title: Purification and biochemical characterisation of endoplasmic reticulum alpha1,2-mannosidase from Sporothrix schenckiil.
abstract: Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, alpha1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one alpha1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound alpha-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-alpha1,2-mannosidase antibodies. The enzyme hydrolysed Man(9)GlcNAc(2) into Man(8)GlcNAc(2) isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This alpha1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised alpha1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi alpha1,2-mannosidases and therefore, the processing of N-glycans by alpha1,2-mannosidases is similar to that present in lower eukaryotes.
authors.affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Scotland, United Kingdom.
authors.forename: Héctor M
authors.initials: HM
authors.lastname: Mora-Montes
authors.forename: Claudia I
authors.initials: CI
authors.lastname: Robledo-Ortiz
authors.forename: Laura C
authors.initials: LC
authors.lastname: González-Sánchez
authors.forename: Adolfo
authors.initials: A
authors.lastname: López-Esparza
authors.forename: Everardo
authors.initials: E
authors.lastname: López-Romero
authors.forename: Arturo
authors.initials: A
authors.lastname: Flores-Carreón
mesh_terms: D004721:Endoplasmic Reticulum / Q000201:enzymology*; D008361:Mannosidases / Q000737:chemistry / Q000302:isolation & purification*; D013175:Sporothrix / Q000145:classification / Q000166:cytology / Q000201:enzymology*
chemical_list: D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase