title: Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha1,2-mannosidase.
abstract: Class I alpha1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities. Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) isomer B (lacking the alpha1, 2-mannose residue of the middle alpha1, 3-arm), whereas other alpha1,2-mannosidases, including Golgi alpha1,2-mannosidases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2). In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha1,2-mannosidase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc. High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man(9)GlcNAc is different from that of other alpha1, 2-mannosidases that remove four mannose from Man(9)GlcNAc. These results demonstrate that Arg(273) is in part responsible for the specificity of the endoplasmic reticulum alpha1,2-mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I alpha1, 2-mannosidases are responsible for the different specificities of these enzymes.
authors.affiliation: McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: P A
authors.initials: PA
authors.lastname: Romero
authors.forename: F
authors.initials: F
authors.lastname: Vallée
authors.forename: P L
authors.initials: PL
authors.lastname: Howell
authors.forename: A
authors.initials: A
authors.lastname: Herscovics
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007930:Leucine; D009682:Magnetic Resonance Spectroscopy; D008361:Mannosidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D001120:Arginine; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D007930:Leucine