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.

β1,3-N-acetylglucosaminyltransferases (B3GNTs) are Golgi-resident glycosyltransferases involved in the biosynthesis of poly-N-acetyl-lactosamine chains. They catalyze the addition of the N-acetylglucosamine to the N-acetyl-lactosamine repeat as a key step of the chain elongation process. Poly-N-acetyl-lactosamine is involved in the immune system in many ways. Particularly, its long chain has been demonstrated to suppress excessive immune responses. Among the characterized B3GNTs, B3GNT2 is the major poly-N-acetyl-lactosamine synthase, and deletion of its coding gene dramatically reduced the cell surface poly-N-acetyl-lactosamine and led to hypersensitive and hyperresponsive immunocytes. Despite the extensive functional studies, no structural information is available to understand the molecular mechanism of B3GNT2, as well as other B3GNTs. Here we present the structural and kinetic studies of the human B3GNT2. Five crystal structures of B3GNT2 have been determined in the unliganded, donor substrate-bound, acceptor substrate-bound, and product(s)-bound states at resolutions ranging from 1.85 to 2.35 Å. Kinetic study shows that the transglycosylation reaction follows a sequential mechanism. Critical residues involved in recognition of both donor and acceptor substrates as well as catalysis are identified. Mutations of these invariant residues impair B3GNT2 activity in cell assays. Structural comparison with other glycosyltransferases such as mouse Fringe reveals a novel N-terminal helical domain of B3GNTs that may stabilize the catalytic domain and distinguish among different acceptor substrates.