Heterologous expression and biochemical characterization of an alpha1,2-mannosidase encoded by the Candida albicans MNS1 gene.
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 alpha1,2-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 alpha1,2-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 alpha1,2-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 alpha1,2-mannosidases in other biological systems as well.
Mora-Montes
Héctor M
HM
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, México.
López-Romero
Everardo
E
Zinker
Samuel
S
Ponce-Noyola
Patricia
P
Flores-Carreón
Arturo
A
D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D005800:Genes, Fungal; D008361:Mannosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D000906:Antibodies; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase