Glycobiology vol 7 no 5 pp. 587-595, 1997
© 1997
research-article |
Partial purification and substrate specificity of heparan sulfate 
-N-acetylglucosaminyltransferase I: synthesis, NMR spectroscopic characterization and in vitro assays of two aryl tetrasaccharides
Department of Biochemistry and Molecular Genetics, and Comprehensive Cancer Center, University of Alabama at Birmingham Birmingham, AL 35294, USA
2Present address: Department of Developmental and Cell Biology, University of California at Irvine Irvine, CA 92717
3Present address: Division of Cellular and Molecular Medicine, Glycobiology Progam, UCSD Cancer Center, University of California San Diego, La Jolla, CA 92093-0687
1To whom correspondence should be addressed
Studies of heparan sulfate biosynthesis on ß-D-xylosides have led to the hypothesis that heparan sulfate 
-N-acetylglucosaminyltransferase I (-GlcNAc-TI) recognizes structures at the reducing end of the proteoglycan linkage tetrasaccharide. We report here the in vivo and in vitro testing of this hypothesis using four synthetic substrates, benzyl- and 2-naphthalenemethanyl-ß-D-xylosides, and two proteoglycan linkage tetrasaccharides containing benzyl alcohol or naphthalmethanol aglycones, viz., GlcAß(1 
3)Galß(1 3)Galß(1 4)Xylß-O-Bn (BNT) and GlcAß(1 3)Galß(1 3)Galß(1 4)Xylß-O-NM (NMT). The aryl tetrasaccharides were chemically synthesized and the 1H and 13C resonances were assigned by two-dimensional NMR spectroscopy. The inter-residue spatial constraints, determined by the 2D NOESY data, revealed essentially identical conformations for the interglycosidic linkages and Xyl-O-CH2Ar linkages in both compounds. Interestingly, the aromatic rings in both tetrasaccharides undergo rapid internal rotation across the CH2-Ar bond. These tetrasaccharides were used to assay heparan sulfate -GlcNAc-TI from homogenates of wild-type CHO cells. -GlcNAc-TI was also purified 
900-fold from rat liver and assayed with BNT and NMT. At nearly all concentrations tested, -GlcNAc-TI activity from both CHO cell homogenates and rat liver was greater with the NMT. When fed to CHO cells, benzyl-ß-D-xyloside primed heparan sulfate poorly relative to 2-naphthalenemethanyl-ß-D-xyIoside. Thus, the in vitro enzyme activity is consistent with the in vivo priming data that suggests that -GlcNAc-TI can directly recognize structure at the reducing end of the linkage tetrasaccharide. These studies provide an in vivo basis for the possible role of core protein sequences in the biosynthesis of specific glycosaminoglycans.
heparan sulfate -N-acetylglucosaminyltransferase I
linkage tetrasaccharide
NMR
proteoglycan
substrate specificity
synthesis
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