Cloning and expression of a specific human alpha 1,2-mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis.
We report the isolation of a novel human cDNA encoding a type II membrane protein 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 secreted protein in Pichia pastoris. The recombinant enzyme removes a single mannose residue from Man9GlcNAc and [1H]-NMR analysis indicates that the only product is Man8GlcNAc isomer B, the form lacking the middle-arm terminal alpha 1,2-mannose. Calcium is required for enzyme activity and both 1-deoxymannojirimycin and kifunensine inhibit the human alpha 1,2-mannosidase. The properties and specificity of this human alpha 1,2-mannosidase are identical to the endoplasmic reticulum alpha 1,2-mannosidase from Saccharomyces cerevisiae and differ from those of previously cloned Golgi alpha 1,2-mannosidases that remove up to four mannose residues from Man9GlcNAc2 during N-glycan maturation. Northern blot analysis showed that all human tissues examined express variable amounts of a 3 kb transcript. This highly specific alpha 1,2-mannosidase is likely to be involved in glycoprotein quality control since there is increasing evidence that trimming of Man9GlcNAc2 to Man8GlcNAc2 isomer B in yeast cells is important to target misfolded glycoproteins for degradation.
Tremblay
L O
LO
McGill Cancer Centre, Montréal, Québec, Canada.
Herscovics
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase