Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Memórias do Instituto Oswaldo Cruz

Héctor M Mora-montes²  Everardo López-romero²  Samuel Zinker¹  Patricia Ponce-noyola²  Arturo Flores-carreón² 

[1] ,Universidad de Guanajuato Facultad de Química Instituto de Investigación en Biología Experimental,México

关键词: 1;    2-mannosidase;    heterologous expression;    glycosyl hydrolase family 47;    Candida albicans;    polyclonal antibodies;    N-glycosylation;   

DOI  :  10.1590/S0074-02762008000700016

来源: SciELO

【 摘 要 】

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

【 授权许可】

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