Characterization of a cDNA encoding a novel human Golgi α1,2-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 alpha 1,2-mannosidases was isolated. The enzymatic properties of the novel alpha 1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein. alpha 1,2-Mannosidase IC sequentially hydrolyzes the alpha 1,2-linked mannose residues of [H-3]mannose-labeled Man(9)GlcNAc to form [H-3]Man(6)GlcNAc and a small amount of [H-3]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 alpha 1,2-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 alpha 1,2-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 alpha 1,2-mannosidases LA and IB. The full-length alpha 1,2-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 alpha 1,2-mannosidases IA and IB. Therefore, the human genome contains at least three differentially regulated Golgi alpha 1,2-mannosidase genes encoding enzymes with similar, but not identical specificities.