Chondroitin

N-linked oligosaccharides on chondroitin 6-sulfotransferase-1 are required for production of the active enzyme, Golgi localization and the sulfotransferase activity toward keratan sulfate.

Yusa A, Kitajima K, Habuchi O.

Department of Chemistry, Aichi University of Education, Kariya, Aichi 448-8542.

We have shown prev

iously that purified chondroitin 6-sulfotransferase-1(C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides, and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however functional roles of the N-glycans has remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity, but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, while deletion of the fifth site resulted in a marked loss of the KSST activity. Wild type recombinant C6ST-1 showed a typical Golgi-localization, while M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an ER-resident protein. Unlike wild type recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi-localization, thereby cause the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.

PMID: 16720579 [PubMed - as supplied by publisher]