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

• Published in: Memórias do Instituto Oswaldo Cruz 2010-04, Vol.105 (1)
• Main Authors: Mora-Montes, Héctor M, Robledo-Ortiz, Claudia I, González-Sánchez, Laura C, López-Esparza, Adolfo, López-Romero, Everardo, Flores-Carreón, Arturo
• Format: Article
• Language: English
• Subjects: α1,2-mannosidase - N-glycosylation - Sporothrix schenckii - endoplasmic reticulum
• ISSN: 1678-8060
• DOI: 10.1590/S0074-02762010000100012

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, α1,2-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 α1,2-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 α-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-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases is similar to that present in lower eukaryotes.