Differential processing and secretion of the melanoma-associated ME20 antigen.
A murine monoclonal antibody, ME20, with high selectivity for melanomas, has been utilized to isolate a unique membrane-bound (designated ME20-M) and secreted (designated ME20-S) antigen from H3606 human melanoma cells. ME20-M was purified from the cell lysate and ME20-S from the conditioned medium of H3606 cells by immunoaffinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weights were 105,000 and 76,000, respectively. Analyses of ME20-M and ME20-S by amino acid sequencing identified the processing sites. Signal peptide cleavage occurs at Thr-24 of pro-ME20 antigen, yielding ME20-M (25 to 661). In addition, proteolytic processing of the precursor at Val-467 yields ME20-S (25 to 467). We report the characterization of Asn-linked glycosylation sites in ME20-M and ME20-S to determine the involvement of oligosaccharides in the proteolytic processing of pro-ME20 antigen. Tryptic peptide maps of ME20-M and ME20-S were prepared and the glycosylation sites identified by sequence analyses. 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 oligosaccharides at the same sites. To assess the role of oligosaccharides in the processing of the ME20 antigen, we tested the effect of the oligosaccharide processing modifier deoxymannojirimycin, a compound that inhibits synthesis of hybrid- and complex-type oligosaccharides. Deoxymannojirimycin 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 secretion.
Maresh
G A
GA
Bristol Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Wang
W C
WC
Beam
K S
KS
Malacko
A R
AR
Hellström
I
I
Hellström
K E
KE
Marquardt
H
H
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000302:isolation & purification / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002462:Cell Membrane / Q000276:immunology; D017077:Culture Media, Conditioned; D006031:Glycosylation; D006801:Humans; D008545:Melanoma / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D014357:Trypsin; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D017077:Culture Media, Conditioned; D010446:Peptide Fragments; D017485:1-Deoxynojirimycin; D001216:Asparagine; D014357:Trypsin