The long-term objective of this project is to elucidate the enzymatic reactions by which xylose-linked proteoglycans are assembled. Progress in this area has been hampered by lack of substrates, and, to remedy this situation in part, we propose to prepare a number of compounds suitable for this purpose. The main focus will be on substrates for the enzymes involved in the formation of the carbohydrate-protein linkage region and the first repeating disaccharide unit of the xylose-linked proteoglycans. Substrates of two types will be prepared: (1) serine-linked oligosaccharides will be synthesized chemically and (2) macromolecular substrates which are more akin to the natural substrates will be prepared by combined enzymatic and chemical degradation of chondroitin sulfate proteoglycan. Compounds in the first category include two serine-linked pentasaccharides from the linkage regions of chondroitin sulfate/dermatan sulfate and heparin/heparan sulfate, respectively. The general structure of the two compounds is: HexNAc-(1-4)-beta-GlcUA-(1-3)-beta-gal-(1- 3)-beta-Gal-(1-4)-beta-Xyl-(1-3)-O-L-Serine, in which HexNAc is beta-GalNAc or alpha-Glc-NAc. Xylose-containing glycopeptides with alternating serine and glycine residues and two compounds with phosphorylated xylose will also be synthesized, i.e. Xyl(2-P)-Ser and Gal-Xyl(2-P)-Ser. Since the newly discovered HNK-1 antigen, first detected on human natural killer lymphocytes but also widely distributed in nervous tissue, is structurally related to the proteoglycans, some aspects of its biosynthesis will also be examined. Specifically, the HNK-1 epitope, glucuronic acid 3- sulfate, and the proteoglycan-related terminal disaccharide, beta- GlcUA(3-S)-(1-3)-Gal, will be synthesized chemically and will be used in studies of the biosynthesis of the antigen. The primary objective of this work is to determine whether the glucuronosyltransferase which catalyzes transfer to galactose during formation of the antigen is identical to the enzyme involved in the formation of the proteoglycan linkage region. Enzyme sources for the testing of all substrates will be embryonic chick brain and embryonic chick cartilage in view of the prominent occurrence of the HNK-1 antigen in nervous tissue and the well- established use of chick cartilage in studies of proteoglycan biosynthesis. The importance of this project for the study of human disease is apparent from the results of our recent collaboration with Kresse and Quentin of the Univ. of Munster, which demonstrate that a patient with a progeria-like syndrome is deficient in galactosyltransferase I. This finding is the first demonstration of a genetic defect in proteoglycan biosynthesis in humans.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Physiological Chemistry Study Section (PC)
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University of Alabama Birmingham
Schools of Medicine
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Hagner-McWhirter, A; Hannesson, H H; Campbell, P et al. (2000) Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates. Glycobiology 10:159-71
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