Cloning and expression of a novel UDP-GlcNAc :alpha-D-mannoside beta 1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc :alpha-3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I

A TBLASTN search with human UDP-GlcNAc:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1. 101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34 %, identity over 340 amino acids). The recombinant protein converted Man(alpha1-6)[Man(alpha1-3)]Man(beta1-)O-octyl to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)] Man(beta1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)] Man(beta1-)O-octyl (the substrate for beta-1,2-N-acetylglucosaminyltransferase II), Man(alpha1-)O-benzyl [with K-m values of approximate to 0.3 and > 30 mM for UDP-GlcNAc and Man(alpha1-)O-benzyl respectively] and the glycopeptide CYA[Man(alpha1-)O-T]AV (K-m similar to 12 mM). The product formed with Man(alpha1-)O-benzyl was identified as GlcNAc(beta1-2)Man(alpha1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:alpha-D-mannoside beta-1,2-N-acetylglucosaniitiyltraiasferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3 kb message with a wide tissue distribution. The cDNA has a 1980 by open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(beta1-)O-octyl, Man(beta1-)O-p-nitrophenyl and GlcNAc(beta1-2)Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1- 4)GlcNAc(beta1-4)GlcNAc(beta1-)O-Asn were not acceptors, indicating that GnT 1.2 is specific for a-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(alpha1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT 1.2 may be responsible for the synthesis of the G1cNAc(beta1-2)Man(alpha1-)O-Ser/Thr moiety on alpha-dystroglycan and other O-mannosylated proteins.