Purification and biochemical characterisation of endoplasmic reticulum α1,2-mannosidase from Sporothrix schenckii

摘要

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentousfungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complexand are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae containonly one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is knownabout the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in thedimorphic fungus Sporothrix schenckii . Here, a membrane-bound α-mannosidase from S. schenckii was solubilisedusing a high-temperature procedure and purified by conventional methods of protein isolation. Analyticalzymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein wasrecognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomerB and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, withthe catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localisedα1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentousfungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidasesis similar to that present in lower eukaryotes.