Title : Characterization and dynamics of O-linked glycosylation of human
cytokeratin 8 and 18
Abstract :
- 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