Glycobiology Advance Access originally published online on September 26, 2003
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Glycobiology, 2003, Vol. 13, No. 12 941-953
© 2003 Oxford University Press
Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation
3 INSERM U-559/UEA-3289 Université de la Méditerranée, 27 Bd. J. Moulin, 13385 Marseille Cedex 5, France; and 4 Glycobiology Program, Cancer Research Center, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037
Received on June 25, 2003; revised on August 25, 2003; accepted on August 28, 2003
Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 ß1,3-N-acetylglucosaminyltransferase (C1-ß3GnT) and core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, 
1,2-fucosyltransferase-I (FucT-I) and 1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, 2,3-sialyltransferase-I (ST3Gal-I) and 2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-ß3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.
1 Present address: Corvas International Inc., San Diego, CA 92121
2 To whom correspondence should be addressed; e-mail: minoru{at}burnham.org
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