Abstract
The lumen of the Golgi apparatus is the subcellular site where galactose is transferred, from UDP-galactose, to the oligosaccharide chains of glycoproteins, glycolipids, and proteoglycans. The nucleotide sugar, which is synthesized in the cytosol, must first be transported into the Golgi lumen by a specific UDP-galactose transporter. Previously, a mutant polarized epithelial cell (MDCKII-RCAr) with a 2% residual rate of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., Rodriguez-Boulan, E., and Simons, K. (1988) J. Biol. Chem. 263, 16283-16290). The mutant has an enrichment in glucosyl ceramide and cell surface glycoconjugates bearing terminal N-acetylglucosamine, as well as a 75% reduction in sialylation of cell surface glycoproteins and glycosphingolipids. We have now studied the biosynthesis of galactose containing proteoglycans in this mutant and the corresponding parental cell line. Wild-type Madin-Darby canine kidney cells synthesize significant amounts of chondroitin sulfate, heparan sulfate, and keratan sulfate, while the above mutant synthesizes chondroitin sulfate and heparan sulfate but not keratan sulfate, the only proteoglycan containing galactose in its glycosaminoglycan polymer. The mutant also synthesizes chondroitin 6-sulfate rather than only chondroitin 4-sulfate as wild-type cells. Together, the above results demonstrate that the Golgi membrane UDP-galactose transporter is rate-limiting in the supply of UDP-galactose into the Golgi lumen; this in turn results in selective galactosylation of macromolecules. Apparently, the Km for galactosyltransferases involved in the synthesis of linkage regions of heparan sulfate and chondroitin sulfate are significantly lower than those participating in the synthesis of keratan sulfate polymer, glycoproteins, and glycolipids. The results also suggest that the 6-O-sulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate.
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