Two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate.
We report that two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate in Chinese hamster ovary cells. The first enzyme initiates heparan sulfate biosynthesis and can be measured by the transfer of GlcNAc from UDP-GlcNAc to GlcUA beta 1-3Gal beta 1-O-naphthalenemethanol. The second enzyme catalyzes the polymerization of heparan sulfate and can be measured by the transfer of GlcNAc from UDP-GlcNAc to the nonreducing terminal GlcUA present in oligosaccharide fragments prepared from the Escherichia coli K5 capsular polysaccharide, N-acetylheparosan. Kinetic characterization of the initiating GlcNAc-transferase (alpha-GlcNAc-TI) indicates an apparent Km for UDP-GlcNAc of 36 +/- 4 microM. The apparent Km for UDP-GlcNAc of the polymerizing GlcNAc-transferase (alpha-GlcNAc-TII) is 230 +/- 30 microM. Both enzymes have broad pH optima and require a divalent cation for activity. alpha-GlcNAc-TI can use both Mn2+ and Ca2+, while alpha-GlcNAc-TII will use only Mn2+. Chinese hamster ovary cells deficient in the synthesis of heparan sulfate and lacking alpha-GlcNAc-TII activity and S49 Thy 1-a lymphoma cells deficient in alpha GlcNAc addition to phosphatidylinositol have wild-type alpha-GlcNAc-TI activity. Thus, distinct alpha-GlcNAc-transferases catalyze the initiation and polymerization of heparan sulfate.
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 35294-0005.
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D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002384:Catalysis; D006224:Cricetinae; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism
D006497:Heparan Sulfate; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I