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.
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November 2002
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Research Article|
November 15 2002
N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase Available to Purchase
Matthias ECKHARDT;
Matthias ECKHARDT
1
Institut für Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universität Bonn, Nussallee 11, D-53115 Bonn, Germany
1To whom correspondence should be addressed (e-mail [email protected]).
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Simon N. FEWOU;
Simon N. FEWOU
Institut für Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universität Bonn, Nussallee 11, D-53115 Bonn, Germany
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Ivonne ACKERMANN;
Ivonne ACKERMANN
Institut für Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universität Bonn, Nussallee 11, D-53115 Bonn, Germany
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Volkmar GIESELMANN
Volkmar GIESELMANN
Institut für Physiologische Chemie, Rheinische-Friedrich-Wilhelms Universität Bonn, Nussallee 11, D-53115 Bonn, Germany
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Publisher: Portland Press Ltd
Received:
June 19 2002
Revision Received:
August 07 2002
Accepted:
August 13 2002
Accepted Manuscript online:
August 13 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 368 (1): 317–324.
Article history
Received:
June 19 2002
Revision Received:
August 07 2002
Accepted:
August 13 2002
Accepted Manuscript online:
August 13 2002
Citation
Matthias ECKHARDT, Simon N. FEWOU, Ivonne ACKERMANN, Volkmar GIESELMANN; N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase. Biochem J 15 November 2002; 368 (1): 317–324. doi: https://doi.org/10.1042/bj20020946
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