Isolation of Sporothrix schenckii MNT1 and the biochemical and functional characterization of the encoded α1,2-mannosyltransferase activity

• Published in: Microbiology (Society for General Microbiology) 2012-09, Vol.158 (Pt 9), p.2419-2427
• Main Authors: HERNANDEZ-CERVANTES, Arturo, MORA-MONTES, Hector M, ALVAREZ-VARGAS, Aurelio, DIAZ JIMENEZ, Diana F, ROBLEDO-ORTIZ, Claudia I, FLORES-CARREON, Arturo
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Candida albicans - enzymology; Candida albicans - genetics; Cations, Divalent - metabolism; Cloning, Molecular; DNA, Fungal - chemistry; DNA, Fungal - genetics; Enzyme Activators - metabolism; Fundamental and applied biological sciences. Psychology; Gene Deletion; Gene Expression; Genetic Complementation Test; Magnesium - metabolism; Mannosyltransferases - genetics; Mannosyltransferases - metabolism; Microbiology; Miscellaneous; Molecular Sequence Data; Mycology; Pichia - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sporothrix - enzymology; Sporothrix - genetics; Sporothrix - metabolism; Substrate Specificity
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.060392-0

Sporothrix (Sp.) schenckii is a pathogenic fungus that infects humans and animals, and is responsible for the disease named sporotrichosis. The cell wall of this fungus has glycoproteins with a high content of mannose and rhamnose units, which are synthesized by endoplasmic reticulum- and Golgi-localized glycosyltransferases. Little is known about the enzymic machinery involved in the synthesis of these oligosaccharides in Sp. schenckii, or the genes encoding these activities. This is in part because of the lack of an available genome sequence for this organism. Using a partial genomic DNA library we identified SsMNT1, whose predicted product has significant similarity to proteins encoded by members of the Saccharomyces (Sa.) cerevisiae KRE2/MNT1 gene family. In order to biochemically characterize the putative enzyme, SsMNT1 was heterologously expressed in the methylotrophic yeast Pichia pastoris. Recombinant SsMnt1 showed Mn(2+)-dependent mannosyltransferase activity and the ability to recognize as acceptors α-methyl mannoside, mannose, Man(5)GlcNAc(2) oligosaccharide and a variety of mannobiosides. The characterization of the enzymic products generated by SsMnt1 revealed that the enzyme is an α1,2-mannosyltransferase that adds up to two mannose residues to the acceptor molecule. Functional complementation studies were performed in Sa. cerevisiae and Candida albicans mutants lacking members of the KRE2/MNT1 gene family, demonstrating that SsMnt1 is involved in both the N- and O-linked glycosylation pathways, but not in phosphomannan elaboration.