Effects of N-glycosylation on the activity and localization of GlcNAc-6-sulfotransferase 1.
N-Acetylglucosamine-6-sulfotransferase-1 (GlcNAc6ST-1) is a Golgi-resident glycoprotein that is responsible for sulfation of the l-selectin ligand on endothelial cells. Here, we report the sites at which GlcNAc6ST-1 is modified with N-linked glycans and the effects that each glycan has on enzyme activity, specificity, and localization. We determined that glycans are added at three of four potential N-linked glycosylation sites: N196, N410, and N428. The N428 glycan is required for the production of sulfated cell surface glycans: cells expressing a mutant enzyme lacking this glycan were unable to sulfate the sialyl Lewis X tetrasaccharide or a putative extended core 1 O-linked glycan. The N196 and N410 glycans differentially affect sulfation of two different substrates: cells that express an enzyme lacking the N410 glycan are able to sulfate the sialyl Lewis X substrate, but produce reduced levels of a sulfated peripheral lymph node addressin epitope and cells that express an enzyme lacking the N196 glycan are able to produce a sulfated peripheral lymph node addressin epitope, but are impaired in their ability to sulfate sialyl Lewis X. The glycans' effects on enzyme activity may be mediated, in part, by changes in enzyme localization. While most mutants that lacked glycans localized normally within the Golgi, the N428A mutant and a mutant lacking all glycans were also found to localize ectopically. Altered trafficking of mutants may be associated with the mechanisms by which misglycosylated enzyme is degraded.
Desko
Marguerite M
MM
Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
Gross
David A
DA
Kohler
Jennifer J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D016256:Lewis X Antigen / Q000031:analogs & derivatives; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D016415:Sequence Alignment; D000080506:Sialyl Lewis X Antigen / Q000031:analogs & derivatives; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
D000939:Epitopes; D016256:Lewis X Antigen; D009844:Oligosaccharides; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C086949:6'-sulfated sialyl Lewis x