The human UDP‐N‐Acetylglucosamine:α‐6‐d‐Mannoside‐β‐1,2‐N‐Acetylglucosaminyltransferase II Gene (MGAT2): Cloning of Genomic DNA, Localization to Chromosome 14q21, Expression in Insect Cells and Purification of the Recombinant Protein

Jenny Tan, Giacomo A. F. D'agostaro, Brad Bendiak, Folkert Reck, Mohan Sarkar, Jeremy A. Squire, Peng Leong, Harry Schachter

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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‐resolution1H‐NMR spectroscopy and mass spectrometry. Copyright © 1995, Wiley Blackwell. All rights reserved
Original languageEnglish
Pages (from-to)317 - 328
Number of pages12
JournalEuropean Journal of Biochemistry
Volume231
Issue number2
DOIs
Publication statusPublished - 1995
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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