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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS027353-05
Application #
3413620
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1989-04-01
Project End
1994-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Meezan, E; Meezan, E; Meezan, J et al. (1997) Alkylglycosides as artificial primers for glycogen biosynthesis. Cell Mol Biol (Noisy-le-grand) 43:369-81
Roden, L; Yu, H; Jin, J et al. (1997) Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography. Anal Biochem 254:240-8
Manzella, S M; Roden, L; Meezan, E (1995) Dodecyl-beta-D-maltoside as a substrate for glucosyl and xylosyl transfer by glycogenin. Glycobiology 5:263-71
Manzella, S; Ananth, S; Oegema, T R et al. (1995) Inhibition of glycogenin-catalyzed glucosyl and xylosyl transfer by cytidine 5'-diphosphate and related compounds. Arch Biochem Biophys 320:361-8
Roden, L; Jin, J; Yu, H et al. (1995) Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water. Glycobiology 5:167-73
Roden, L; Ananth, S; Campbell, P et al. (1994) Xylosyl transfer to an endogenous renal acceptor. Purification of the transferase and the acceptor and their identification as glycogenin. J Biol Chem 269:11509-13
Campbell, P; Hannesson, H H; Sandback, D et al. (1994) Biosynthesis of heparin/heparan sulfate. Purification of the D-glucuronyl C-5 epimerase from bovine liver. J Biol Chem 269:26953-8
Manzella, S M; Roden, L; Meezan, E (1994) A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl-beta-D-maltoside. Anal Biochem 216:383-91
Meezan, E; Ananth, S; Manzella, S et al. (1994) Xylosyl transfer to an endogenous renal acceptor. Characteristics of the reaction and properties of the product. J Biol Chem 269:11503-8

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