Evidence for carboxyl-terminal processing and glycolipid-anchoring of human carcinoembryonic antigen.
We have investigated the post-translational modification of carcinoembryonic antigen (CEA) for membrane-anchoring in QGP-1 cells derived from a human pancreatic carcinoma. Pulse-chase experiments with [3H]leucine demonstrated that CEA was initially synthesized as a precursor form with Mr 150,000 having N-linked high-mannose-type oligosaccharides, which was then converted to a mature form with Mr 200,000 containing the complex type sugar chains. The mature protein thus labeled was found to be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that CEA is a phosphatidylinositol-linked membrane protein. This was confirmed by metabolic incorporation into CEA of 3H-labeled compounds such as ethanolamine, myo-inositol, palmitic acid, and stearic acid. The 3H-labeled fatty acids incorporated were specifically removed from the protein by nitrous acid deamination as well as by phosphatidylinositol-specific phospholipase C treatment. Since the available cDNA sequence predicts that CEA contains a single methionine residue only in its carboxyl-terminal hydrophobic domain, processing of the carboxyl terminus was examined by pulse-chase experiments with [35S]methionine. It was found that CEA with Mr 150,000 was initially labeled with [35S]methionine but its radioactivity was immediately lost with chase. Taken together, these results suggest that CEA is anchored to the membrane by simultaneously occurring proteolysis of the carboxyl terminus and replacement by the glycophospholipid immediately after the synthesis.
Takami
N
N
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Misumi
Y
Y
Kuroki
M
M
Matsuoka
Y
Y
Ikehara
Y
Y
D002272:Carcinoembryonic Antigen / Q000032:analysis; D002277:Carcinoma / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D006017:Glycolipids / Q000276:immunology / Q000378:metabolism*; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D007163:Immunosorbent Techniques; D008358:Mannose / Q000032:analysis; D008565:Membrane Proteins / Q000276:immunology; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009608:Nitrous Acid; D009844:Oligosaccharides / Q000032:analysis; D010190:Pancreatic Neoplasms / Q000276:immunology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D002272:Carcinoembryonic Antigen; D005227:Fatty Acids; D006017:Glycolipids; D008565:Membrane Proteins; D009844:Oligosaccharides; D010716:Phosphatidylinositols; D014415:Tunicamycin; D008715:Methionine; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D006596:Hexosaminidases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D008358:Mannose; D009608:Nitrous Acid