Oncofetal fibronectin epitope

Details Oncofetal fibronectin epitope Oncofetal fibronectin epitope

1992-10-02

1994-04-05

Lee, Lester L.

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

6 to 7 amino acid residues in defined sequence

530322, A61K 3702, C07K 706

Patent

active

053006305

ABSTRACT:
An onco-developmentally regulated .alpha.-N-acetylgalactosaminyltransferase isolated as a component of a particulate membrane fraction separated from cell and tissue homogenates and having the following characteristics:
(a) Activity in Various Cells and Tissues--present in human fetal lung fibroblasts, hepatoma tissues and placenta, but absent from normal adult liver, lung, kidney and spleen tissues;
(b) Substrate Specificity--acts on polypeptides comprising a sequence val-thr-his-pro-gly-tyr by catalyzing u-N-acetylgalactosaminylation at the thr residue of the sequence;
(c) Requirements for Metal Ion--requires metal ion for activity in 25 mM tris buffer, pH 7.6;
(d) Optimal pH--optimal pH is about 7.6 assayed in hepatoma cell homogenate in tris buffer and at a pH of about 6 to about 7 assayed in hepatoma cell homogenate enzyme activity is higher in 2(N-morpholino)ethanesulfonic acid than in cacodylate buffer; and
(e) Km--apparent Km for UDP - GalNAc is about 48 .mu.M.
A method of detecting tumor cells comprising the steps of:
(1) homogenizing tissue suspected of having tumor cells;
(2) incubating the homogenate with labeled .alpha.-N-acetylgalactosamine (.alpha.-GalNAc) and a polypeptide substrate comprising a sequence of val-thr-his-pro-gly-tyr under conditions sufficient to catalyze .alpha.-N-acetylgalactosaminylation of the thr residue to thereby form labeled polypeptide substrate comprising val-thr(m-GalNAc)-his-pro-gly-tyr; and
(3) determining the presence of said labeled polypeptide substrate.
A diagnostic test kit for detecting tumor cells in a sample suspected of containing tumor cells comprising as separate components:
(a) labeled m-N-acetylgalactosamine; and
(b) polypeptide substrate comprising a sequence of val-thr-his-pro-gly-tyr.

REFERENCES:
patent: 4894326 (1990-01-01), Matsuura et al.
Matsuura et al.; JBC 263(7), pp. 3314-3322, (1988).
Matsuura, H., and S. Hakomori, The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: Its presence in fibronectins from fetal and tumor tissues and its absence in those from normal adult tissues and plasma, Proc. Natl. Acad. Sci USA 82:6517-6521, 1985.
Matsuura, H. et al., The oncofetal structure of human fibronectin defined by monoclonal antibody FDC-6, The Journal of Biolobical Chemistry 263(7):3314-3322, 5 Mar. 1988.
Matsuura, H., et al., An .alpha.-N-acetylgalactosaminylation at the threonine residue of a defined peptide sequence creates the oncofetal peptide epitope in human fibronectin, The Journal of Biological Chemistry 264(18):10472-10476, 25 Jun. 1989.

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