N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase.
3- O -Sulphogalactosylceramide (sulphatide) is a major lipid component of myelin membranes, and is required for proper myelin formation. Sulphatide is synthesized in the Golgi apparatus by galactosylceramide sulphotransferase (CST; EC 2.8.2.11). Murine and human CSTs contain two putative N-glycosylation sites (Asn-66 and Asn-312). The second site is conserved among all galactose 3-O-sulphotransferases cloned to date. In order to study the functional relevance of N-glycosylation, we generated epitope-tagged CST and soluble Protein A-CST fusion proteins lacking both N-glycosylation sites, separately or in combination. Our results show that both sites are glycosylated when CST is expressed in Chinese hamster ovary (CHO) or COS cells. Moreover, transfecting CST mutants lacking both N-glycosylation sites, or only Asn-312, reduced significantly the amount of sulphatide synthesized, whereas substituting Asn-66 with a glutamine residue did not. In contrast, activity in vitro was reduced by approx. 50% in the Asn-66-->Gln (N66Q) mutant, and was almost undetectable in N312Q and N66/312Q transfectants. Furthermore, soluble Protein A-CST expressed in the presence of tunicamycin was almost inactive, and accumulated in transfected cells. Expression of fully active CST in a CHO-glycosylation mutant lacking N-acetylglucosaminyltransferase I demonstrated that condensation of the N-linked pentamannosyl-core structure is sufficient to form a fully active enzyme.
Eckhardt
Matthias
M
Institut für Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universität Bonn, Nussallee 11, D-53115 Bonn, Germany. eckhardt@institut.physiochem.uni-bonn.de
Fewou
Simon N
SN
Ackermann
Ivonne
I
Gieselmann
Volkmar
V
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D006031:Glycosylation; D051379:Mice; D016296:Mutagenesis; D011134:Polysaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013205:Staphylococcal Protein A / Q000235:genetics; D013347:Subcellular Fractions; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014415:Tunicamycin; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase