The human UDP-N-Acetylglucosamine:α-6-d-Mannoside-β-1,2-N-Acetylglucosaminyltransferase II Gene (MGAT2)

Abstract

UDP-GlcNAc:α-6–D-mannoside [GlcNAc to Manα1–6] β-1,2-N-acetylglucosaminyltransferase II (GlcNAc-T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligo-mannbse to complex N-glycans. A 1.2-kb probe from a rat liver cDNA encoding GlcNAc-T II was used to screen a human genomic DNA library in λEMBL3. Southern analysis of restriction endonuclease digests of positive phage clones identified two hybridizing fragments (3.0 and 3.5 kb) which were sub-cloned into pBlueScript. The inserts of the resulting plasmids (pHG30 and pHG36) are over-lapping clones containing 5.5 kb of genomic DNA. The pHG30 insert (3.0 kb) contains a 1341-bp open reading frame encoding a 447-amino-acid protein, 250 bp of G+C-rich 5′-upstream sequence and 1.4kb of 3′-downstream sequence. The pHG36 insert (3.5 kb) contains 2.75 kb of 5′-upstream sequence and 750 bp of the 5′-end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e. a short 9-residue putative cytoplasmic N-terminal domain, a 20-residue hydrophobic non-cleavable putative signal-anchor domain and a 418-residue C-terminal catalytic domain. Northern analysis of human tissues showed a major message at 3 kb and minor signals at 2 and 4.5 kb. There is no sequence similarity to any previously cloned glycosyltransferases including human UDP-GlcNAc:α-3-d-mannoside [GlcNAc to Manα;1–3] β-1,2-N-acetylglucosaminyltransferase I (GlcNAc-T I) which has 445 amino acids with a 418-residue C-terminal catalytic domain. The human GlcNAc-T I and II genes (MGAT1 and MGAT2) map to chromosome bands 5q35 and 14q21, respectively, by fluorescence in situ hybridization. The entire coding regions of human GlcNAc-T I and II are each on a single exon. There is 92% identity between the amino acid sequences of the catalytic domains of human and rat GlcNAc-T II. Southern analysis of restriction enzyme digests of human genomic DNA indicates that there is only a single copy of the MGAT2 gene. The full-length coding region of GlcNAc-T II has been expressed in the baculovirus/Sf9 insect cell system, the recombinant enzyme has been purified to near homogeneity with a specific activity of about 20 μmol · min-1· mg-1· and the product synthesized by the recombinant enzyme has been identified by high-resolution 1H-NMR spectroscopy and mass spectrometry.