Ga. Maresh et al., DIFFERENTIAL PROCESSING AND SECRETION OF THE MELANOMA-ASSOCIATED ME20ANTIGEN, Archives of biochemistry and biophysics, 311(1), 1994, pp. 95-102
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.