Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18.
The glycosylation of human cytokeratin (CK) 8 and 18 was studied after metabolic labeling of HT29 colonic cells with [3H]glucosamine. Labeling of CK8/18 was not inhibited by tunicamycin, suggesting that glycosylation was not N-linked. Acid hydrolysis of CK8 and CK18, purified from [3H]glucosamine-labeled cells, generated free glucosamine. In the presence of UDP-[3H]galactose, galactosyltransferase catalyzed the labeling of cytokeratin 8 and 18. beta-Elimination of the [3H]galactose- labeled CK8/18 generated the disaccharide N-acetyllactosaminitol, indicating that cytokeratin 8 and 18 contain single O-linked N-acetylglucosamine residues. Using chemical analysis, the stoichiometry of glycosylation was found to be 1.5 and 2 molecules of N-acetylglucosamine/protein molecule of CK8 and CK18, respectively. Peptide maps of [3H]glucosamine-labeled CK8/18 showed that multiple peptides were labeled with the amino sugar. The biosynthetic and degradation rates of the carbohydrate moiety were faster than the protein core as determined by metabolic radiolabeling or pulse-chase experiments, respectively. Our results show that CK8 and 18 are glycosylated at multiple sites with a single O-linked N-acetylglucosamine. Furthermore, CK8/18 glycosylation is a dynamic process which is likely to have functional relevance.
Department of Medicine, Palo Alto Veterans Administration Medical Center, California.
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Chou
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Smith
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Omary
D000606:Amino Sugars / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D003106:Colon / Q000166:cytology / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007633:Keratins / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D000606:Amino Sugars; D014415:Tunicamycin; D014531:Uridine Diphosphate Galactose; C000458:N-acetyllactosamine; D007633:Keratins; D008715:Methionine; D005700:Galactosyltransferases; D005944:Glucosamine