JBC Ideal method for primary cell transfection

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Volume 272, Number 36, Issue of September 5, 1997 pp. 22695-22702
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Altered Golgi Localization of Core 2 beta -1,6-N-Acetylglucosaminyltransferase Leads to Decreased Synthesis of Branched O-Glycans

(Received for publication, May 5, 1997, and in revised form, June 30, 1997)

David Skrincosky , Renate Kain , Assou El-Battari , Markus Exner par , Dontscho Kerjaschki par and Minoru Fukuda

From the Glycobiology Program, The Burnham Institute, La Jolla, California 92037, the  Faculty of Medicine, University of the Mediterranean, Marseille, Cedex 5, France, and the par  Institute of Clinical Pathology, University of Vienna, Vienna, A1090, Austria

Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 beta -1,6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of alpha -2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides.

These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than alpha -2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.


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