Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Utility Patent
1998-08-10
2001-01-02
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S320100, C435S325000, C530S350000, C536S023100, C536S023500
Utility Patent
active
06168920
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of extracellular adhesive proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, immune disorders, and developmental disorders.
BACKGROUND OF THE INVENTION
The surface of a cell is rich in transmembrane proteoglycans, glycoproteins, glycolipids, and receptors. These macromolecules mediate adhesion with other cells and with components of the extracellular matrix (ECM). The ECM is comprised of diverse glycoproteins, polysaccharides, and carbohydrate-binding proteins which are secreted from the cell and assembled into an organized meshwork in close association with the cell surface. The interaction of the cell with the surrounding matrix profoundly influences cell shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development.
Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells. (Reviewed in Drickamer, K. and Taylor, M. E. (1993) Annu. Rev. Cell Biol. 9:237-264.) This function is particularly important for activation of the immune response. Lectins mediate the agglutination and mitogenic stimulation of lymphocytes at sites of inflammation. (Lasky, L. A. (1991) J. Cell. Biochem. 45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857.)
Lectins are further classified into subfamilies based on carbohydrate-binding specificity. The galectin subfamily, in particular, includes lectins that bind &bgr;-galactoside carbohydrate moieties in a thioldependent manner. (Reviewed in Hadari, Y. R. et al. (1998) J. Biol. Chem. 270:3447-3453.) Galectins are widely expressed and developmentally regulated. Because all galectins lack an N-terminal signal peptide, it is suggested that galectins are externalized through an atypical secretory mechanism. Two classes of galectins have been defined based on molecular weight and oligomerization properties. Small galectins form homodimers and are about 14 to 16 kilodaltons in mass, while large galectins are monomeric and about 29-37 kilodaltons.
Galectins contain a characteristic carbohydrate recognition domain (CRD). The CRD is about 140 amino acids and contains several stretches of about 1-10 amino acids which are highly conserved among all galectins. A particular 6-amino acid motif within the CRD contains conserved tryptophan and arginine residues which are critical for carbohydrate binding. The CRD of some galectins also contains cysteine residues which may be important for disulfide bond formation. Secondary structure predictions indicate that the CRD forms several &bgr;-sheets.
Galectins play a number of roles in diseases and conditions associated with cell-cell and cell-matrix interactions. For example, certain galectins associate with sites of inflammation and bind to cell surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis. Galectin overexpression is correlated with the metastatic potential of cancers in humans and mice. Moreover, anti-galectin antibodies inhibit processes associated with cell transformation, such as cell aggregation and anchorage-independent growth.
Prostate carcinoma tumor antigen 1 (PCTA-1) is a novel galectin implicated in cancer progression. (Su, Z.-Z. et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:7252-7257.) PCTA-1 was initially identified as the cell surface antigen recognized by a prostate tumor-directed monoclonal antibody, Pro 1.5. PCTA-1 cDNA is 3.85 kilobases and encodes a 317-amino acid protein of about 35 kilodaltons. PCTA-1 is expressed in invasive prostate carcinomas and early-stage prostate cancers, but not in normal prostate or benign prostatic hypertrophic tissue. In addition, PCTA-1 is shed from the surface of cultured prostate cancer cells into the growth media. Together, these results suggest that detection of PCTA-1 may be useful for early diagnosis of prostate cancer and that levels of PCTA-1 in the circulation may correlate with disease progression. In addition, preliminary studies in mice suggest that the monoclonal antibody Pro 1.5 may itself be an effective therapeutic agent against tumor progression.
Fibronectin is another component of the ECM which binds to specific integrin-family transmembrane receptors. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Publishing, New York, N.Y., pp. 986-987.) Fibronectin is found in insoluble form in connective tissue and at the base of epithelia. Fibronectin is a dimer composed of two rod-shaped subunits joined by disulfide bonds. Each subunit is a multidomain glycoprotein of nearly 2500 amino acids, and each domain consists of smaller repeated modules. The main type of module is called the type III fibronectin repeat, which consists of about 90-100 amino acids which form several &bgr;-sheets. The type III repeat is found in at least 45 protein families, most of which are involved in cell adhesion. Some type III repeats contain a specific tripeptide sequence, the RGD motif. This motif mediates the attachment of fibronectin to the integrin receptor. This motif is also found in various other proteins important for cell-cell adhesion and cell-matrix adhesion, including collagen, fibrinogen, and vitronectin.
Fibronectin is involved in a number of important functions including wound healing, blood coagulation, cell adhesion, cell differentiation, cell migration, embryonic development, and tumor metastasis. In addition, the expression of fibronectin is markedly reduced in neoplastically transformed cells. Furthermore, inactivation of both copies of the fibronectin gene causes early embryonic lethality in mice. These knockout mice have multiple morphological defects, including malformation of the notochord, somites, heart, blood vessels, neural tube, and extraembryonic structures.
A novel class of proteins involved in cell adhesion are the leucine-rich repeat proteins. Leucine rich repeats (LLRs) are about 22-28 amino acids in length and are found in various extracellular adhesive glycoproteins. (Rothberg, J. M. et al. (1990) Genes Dev. 4:2169-2187.) LLRs are predicted to form hydrophobic surfaces capable of interacting with membranes. In addition, synthetic LRRs form &bgr;-sheet structures and extended filaments. (Gay, N. J. et al. (1991) FEBS Lett. 291:87-91.) These properties are consistent with a role for LRRs in cell adhesion and cell surface interactions.
The discovery of new extracellular adhesive proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cancer, immune disorders, and developmental disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, extracellular adhesive proteins, referred to collectively as “EXADH” and individually as “EXADH-1” and “EXADH-2.” In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and fragments thereof.
Additionally, the
Corley Neil C.
Guegler Karl J.
Hillman Jennifer L.
Patterson Chandra
Yue Henry
Achutamurthy Ponnathapu
Incyte Genomics Inc.
Incyte Genomics, Inc.
Tung Peter P.
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