Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Memórias do Instituto Oswaldo Cruz

Héctor M Mora-montes²  Claudia I Robledo-ortiz¹  Laura C González-sánchez¹  Adolfo López-esparza¹  Everardo López-romero¹  Arturo Flores-carreón¹ 

[1] ,University of Aberdeen Institute of Medical Sciences School of Medical SciencesScotland,United Kingdom

关键词: 1;    2-mannosidase;    N-glycosylation;    Sporothrix schenckii;    endoplasmic reticulum;   

DOI  :  10.1590/S0074-02762010000100012

来源: SciELO

【 摘 要 】

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.

【 授权许可】

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