Characterization of a cDNA encoding a novel human Golgi alpha 1, 2-mannosidase (IC) involved in N-glycan biosynthesis.
A human cDNA encoding a 70.9-kDa type II membrane protein with sequence similarity to class I alpha1,2-mannosidases was isolated. The enzymatic properties of the novel alpha1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein. alpha1,2-Mannosidase IC sequentially hydrolyzes the alpha1,2-linked mannose residues of [(3)H]mannose-labeled Man(9)GlcNAc to form [(3)H]Man(6)GlcNAc and a small amount of [(3)H]Man(5)GlcNAc. The enzyme requires calcium for activity and is inhibited by both 1-deoxymannojirimycin and kifunensine. The order of mannose removal was determined by separating oligosaccharide isomers formed from pyridylaminated Man(9)GlcNAc(2) by high performance liquid chromatography. The terminal alpha1,2-linked mannose residue from the middle branch is the last mannose removed by the enzyme. This residue is the mannose cleaved from Man(9)GlcNAc(2) by the endoplasmic reticulum alpha1, 2-mannosidase I to form Man(8)GlcNAc(2) isomer B. The order of mannose hydrolysis from either pyridylaminated Man(9)GlcNAc(2) or Man(8)GlcNAc(2) isomer B differs from that previously reported for mammalian Golgi alpha1,2-mannosidases IA and IB. The full-length alpha1,2-mannosidase IC was localized to the Golgi of MDBK and MDCK cells by indirect immunofluorescence. Northern blot analysis showed tissue-specific expression of a major transcript of 3.8 kilobase pairs. The expression pattern is different from that of human Golgi alpha1,2-mannosidases IA and IB. Therefore, the human genome contains at least three differentially regulated Golgi alpha1, 2-mannosidase genes encoding enzymes with similar, but not identical specificities.
McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada.
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Herscovics
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008351:Mannans / Q000737:chemistry / Q000378:metabolism; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D009928:Organ Specificity; D010843:Pichia; D011134:Polysaccharides / Q000096:biosynthesis*; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D004791:Enzyme Inhibitors; D008351:Mannans; D009844:Oligosaccharides; D011134:Polysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C057328:mannosyl(9)-N-acetylglucosamine; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase