Cloning and expression of a specific human alpha 1,2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B during N-glycan biosynthesis

We report the isolation of a novel human cDNA encoding a type II membrane p rotein of 79.5 kDa with amino acid sequence similarity to Class I alpha 1,2 -mannosidases. The catalytic domain of the enzyme was expressed as a secret ed protein in Pichia pastoris. The recombinant enzyme removes a single mann ose residue from Man(9)GlcNAc and [H-1]-NMR analysis indicates that the onl y product is Man(8)GlcNAc isomer B, the form lacking the middle-arm termina l alpha 1,2-mannose. Calcium is required for enzyme activity and both 1-deo xymannojirimycin and kifunensine inhibit the human alpha 1,2-mannosidase. T he properties and specificity of this human alpha 1,2-mannosidase are ident ical to the endoplasmic reticulum alpha 1,2-mannosidase from Saccharomyces cerevisiae and differ from those of previously cloned Golgi alpha 1d,2-mann osidases that remove up to four mannose residues from Man(9)GlcNAc(2) durin g N-glycan maturation. Northern blot analysis showed that all human tissues examined express variable amounts of a 3 kb transcript. This highly specif ic alpha 1,2-mannosidase is likely to be involved in glycoprotein quality c ontrol since there is increasing evidence that trimming of Man(9)GlcNAc(2) to Man(9)GlcNAc(2) isomer B in yeast cells is important to target misfolded glycoproteins for degradation.