Biosynthesis and glycosylation of the carcinoembryonic antigen.
Human gastric adenocarcinoma MKN-45 cells were found to synthesize actively carcinoembryonic antigen (CEA). The biosynthesis and carbohydrate processing of CEA were studied in these cells by means of metabolic labelling followed by immunoadsorption with a specific polyclonal-antibody preparation and gel electrophoresis. Pulse-chase studies with [14C]leucine and [3H]mannose (shortest pulse 3 min) showed that N-linked oligosaccharide side chains are added to the protein co-translationally, producing a high-mannose immature CEA; the average molecular mass of this form is 145 kDa. The protein is later translocated to the Golgi apparatus and here undergoes additional processing; these modifications are visible in our system as a broadening of the CEA band and require about 4 h. The upper limit of mature CEA band reaches 200 kDa, but radioactivity is maximally incorporated at 168 kDa. The extent of co-translational glycosylation was measured by treating the cells with tunicamycin; in the presence of this inhibitor, a 74 kDa aglyco-CEA was produced and was still recognized by the antibody. Monensin, an ionophore which interferes with glycoprotein maturation and terminal sugar addition, blocked broadening of the CEA band, producing a sharp 141 kDa peak. In conclusion, CEA appears to be synthesized as a 145 kDa high-mannose immature form, the protein core accounting for about half of its molecular mass. Full maturation results in a broad band at 168 kDa.
Mottola-Hartshorn
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C
Sclavo Research Center, Siena, Italy.
Lorenzoni
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P
Ceccarini
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C
D000906:Antibodies / Q000032:analysis; D002272:Carcinoembryonic Antigen / Q000032:analysis*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007163:Immunosorbent Techniques; D008985:Monensin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology; D014415:Tunicamycin / Q000494:pharmacology
D000906:Antibodies; D002272:Carcinoembryonic Antigen; D014415:Tunicamycin; D008985:Monensin