Topography of Glycosylation in UDP-Xylose Production and Xylosylation

Glycosylation is a major modification of the proteins in eukaryotic cells. As compared with other protein modifications such as phosphorylation the glycosylation process is much more complex. The stereospecificity of the sugars and the large variations in the core carbohydrate structure require very complex enzymatic processes in an orderly manner. The first step in the formation of a chondroitin sulfate proteoglycan is the addition of a xylose to a serine residue on the protein core forming the protein-carbohydrate linkage. The enzyme involved is xylosyltransferase, a soluble enzyme. It interacts with the core protein and appears to recognize specific sequences containing glycine residues next to serine acceptor. The enzyme also interacts with and binds to the next enzyme in the synthetic sequence, the first galactosyltransferase, an enzyme located in the smooth ER. The third step, the addition of the second galactose residue is catalyzed by a distinct separate galactosyltransferase. The fourth step, the addition of the first glucuronic acid residue, is catalyzed by a glucuronosyltransferase, a different enzyme from the one involved in the chain elongation. It recognizes the acceptor galactose of the linkage region. The subsequent steps in the chain elongation are the alternating addition of Nacetylgalactosamine and glucuronic acid to the growing chain, catalyzed by two distinct transferases. These enzymes are membrane-bound. The final step is the transfer of the sulfate group from PAPS to the chain at positions 4 and 6 of the sugar. This reaction is catalyzed by two separate enzymes with strict specificities. Although we have the knowledge of the individual steps in the synthesis of CSPG, we have very little knowledge about the precise topology of the synthesis. Since the glycosylations of other glycoproteins take place in ER and Glogi, it is assumed that xylosylation in CSPG synthesis takes place in a similar fashion. In the past, many investigators have utilized various techniques and cell types to address this issue, e.g., subcellular fractionation followed by measurements of the enzyme activities in the various fractions, immunoelectron-microscopy using specific antibodies and the kinetic labeling of the GAG with specific radiolabeled precursors. These observations have made important contributions to our knowledge about the biosynthesis of GAG. However, the flow of the steps, the location of the enzyme and their substrates remain un-