Transport of UDP-Galactose into the Golgi Lumen Regulates the Biosynthesis of Proteoglycans

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-RCA r ) with a 2% residual rate of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., RodriguezBoulan, 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 K m 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-Osulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate.Proteoglycans are complex macromolecules consisting of a protein core to which glycosaminoglycans are covalently linked. Their strategic localization in the plasma membrane and extracellular matrix makes them important intermediates between cells and their environment (1, 2). They have been implicated to play a role in cell-cell (3) and cell-matrix interactions (4), organization of basement membranes (5), control of macromolecules' diffusion (6), and also interactions with a variety of ligands such as growth factors, hormones, and neurotransmitters (7).In most GAGs, 1 the repeating disaccharide units are composed of one amino sugar and one uronic acid, the only exception being keratan sulfate in which galactose replaces the sugar acid. Most GAGs are attached to serine of the core protein by a tetrasaccharide of xylose-galactose-galactose-glucuronic acid (8, 9). Keratan sulfate is an exception; keratan sulfate I, from cornea, is N-linked to proteins and keratan sulfate II, from skeletal tis...

Inhibition of Golgi Apparatus Glycosylation Causes Endoplasmic Reticulum Stress and Decreased Protein Synthesis

“…Previous results with cells in tissue culture (20) and multicellular organisms (21) from this and other laboratories showed that partial inhibition of transport of nucleotide sugars into the Golgi apparatus lumen resulted in a global decrease of the corresponding sugar in glycoconjugates (20). This decrease could be selective, for example, in a mutant MDCK cell line in which we found that even though there was a 80 -90% decrease in UDP-galactose transport, synthesis of some but not all galactose-containing glycosaminoglycans was affected (20).…”

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

“…The specificity may be due to channeling of UDP-Gal to specific galactosyltransferases or to different galactosyltransferases having different K m values for UDP-Gal. In a classic study of mammalian UDP-Gal transport, some proteoglycans were affected by a 98% reduction in transporter activity, whereas other proteoglycans were unaffected, and differences in K m were suggested as the most likely explanation (37).…”

SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP- N - acetylgalactosamine, and UDP-galactose

“…The reduced availability of a nucleotide sugar can have a differential effect on the biosynthesis of different glycoconjugates. Thus, an MDCK mutant 98% deficient in UDP-Gal transport into the Golgi lumen shows significantly reduced levels of galactoproteins, galactosphingolipids, and keratan sulfate, a glycosaminoglycan that contains Gal in its polymeric repeating disaccharide units, but normal amounts of heparan sulfate and chondroitin sulfate, which have Gal only in the proteoglycan linkage region (12,13). This differential effect might reflect a lower K m for the galactosyltransferases involved in the biosynthesis of the linkage region as compared with those involved in the biosynthesis of keratan sulfate, galactoproteins, and galactosphingolipids.…”

“…Yeast, Leishmania donovani, and mammalian cell line mutants impaired in the transport of specific nucleotide sugars into the Golgi lumen have severe deficiencies of the corresponding sugar in their macromolecules (11), demonstrating the essential role of the transport process in glycosylation (11)(12)(13)(14)(15). In addition, fibroblasts from a patient with a clinical phenotype resembling that of leukocyte adhesion deficiency II are defective in GDP-fucose (Fuc) transport into the Golgi (16).…”