Overexpression of the Golgi-localized enzyme α-mannosidase IIx in Chinese hamster ovary cells results in the conversion of hexamannosyl-N-acetylchitobiose to tetramannosyl-N-acetylchitobiose in the N-glycan-processing pathway

Abstract

Golgi α-mannosidase II is an enzyme that processes the intermediate oligosaccharide Gn1M5Gn2 to Gn1M3Gn2 during biosynthesis of N-glycans. Previously, we isolated a cDNA encoding a protein homologous to α-mannosidase II and designated it α-mannosidase IIx. Here, we show by immunocytochemistry that α-mannosidase IIx resides in the Golgi in HeLa cells. When coexpressed with α-mannosidase II, α-mannosidase IIx colocalizes with α-mannosidase II in COS cells. A protein A fusion of the catalytic domain of α-mannosidase IIx hydrolyzes a synthetic substrate, 4-umbelliferyl-α-D-mannoside, and this activity is inhibited by swainsonine. [3H]glucosamine-labeled Chinese hamster ovary cells overexpressing α-mannosidase IIx show a reduction of M6Gn2 and an accumulation of M4Gn2. Structural analysis identified M4Gn2 to be Manα1→6(Manα1→2Manα1→3) Manβ1→4GlcNAcβ1→4Glc-NAc. The results suggest that α-mannosidase IIx hydrolyzes two peripheral Manα1→6 and Manα1→3 residues from [(Manα1→6)(Manα1→3)Manα1→6] (Manα1→2Manα1→3)Manβ1→4GlcNAcb1→4GlcNAc, during N-glycan processing.