Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-08-02
2002-07-16
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06420154
ABSTRACT:
BACKGROUND OF THE INVENTION
Disintegrins have been shown to bind cell surface molecules, including integrins, on the surface of various cells, such as platelets, fibroblasts, tumor, endothelial, muscle, neuronal, bone, and sperm cells. Disintegrins are unique and potentially useful tools for investigating cell-matrix and cell-cell interactions. Additionally, they have been useful in the development of antithrombotic and antimetastatic agents due to their anti-adhesive, anti-migration of certain tumor cells, and anti-angiogenesis activities.
Families of proteins which have disintegrin domains include ADAMs (A Disintegrin and Metalloprotease), MDCs (Metalloprotease/Disintegrin/Cysteine-rich) and SVMPs (Snake Venom Metalloprotease).
For a review of ADAMs, see Wolfsberg and White,
Developmental Biology
, 180:389-401, 1996. ADAMs have been shown to exist as independent functional units as well as in conjunction with other members of this family in heterodimeric complexes. Some members of the family have multiple isoforms which may have resulted from alternative splicing. ADAMs proteins have been shown to have adhesive as well as anti-adhesive functions in their extracellular domains. Some members of the ADAMs family have very specific tissue distribution while others are widely distributed. Not all members of this family are capable of manifesting all of the potential functions represented by the domains common to their genetic structure.
The ADAMs are characterized by having a propeptide domain, a metalloprotease-like domain, a disintegrin-like domain, a cysteine-rich domain, an EGF-like domain, and a cytoplasmic domain.
A prototypical example 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 muscle cell fusion and differentiation (Gilpin et al.,
J. Biol. Chem
. 273:157-166, 1998). Other ADAMs involved in fusion are ADAM 1, and ADAM 2 which form a heterodimer (fertilin) and are involved in sperm/egg fusion (Wolfsberg and White, supra).
The SVMP family is represented by three classes (P-I, P-II, and P-III). All three classes contain propeptide and metalloprotease domains. The P-II and P-III classes also contain a disintegrin domain, and the P-III class further contains a cysteine-rich domain. These domains are similar in sequence to those found in the ADAMs. Some members of the SVMP family have a conserved “RGD” amino acid sequence. This tripeptide has been shown to form a hairpin loop whose conformation can disrupt the binding of fibrinogen to activated platelets. This “RGD” sequence may be substituted by RSE, MVD, MSE, and KGD in P-II SVMPs, and by MSEC (SEQ ID NO:14), RSEC (SEQ ID NO:15), IDDC (SEQ ID NO:16), and RDDC (SEQ ID NO:17) (a tripeptide along with a carboxy-terminal cysteine residue) in P-III SVMPs. Thus, these sequences may be responsible for integrin binding in the P-II and P-III SVMPs.
A prototypical example of a SVMP is jararhagin, which mediates platelet aggregation by binding to the platelet a
2
subunit (GPIa) via the disintegrin domain followed by proteolysis of the b
1
subunit (GPIIA) (Huang and Liu,
J. Toxicol-Toxin Reviews
16: 135-161, 1997).
The proteins of the Metalloprotease/Disintegrin/Cysteine-rich family are involved in diverse tasks, ranging from roles in fertilization and muscle fusion, TNFa release from plasma membranes, intracellular protein cleavage, and essential functions in neuronal development (Blobel, C. P.
Cell
90:589-592, 1997). This family is also characterized by the metalloprotease, disintegrin and cysteine-rich domains, as described above.
Members of the DP family of proteins which have been shown to be therapetuically useful include eptifibatide (Integrilin®, made by COR Therapeutics, Inc. and Key Pharmaceuticals, Inc.) which is useful as an anti-clotting agent for acute coronary syndrome, and contortrostatin, which inhibits &bgr;
1
Integrin-mediated human metastatic melanoma cell adhesion and blocks experimental metastasis (Trikha, M. et at.,
Cancer Research
54: 4993-4998, 1994) and inhibits platelet aggregation (Clark, E. A. et al.,
J. Biol. Chem
. 269 (35):21940-21943, 1994).
The present invention provides a novel member of the Disintegrin Proteases and related compositions whose uses will be apparent to those skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
Within one aspect the invention provides an isolated polypeptide molecule comprising residues 475 to 488 of SEQ ID NO:2. Within an embodiment, the isolated polypeptide molecule has one amino acid substitution. Within another embodiment, the isolated polypeptide molecule has two amino acid substitutions. Within another embodiment, the isolated polypeptide molecule comprises residues 420 to 495 of SEQ ID NO:2. Within another embodiment, the isolated polypeptide molecule is selected from the group consisting of: a) a polypeptide molecule comprising residues 208 to 495 of SEQ ID NO:2; b) a polypeptide molecule comprising residues 31 to 495 of SEQ ID NO:2; c) a polypeptide molecule comprising residues 1 to 495 of SEQ ID NO:2; d) a polypeptide molecule comprising residues 1 to 802 of SEQ ID NO:2; and e) a polypeptide molecule comprising residues 1 to 812 of SEQ ID NO:4. Within another embodiment, the invention provides an isolated polynucleotide molecule encoding the polypeptide. Within another embodiment, the invention provides the isolated polypeptide molecule comprising residues 475 to 488 of SEQ ID NO:2, wherein at least nine contiguous amino acid residues of SEQ ID NO:2 or SEQ ID NO:4 are operably linked via a peptide bond or polypeptide linker to a second polypeptide selected from the group consisting of maltose binding protein, an immunoglobulin constant region, and a polyhistidine tag.
Within another aspect, the invention provides an isolated polypeptide molecule, wherein the polypeptide molecule is selected from the group consisting of: a) a polypeptide molecule comprising residues 208 to 410 of SEQ ID NO:2; b) a polypeptide molecule comprising residues 497 to 802 of SEQ ID NO:2; c) a polypeptide molecule comprising residues 31 to 200 of SEQ ID NO:2; d) a polypeptide molecule comprising residues 497 to 701 of SEQ ID NO:4; e) a polypeptide molecule comprising residues 702 to 724 of SEQ ID NO:4; f) a polypeptide molecule comprising residues 725 to 812 of SEQ ID NO:4; g) a polypeptide molecule comprising residues 208 to 495 of SEQ ID NO:2; h) a polypeptide molecule comprising residues 31 to 495 of SEQ ID NO:2; i) a polypeptide molecule comprising residues 1 to 495 of SEQ ID NO:2; j) a polypeptide molecule comprising residues 208 to 802 of SEQ ID NO:2; k) a polypeptide molecule comprising residues 31 to 802 of SEQ ID NO:2; 1) a polypeptide molecule comprising residues 1 to 802 of SEQ ID NO:2; m) a polypeptide molecule comprising residues 420 to 812 of SEQ ID NO:4; n) a polypeptide molecule comprising residues 204 to 812 of SEQ ID NO:4; o) a polypeptide molecule comprising residues 31 to 812 of SEQ ID NO:4; p) a polypeptide molecule comprising residues 1 to 812 of SEQ ID NO:4; q) a polypeptide molecule comprising residues 725 to 812 of SEQ ID NO:4; r) a polypeptide molecule comprising residues 497 to 724 of SEQ ID NO:4; s) a polypeptide molecule comprising residues 420 to 724 of SEQ ID NO:4; t) a polypeptide molecule comprising residues 208 to 724 of SEQ ID NO:4; u) a polypeptide molecule comprising residues 31 to 724 of SEQ ID NO:4; and v) a polypeptide molecule comprising residues 1 to 724 of SEQ ID NO:4. Within an embodiment is provided an isolated polynucleotide molecule encoding the polypeptide. Within an embodiment, is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding the polypeptide; and a transcription terminator. Within an embodiment, the DNA segment further encodes an affinity tag. Within another embodiment, the expression vector is introduced into a cultured cell and the cell expresses the polypeptide encoded by the DNA segment. Within a further embodiment, the invention
Baindur Nand
Bishop Paul D.
Sheppard Paul O.
Achutamurthy Ponnathapu
Adams Robyn
Pak Yong
Zymogenetics Inc.
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