Purification of an autoantigenic 75-kDa human melanosomal glycoprotein.
Only a few autoantigenic human tumor antigens have been purified and characterized. We employed the monoclonal antibody (MAb) TA99 to isolate, purify and partially characterize an autoantigenic intracellular glycoprotein, gp75, from human melanoma cells. The gp75 antigen is the most abundant glycoprotein expressed in human melanocytes and pigmented melanomas and is the human homologue of the mouse brown locus gene product. Differential solubilization of melanoma membrane fraction and subcellular fractionation of pigmented melanoma cells showed that gp75 is an integral membrane protein localized to melanosomes. The gp75 glycoprotein eluted as a broad peak during ion exchange chromatography and appeared as a protein with broad pI (pI 5.5-5.9), consistent with charge microheterogeneity. gp75 also exhibited heterogeneity of binding to concanavalin A. Tyrosine hydroxylase (tyrosinase) activity co-purified with gp75 during membrane solubilization and anion exchange and Con A chromatography. However, most tyrosine hydroxylase activity could be dissociated from gp75 antigen during MAb TA99 affinity chromatography. TA99 did not immunoprecipitate or deplete tyrosine hydroxylase activity from lysates of human melanoma cells. Attempts to obtain N-terminal amino acid sequence of purified gp75 were not successful due to blocked N-terminus. Amino acid composition of gp75 was similar to that of tyrosinase. Physicochemical similarities and limited identity in the primary structure between gp75 and tyrosinase support the conclusion that the gp75 antigen does not exhibit tyrosine hydroxylase activity, but is a member of a tyrosinase-related family of proteins.
Vijayasaradhi
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Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Houghton
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D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D001324:Autoantigens / Q000302:isolation & purification*; D002462:Cell Membrane / Q000276:immunology; D002845:Chromatography; D003208:Concanavalin A; D004563:Electrochemistry; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D007163:Immunosorbent Techniques; D007526:Isoelectric Point; D008544:Melanocytes / Q000737:chemistry*; D008545:Melanoma / Q000276:immunology*; D008970:Molecular Weight; D014446:Tyrosine 3-Monooxygenase / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D001324:Autoantigens; D006023:Glycoproteins; D003208:Concanavalin A; D014446:Tyrosine 3-Monooxygenase