DIFFERENTIAL PROCESSING AND SECRETION OF THE MELANOMA-ASSOCIATED ME20ANTIGEN

A murine monoclonal antibody, ME20, with high selectivity for melanoma s, has been utilized to isolate a unique membrane-bound (designated ME 20-M) and secreted (designated ME20-S) antigen from H3606 human melano ma cells. ME20-M was purified from the cell lysate and ME20-S from the conditioned medium of H3606 cells by immunoaffinity chromatography an d sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appar ent molecular weights were 105,000 and 76,000, respectively. Analyses of ME20-M and ME20-S by amino acid sequencing identified the processin g sites. Signal peptide cleavage occurs at Thr-24 of pro-ME20 antigen, yielding ME20-M (25 to 661). In addition, proteolytic processing of t he precursor at Val-467 yields ME20-S (25 to 467). We report the chara cterization of Asn-linked glycosylation sites in ME20-M and ME20-S to determine the involvement of oligosaccharides in the proteolytic proce ssing of pro-ME20 antigen. Tryptic peptide maps of ME20-M and ME20-S w ere prepared and the glycosylation sites identified by sequence analys es. Oligosaccharides were enzymatically released and characterized by high-performance anion-exchange chromatography. We found high-mannose- type structures at Asn-57, Asn-82, and Asn-87 of ME20-M, whereas ME20- S contained 73% complex-type and 27% high-mannose-type oligosaccharide s at the same sites. To assess the role of oligosaccharides in the pro cessing of the ME20 antigen, we tested the effect of the oligosacchari de processing modifier deoxymannojirimycin, a compound that inhibits s ynthesis of hybrid- and complex-type oligosaccharides. Deoxymannojirim ycin had no effect on the synthesis and relative rate of synthesis of ME20-M, but markedly reduced the synthesis of ME20-S without affecting the rate of secretion. The reported results suggest that carbohydrate maturation of the ME20 antigen may be important for processing and se cretion. (C) 1994 Academic Press, Inc.