UDP-GlcNAc:Gal beta 1-4Glc(NAc) beta 1-3N-acetylglucosaminyltransferase. Identification and characterization in human serum.

Abstract

A ,f31-3-N-Acetylglucosaminyltransferase has been detected in human serum which transfers N-acetylglucosamine residues from UDP-GlcNAc to terminal Gal/31-4Glc(NAc) structures in oligosaccharides, glycoproteins, glycolipids, and proteoglycans.The product of the transferase reaction with lactose as acceptor was identified by methylation analysis and mass spectrometry as GlcNAcB1-3GalB1-4Glc.The B-linkage of the GlcNAc in the synthesized trisaccharide was confirmed by the action of the specific enzymes 8hexosaminidase and 8-N-acetylglucosaminide 81-4galactosyltransferase. Kinetic parameters were determined for UDP-GlcNAc, lactose, and N-acetyllactosamine.The enzyme requires Mn2+ ions for maximal activity and shows a pH optimum between 6 and 8.Using a wide variety of synthetic and natural oligosaccharides, the substrate specificity of the B1-3Nacetylglucosaminyltransferase was investigated.The enzyme was found to recognize specifically the free terminal structure Gal@l-4Glc(NAc).The substrate specificity was found to be equally stringent for glycoconjugates.Among the glycoproteins and glycolipids tested as acceptors, N-acetylglucosamine was incorporated only into those containing free terminal Galbl-4Glc(NAc) structures.When the terminal galactose residues were partially removed, the transfer of Nacetylglucosamine was strongly reduced.N-acetylglucosamine residues occur in glycoproteins, glycolipids, and proteoglycans in a variety of structures (1).One of the linkages most commonly found in all three classes of glycoconjugates is GlcNAcpl-3Gal.Repeating units of this disaccharide are present in certain mucins, membrane glycoproteins, and polyglycosylceramides where they are associated with the Ii-antigenic structures and serve as precursors of the ABH, Lewis, and PI blood group antigens (2).Among the proteoglycans, the keratan sulfate carries the GlcNAcpl-3Gal units (3).In general, the biosynthesis of glycans is accomplished by the sequential action of glycosyltransferases which transfer sugar residues from a sugar nucleotide to an oligosaccharide chain (1).In spite of the relative abundance of the GlcNAcp1-3Gal linkage in glycoconjugates, a specific trans-