Lys Gly Gly Glu HTS Cys Tyr Tyr His Gly Ser He 65 70 75 aga ggc gtc aaa gac tcc aag gtg gct ctg tea acc tgc aat gga ctt 287 Arg Gly Val Lys Asp Ser Lys Val Ala Leu Ser Thr Cys Asn Gly Leu 80 85 90 95
cat ggc atg ttt gaa gat gat acc ttc gtg tat atg ata gag cea cta 335 His Gly Met Phe Glu Asp Asp Thr Phe Val Tyr Met He Glu Pro Leu 100 105 110
gag ctg gtt cat gat gag aaa age here ggt cga cea cat ata ata cag 383 Glu Leu Val Hi s Asp Glu Lys Ser Thr Gly Arg Pro His He He Gln 115 120 125 aaa acc ttg gca gga cag tat tct aag caa atg aag aat etc act atg 431 Lys Thr Leu Wing Gly Gln Tyr Ser Lys Gln Met Lys Asn Leu Thr Met 130 135 140
gaa aga ggt gac cag tgg ccc ttt etc tct gaa tta cag tgg ttg aaa 479 Glu Arg Gly Asp Gln Trp Pro Phe Leu Ser Glu Leu Gln Trp Leu Lys 145 150 155
aga agg aag aga gca gtg aat cea tea cgt ggt ata ttt gaa gaa atg 527 Arg Arg Lys Arg Ala Val Asn Pro Ser Arg Gly He Phe Glu Glu Met 160 165 170 175
aaa tat ttg gaa ctt atg att ggt aat gat falls aaa acg tat aag aag 575 Lys Tyr Leu Glu Leu Met He Gly Asn Asp His Lys Thr Tyr Lys Lys 180 185 190 cat cgc tct tct cat gca cat acc acac aac ttt gca aag tcc gtg gtc 623 His Arg Ser Ser His Wing His Thr Asn Asn Phe Wing Lys Ser Val Val 195 200 205
aac ctt gtg gat tct att tac aag gag cag etc aac acc agg gtt gtc 671 Asn Leu Val Asp Ser He Tyr Lys Glu Gln Leu Asn Thr Arg Val Val 210 215 220
ctg gtg gct gta gag acc tgg act gag aag gat cag att gac ate acc 719 Leu Val Wing Val Glu Thr Trp Thr Glu Lys Asp Gln He Asp He Thr 225 230 235
acc aac ect gtg cag atg etc cat gag ttc tea aaa tac cgg cgc 767 Thr Asn Pro Val Gln Met Leu His Glu Phe Ser Lys Tyr Arg Gln Arg 240 245 250 255 att aag cag cat gct gat gct gtg falls etc ate tcg cgg gtg here ttt 815 He Lys Gln His Wing Asp Wing Val His Leu He Ser Arg Val Thr Phe 260 265 270 falls tat aag aga age agt ctg agt tac ttt gaa ggt gtc tgt tct cgc 863 His Tyr Lys Arg Ser Ser Leu Ser Tyr Phe Glu Gly Val Cys Ser Arg 275 280 285
here aga gga gtt ggt gtg aat gag tat ggt ctt cea atg gca gtg gca 911 Thr Arg Gly Val Gly Val Asn Glu Tyr Gly Leu Pro Met Ala Wing Ala 290 295 300 caá gta tta tcg cag age ctg gct caac aac ctt gga ate ca Tgg Gaa 959 Gm Val Leu Ser Gln Ser Leu Ala Gln Asn Leu Gly He Gln Trp Glu 305 310 315 ect tct age aga aag cea aaa tgt gac tgc here gaa tcc tgg ggt ggc 1007 Pro Ser Ser Arg Lys Pro Lys Cys Asp Cys Thr Glu Ser Trp Gly Gly 320 325 330 335 tgc ate atg gag gaa here ggg gtg tcc cat tct cga aaa ttt tea aag 1055 Cys He Met Glu Glu Thr Gly Val Ser His Ser Arg Lys Phe Ser Lys 340 345 350 tgc age att ttg gag tat aga gac ttt tta cag aga gga ggt gga gcc 1103 Cys Ser He Leu Glu Tyr Arg Asp Phe Leu Gln Arg Gly Gly Gly Wing 355 360 365 tgc ctt ttc aac agg cea here aag cta ttt gag ccc acg gaa tgt gga 1151 Cys Leu Phe Asn Arg Pro Thr Lys Leu Phe Glu Pro Thr Glu Cys Gly 370 375 380 aat gga tac gtg gaa gct ggg gag gag tgt gat tgt ggt ttt cat gtg 1199 Asn Gly Tyr Val Glu Ala Gly Glu Glu C ys Asp Cys Gly Phe His Val 385 390 395 gaa tgc tat gga tta tgc tgt aag aaa tgt tcc etc tcc aac ggg gct 1247 Glu Cys Tyr Gly Leu Cys Cys Lys Cys Ser Leu Ser Asn Gly Wing 400 405 410 415
falls tgc age gac ggg ccc tgc tgt aac aat acc tea tgt ctt ttt cag 1295 His Cys Ser Asp Gly Pro Cys Cys Asn Asn Thr Ser Cys Leu Phe Gln 420 425 430
cea cga ggg tat gaa tgc cgg gat gct gtg aac gag tgt gat att act 134: Pro Arg Gly Tyr Glu Cys Arg Asp Wing Val Asn Glu Cys Asp He Thr 435 440 445
gaa tat tgt act gga gac tct ggt cag tgc cea cea aat ctt cat aag 1391 Glu Tyr Cys Thr Gly Asp Ser Gly Gln Cys Pro Pro Asn Leu His Lys 450 455 460
ca gac gga tat gca tgc aat caa aat cag ggc cgc tgc tac aat ggc 1439 Gln Asp Gly Tyr Ala Cys Asn Gln Asn Gln Gly Arg Cys Tyr Asn Gly 465 470 475
gag tgc aag acc aga gac aac cag tgt cag tac ate tgg gga here aag 1487 Glu Cys Lys Thr Arg Asp Asn Gln Cys Gln Tyr He Trp Gly Thr Lys 480 485 490 495 gct gca ggg tct gac aag ttc tgc tat gaa aag ctg aat here gaa ggc 1535 Ala Ala Gly Ser Asp Lys Phe Cys Tyr Glu Lys Leu Asn Thr Glu Gly 500 505 510
act gag aag gga aac tgc ggg aag gat gga gac cgg tgg att cag tgc 1583 Thr Glu Lys Gly Asn Cys Gly Lys Asp Gly Asp Arg Trp He Gln Cys 515 520 525
age aaa cat gat gtg ttc tgt gga ttc tta etc tgt acc aat ctt act 1631 Ser Lys His Asp Val Phe Cys Gly Phe Leu Leu Cys Thr Asn Leu Thr 530 535 540
cga gct cea cgt att ggt caa ctt cag ggt gag ate att cea act tcc 1679 Arg Ala Pro Arg He Gly Gln Leu Gln Gly Glu He He Pro Thr Ser 545 550 555
ttc tac cat ca ggc cgg gtg att gac tgc agt ggt gcc cat gta gtt 1727 Phe Tyr His Gln Gly Arg Val He Asp Cys Ser Gly Ala His Val Val 560 565 570 575
tta gat gat gat gat acg gat gtg ggc tat gta gaa gat gga acg cea tgt 1775 Leu Asp Asp Asp Thr Asp Val Gly Tyr Val Glu Asp Gly Thr Pro Cys 580 585 590
ggc ccg tct atg atg tgt tta gat cgg aag tgc cta ca gt cacc gcc 182: Gly Pro Met Met Met Cys Leu Asp Arg Lys Cys Leu Gln He Gln Ala 595 600 605
cta aat atg age age tgt cea etc gat tcc aag ggt aaa gtc tgt tcg 1871 Leu Asn Met Ser Ser Cys Pro Leu Asp Ser Lys Gly Lys Val Cys Ser 610 615 620 ggc cat ggg gtg tgt agt aat gaa gcc acc tgc att tgt gat ttc acc 1919 Gly His Gly Val Cys Ser Asn Glu Wing Thr Cys He Cys Asp Phe Thr 625 630 635
tgg gca ggg here gat tgc agt ate cgg gat cea gtt agg aac ctt falls 1967 Trp Wing Gly Thr Asp Cys Ser He Arg Asp Pro Val Arg Asn Leu His 640 645 650 655
ccc ccc aag gat gaa gga ccc aag ggt ttg tgt gat ttt ggt ttc aat 2015 Pro Pro Lys Asp Glu Gly Pro Lys Gly Leu Cys Asp Phe Gly Phe Asn 660 665 670
tea tgg aat act gaa ttc gtt gac act gtt cea atg fall cag tat aac 2063 Ser Trp Asn Thr Glu Phe Val Asp Thr Val Pro Met His Gln Tyr Asn 675 680 685
att cta att gac tta aga gga gac here taagaatatc ngtttttgcc 2110 He Leu He Asp Leu Arg Gly Asp Thr 690 695
tttaaagtat ataatttatg ttactgccaa attaaggatt ctgatatatc atatttttaa 2170 aatgtgtttg aattacttct tagtctagaa ctgagattgg gaagaagtaa atatacacat 2230 tttctttaat acagtattct ttttctcttt aaacetta 2268
< 210 > 2 < 211 > 696 < 212 > PRT < 213 > Homo sapiens
< 400 > 2 Thr Val Leu Glu Phe Gly Thr Arg Leu Asp Thr Lys Wing Arg His Gln 1 5 10 15 Gln Lys i-hs Asn Lys Wing Val His Leu Wing Gln Wing Being Phe Gln He 20 25 30 Glu Wing Phe Gly Ser Lys Phe He Leu Asp Leu He Leu Asn Asn Gly 35 40 45
faith. K tti y. *. * r * Sl & Leu Leu Ser Being Asp Tyr Val Glu He His Tyr Glu Asn Gly Lys Pro 50 55 60 Gln Tyr Ser Lys Gly Gly Glu His Cys Tyr Tyr His Gly Ser He Arg 65 70 75 80 Gly Val Lys Asp Ser Lys Val Ala Leu Ser Thr Cys Asn Gly Leu His 85 90 95 Gly Met Phe Glu Asp Asp Thr Phe Val Tyr Met He Glu Pro Leu Gl u 100 105 110 Leu Val His Asp Glu Lys Ser Thr Gly Arg Pro His He He Gl n Lys 115 120 125 Thr Leu Wing Gly Gln Tyr Ser Lys Gl n Met Lys Asn Leu Thr Met Gl u 130 135 140 Arg Gly Asp Gln Trp Pro Phe Leu Ser Gl u Leu G n Trp Leu Lys Arg 145 150 155 160 Arg Lys Arg Ala Val Asn Pro Ser Arg Gly He Phe Glu Glu Met Lys 165 170 175 Tyr Leu Glu Leu Met He Gly Asn Asp His Lys Thr Tyr Lys Lys His 180 185 190 Arg Ser Ser His Wing His Thr Asn Asn Phe Wing Lys Ser Val Val Asn 195 200 205 Leu Val Asp Ser He Tyr Lys Glu Gln Leu Asn Thr Arg Val Val Leu 210 215 220 Val Wing Val Glu Thr Trp Thr Glu Lys Asp Gln He Asp He Thr Thr 225 230 235 240 Asn Pro Val Gln Met Leu His Glu Phe Ser Lys Tyr Arg Gln Arg He 245 250 255 Lys Gln His Wing Asp Wing Val His Leu He Ser Arg Val Thr Phe His 260 265 270 Tyr Lys Arg Ser Ser Leu Ser Tyr Phe Glu Gly Val Cys Ser Arg Thr 275 280 285 Arg Gly Val Gly Val Asn Gl u Tyr Gly Leu Pro Met Ala Ala Ala Gln 290 295 300 Val Leu Ser Gln Ser Leu Al a Gl n Asn Leu Gly He Gln Trp Glu Pro 305 310 315 320 Ser Ser Arg Lys Pro Lys Cys Asp Cys Thr Glu Ser Trp Gly Gly Cys 325 330 335 He Met Glu Glu Thr Gly Val Ser His Ser Arg Lys Phe Ser Lys Cys 340 345 350 Ser lie Leu Glu Tyr Arg Asp Phe Leu Gln Arg Gly Gly Gly Ala Cys 355 360 365 Leu Phe Asn Arg Pro Thr Lys Leu Phe Glu Pro Thr Glu Cys Gly Asn 370 375 380 Gly Tyr Val Glu Wing Gly Glu Glu Cys Asp Cys Gly Phe Hi s Val Glu 385 390 395 400
Cys Tyr Gly Leu Cys Cys Lys Lys Cys Ser Leu Ser Asn Gly Wing His 405 410 415
Cys Ser Asp Gly Pro Cys Cys Asn Asn Thr Ser Cys Leu Phe Gln Pro 420 425 430 Arg Gly Tyr Glu Cys Arg Asp Ala Val Asn Glu Cys Asp He Thr Glu 435 440 445 Tyr Cys Thr Gly Asp Ser Gly Gln Cys Pro Pro Asn Leu His Lys Gln
450 455 460 Asp Gly Tyr Wing Cys Asn Gln Asn Gln Gly Arg Cys Tyr Asn Gly Glu 465 470 475 480 Cys Lys Thr Arg Asp Asn Gln Cys Gln Tyr He Trp Gly Thr Lys Wing 485 490 495
Wing Gly As Asp Lys Phe Cys Tyr Glu Lys Leu Asn Thr Glu Gly Thr 500 505 510 Glu Lys Gly Asn Cys Gly Lys Asp Gly Asp Arg Trp He Gln Cys Ser 515 520 525 Lys His Asp Val Phe Cys Gly Phe Leu Leu Cys Thr Asn Leu Thr Arg
530 535 540 Pro Wing Arg He Gly Gln Leu Gln Gly Glu He He Pro Thr Ser Phe 545 550 555 560 Tyr His Gln Gly Arg Val He Asp Cys Ser Gly Wing His Val Val Leu 565 570 575 Asp Asp Asp Thr Asp Val Gly Tyr Val Glu Asp Gly Thr Pro Cys Gly 580 585 590 Pro Met Met Cys Leu Asp Arg Lys Cys Leu Gln He Gln Ala Leu 595 600 605 Asn Met Ser Ser Cys Pro Leu Asp Ser Lys Gly Lys Val Cys Ser Gly
610 615 620 His Gly Val Cys Ser Asn Glu Wing Thr Cys He Cys Asp Phe Thr Trp 625 630 635 640 Wing Gly Thr Asp Cys Ser He Arg Asp Pro Val Arg Asn Leu His Pro 645 650 655 Pro Lys Asp Glu Gly Pro Lys Gly Leu Cys Asp Phe Gly Phe Asn Ser 660 665 670 Trp Asn Thr Glu Phe Val Asp Thr Val Pro Met His Gln Tyr Asn He 675 680 685 Leu He Asp Leu Arg Gly Asp Thr 690 695 < 210 > 3 < 211 > 2088 < 213 > Artificial Sequence
< 220 >
- *. * < 223 Degenerative amino acid sequence zdintl
< 221 > vanation < 222 > (1) .. (2088) < 223 > n is any nucleotide
< 221 > m? sc_feature < 222 > (1) ... (2088) < 223 > n = A.T.C or G < 400 > 3 acngtnytng arttyggnac nmgnytngay acnaargcnm gncaycarca raarcayaay 60 aargcngtnc ayytngcnca rgcnwsntty carathgarg cnttyggnws naarttyath 120 ytngayytna thytnaayaa yggnytnytn wsnwsngayt aygtngarat hcaytaygar 180 aayggnaarc cncartayws naarggnggn garcaytgyt aytaycaygg nwsnathmgn 240 ggngtnaarg aywsnaargt ngcnytnwsn acntgyaayg gnytncaygg natgttygar 300 gaygayacnt tygtntayat gathgarccn ytngarytng tncaygayga raarwsnacn 360 ggnmgnccnc ayathathca raaracnytn gcnggncart aywsnaarca ratgaaraay 420 ytnacnatgg armgnggnga ycartggccn ttyytnwsng arytncartg gytnaarmgn 480 mgnaarmgng cngtnaaycc nwsnmgnggn athttygarg aratgaarta yytngarytn 540 aygaycayaa atgathggna racntayaar aarcaymgnw snwsncaygc ncayacnaay 600 aayttygcna arwsngtngt naayytngtn gaywsnatht ayaargarca rytnaayacn 660 mgngtngtny tngtngcngt ngaracntgg acngaraarg aycarathga yathacnacn 720 aratgytnca aayccngtnc ygarttywsn aartaygnc argnathaa rcarcaygcn 780 ayytnathws gaygcngtnc nmgngtnacn ttycaytaya armgnwsnws nytnwsntay 840 ttygarggng tntgywsng nacngnggn gtnggngtna aygartaygg nytnccnatg 900 gcngtngcnc argtnytnws ncarwsnytn gcncaraayy tnggnathca rtgggarccn 960 wsnwsnmgna arecnaartg ygaytgyacn garwsntggg gnggntgyat hatggargar 1020 acnggngtnw sncaywsnmg naarttywsn aartgywsna thytngarta ymgngaytty 1080 ytncarmgng gnggnggngc ntgyytntty aaymgnccna cnaarytntt ygarecnaen 1140 gartgyggna ayggntaygt ngargcnggn gargartgyg aytgyggntt ycaygtngar 1200 tgytayggny tntgytgyaa raartgywsn ytnwsnaayg gngcncaytg ywsngayggn 1260 ayaayacnws ccntgytgya ntgyytntty carccn gng gntaygartg ymgngaygcn 1320 gtnaaygart gygayathac ngartaytgy acnggngayw snggncartg yccnccnaay 1380 argayggnta ytncayaarc ygcntgyaay caraaycarg gngntgyta yaayggngar 1440 gngayaayca tgyaaracnm rtgycartay athtggggna cnaargenge nggnwsngay 1500 aarttytgyt aygaraaryt naayacngar ggnaengara arggnaaytg yggnaargay 1560 ggngaymgnt ggathcartg ywsnaarcay gaygtnttyt gyggnttyyt nytntgyacn 1620 aayytnacn gngcnccng nathggncar ytncarggng arathathee nacnwsntty 1680 tayeayearg gnmgngtnat hgaytgywsn ggngcncayg tngtnytnga ygaygayacn 1740 gaygtnggnt aygtngarga yggnacnccn tgyggnccnw snatgatgtg yytngaygn 1800 aartgyytnc arathearge nytnaayatg wsnwsntgyc cnytngayws naarggnaar 1860 gtntgywsng gncayggngt ntgywsnaay gargenaent gyathtgyga yttyacntgg 1920 gcnggnacng aytgywsnat hmgngayccn gtnmgnaayy tncayccncc naargaygar 1980 gnytntgyga ggnccnaarg yttyggntty aaywsntgga ayaengartt ygtngayacn 2040
jwan? ri * - * • y. ** A? mB? u £. • gtnccnatgc ayeartayaa yathytnath gayy gng gngayacn 2088 < 210 > 4 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > ol igonucleotide - 17991 < 400 > 4 gctatttgag cccacggaat gtg 23 < 210 > 5 < 211 > 23 < 212 > DNA 213 > Artificial Sequence
< 220 > < 223 > oligonucleotide ZC17992 < 400 > 5 actgaccaga gtctcccagt here 23 < 210 > 6 < 211 > 20 < 212 > DNA < 213 Artificial Sequence
< 220 > < 223 > oligonucleotide: ZC13006 < 400 > 6 ggctgtcctc taagcgtcac 20 < 210 > 7 < 211 > 6 < 212 > PRT v < 213 Artificial Sequence
< 220 > < 223 > Antigenic Peptide
< 400 > 7
Lys Arg Arg Lys Arg Ala 1 5 < 210 > 8 < 211 > 6 < 212 > PRT 213 > Artificial Sequence
< 220 > < 223 > Antigenic peptide < 400 > 8 Leu Lys Arg Arg Lys Arg 1 5 < 210 > 9 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence
< 220 > < 223 > Antigenic peptide < 400 > 9 Gly Lys Asp Gly Asp Arg 1 5 < 210 > 10 < 211 > 6 < 212 > PRT 213 > Artificial Sequence
< 220 > < 223 Antigenic peptide
< 400 > 10 Lys Asp Glu Gly Pro Lys 1 5 < 210 > 11 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence
< 220 >
< 223 Antigenic peptide
< 400 > 11 Lys Lys His Arg Ser Ser 1 5 < 210 > 12 < 211 > 18 < 212 > DNA < 213 Artificial Sequence
< 220 > < 223 > First oligonusleotide ZC20.843
< 400 > 12 tcctggtggc tgtagaga 18
< 210 > 13 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > First oligonucleotide ZC20, 844
< 400 > 13 tgccggtatt ttgagaac 8
Claims (27)
1. A molecule isolated from polypeptide, characterized in that it comprises a contiguous sequence of 14 amino acids of SEQ ID NO: 2.
2. The isolated polypeptide molecule according to claim 1, characterized in that the polypeptide molecule comprises residues 437 to 450 of SEQ ID NO: 23.
The isolated polypeptide molecule according to claim 1, characterized in that the polypeptide molecule is between 82 and 232 amino acids in length.
4. The isolated polypeptide molecule according to claim 3, characterized in that the polypeptide molecule is residues 164 to 382 of SEQ ID NO: 2.
5. The isolated polypeptide molecule according to claim 3, characterized in that the polypeptide molecule is residues 383 to 464 of SEQ ID NO: 2. Battutf ^
6. The isolated polypeptide molecule according to claim 3, characterized in that the polypeptide molecule is the waste 465 to 696 of SEQ ID NO: 2.
7. A molecule isolated from polypeptide, characterized in that it is selected from the group consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO: 2. b) a polypeptide molecule comprising residues 383 to 464 of SEQ ID NO: 2. c) a polypeptide molecule comprising residues 465 to 696 of SEQ ID NO: 2. d) a polypeptide molecule comprising residues 438 to 449 of SEQ ID NO: 2. e) a polypeptide molecule comprising residues 164 to 464 of SEQ ID NO: 2. f) a polypeptide molecule comprising residues 164 to 696 of SEQ ID NO: 2. g) a polypeptide molecule comprising residues 383 to 696 of SEQ ID NO: 2. h) a polypeptide molecule comprising residues 164 to 449 of SEQ ID NO: 2. i) a polypeptide molecule comprising residues 438 to 696 of SEQ ID NO: 2; Y j) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO: 2.
8. A molecule isolated from a polynucleotide that encodes a polypeptide molecule, characterized in that the molecule comprises a contiguous sequence of 14 amino acids of SEQ ID NO: 2.
9. The isolated polynucleotide molecule according to claim 8, characterized in that the polypeptide molecule comprises residues 437 to 450 of SEQ ID NO: 2.
10. The isolated nucleotide molecule according to claim 8, characterized in that the polypeptide molecule is between 82 and 222 amino acids in length.
11. The isolated polynucleotide molecule according to claim 10, characterized in that the polypeptide molecule is residues 164 to 382 of SEQ ID NO: 2.
12. The isolated polynucleotide molecule according to claim 10, characterized in that the polypeptide molecule is 10 residues 383 to 464 of SEQ ID NO: 2.
13. The isolated polynucleotide molecule according to claim 10, characterized in that the polypeptide molecule is 15 residues 465 to 696 of SEQ ID NO: 2.
14. An isolated polynucleotide molecule encoding a polypeptide, characterized in that the polypeptide molecule is selected from the group 20 consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO: 2. B) a polypeptide molecule comprising residues 383 to 464 of SEQ ID NO: 2. c) a polypeptide molecule comprising residues 465 to 696 of SEQ ID NO: 2. d) a polypeptide molecule comprising the residues 438 to 449 of SEQ ID NO: 2. e) a polypeptide molecule comprising residues 164 to 464 of SEQ ID NO: 2. F) a polypeptide molecule comprising residues 164 to 696 of SEQ ID NO: 2. g) a polypeptide molecule comprising residues 383 to 696 of SEQ ID NO: 2. 15 h) a polypeptide molecule comprising residues 164 to 449 of SEQ ID NO: 2. i) a polypeptide molecule comprising the residues 438 to 696 of SEQ ID NO: 2; Y j) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO: 2.
15. An isolated polynucleotide encoding a fusion protein having a first segment and a second segment, characterized in that the ^ ¡^^ kíA ^ É ^^^. * '= Iy ^ S ^ &'; and? Iyy. and - first polypeptide segment encoding a one protease domain and the second segment comprises a second polynucleotide encoding a polypeptide having a contiguous sequence of 14 amino acids between residues 383 and 464 of SEQ ID NO: 2, and wherein the first segment is positioned in the amino terminal of the second segment.
16. The isolated polynucleotide according to claim 15, characterized in that the protease domain is selected from the group consisting of: a) a protease domain that is a member of the disintegrin proteases; and b) a protease domain that is at least 80% identical to amino acid residues 164 to 382 of SEQ ID NO: 2.
17. An isolated polynucleotide molecule that encodes a polypeptide molecule, characterized in that the polynucleotide molecule is selected from the group consisting of: a) a nucleic acid molecule encoding a polypeptide molecule that is at least 80% identical to residues 383 to 464 of SEQ ID NO: 2; and b) a polynucleotide molecule that is complementary to a).
18. The isolated polynucleotide molecule according to claim 17, characterized in that the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80% identical to residues 383 to 696 of SEQ ID NO: 2; and b) a polynucleotide molecule that is complementary to a).
19. The isolated polynucleotide molecule according to claim 17, characterized in that the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule encoding a polypeptide molecule that is at least 80% identical to residues 1 to 696 of SEQ ID NO: 2; and b) a polynucleotide molecule that is complementary to a).
20. An expression vector characterized in that it comprises the following elements operably linked. a) a transcription promoter; b) a DNA segment encoding the polypeptide of claim 1; and c) a transcription terminator
21. The expression vector according to claim 20, characterized in that the DNA segment also encodes an affinity tag.
22. A cultured cell into which an expression vector according to claim 21 has been introduced, characterized in that the cell expresses the polypeptide encoded by the DNA segment.
23. A method for the production of a polypeptide comprising the culture of a cell according to claim 22, characterized in that the cell expresses the polypeptide encoded by the DNA segment and the recovery of the polypeptide.
24. A method for the modulation of cell-cell interactions by combining the polypeptide according to claim 1, with cells in vivo and in vitro.
25. A method for the modulation of cell-to-cell interactions according to claim 24, characterized in that the cells are derived from tissues selected from the group consisting of: 15 a) heart tissues b) brain tissues c) spinal tissues; and d) skeletal muscle tissues.
26. An isolated polypeptide molecule comprising a contiguous amino acid sequence, characterized in that the contiguous amino acid sequence is selected from the group consisting of: a) SEQ ID NO: 7; b) SEQ ID NO: 8; c) SEQ ID NO 9;
27. An isolated polynucleotide molecule that encodes an isolated polypeptide molecule of claim 26.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/113,883 | 1998-07-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA01000349A true MXPA01000349A (en) | 2001-11-21 |
Family
ID=
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