Isolation and functional characterization of Sporothrix schenckii ROT2, the encoding gene for the endoplasmic reticulum glucosidase II.

The N-linked glycosylation is a ubiquitous protein modification in eukaryotic cells. During the N-linked glycan synthesis, the precursor Glc(3)Man(9)GlcNAc(2) is processed by endoplasmic reticulum (ER) glucosidases I, II and α1,2-mannosidase, before transporting to the Golgi complex for further structure modifications. In fungi of medical relevance, as Candida albicans and Aspergillus, it is well known that ER glycosidases are important for cell fitness, cell wall organization, virulence, and interaction with the immune system. Despite this, little is known about these enzymes in Sporothrix schenckii, the causative agent of human sporotrichosis. This limited knowledge is due in part to the lack of a genome sequence of this organism. In this work we used degenerate primers and inverse PCR approaches to isolate the open reading frame of S. schenckii ROT2, the encoding gene for α subunit of ER glucosidase II. This S. schenckii gene complemented a Saccharomyces cerevisiae rot2Δ mutant; however, when expressed in a C. albicans rot2Δ mutant, S. schenckii Rot2 partially increased the levels of α-glucosidase activity, but failed to restore the N-linked glycosylation defect associated to the mutation. To our knowledge, this is the first report where a gene involved in protein N-linked glycosylation is isolated from S. schenckii.

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

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.

Purification and biochemical characterisation of endoplasmic reticulum alpha1,2-mannosidase from Sporothrix schenckiil.

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, alpha1,2-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 alpha1,2-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 alpha-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-alpha1,2-mannosidase antibodies. The enzyme hydrolysed Man(9)GlcNAc(2) into Man(8)GlcNAc(2) isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This alpha1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised alpha1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi alpha1,2-mannosidases and therefore, the processing of N-glycans by alpha1,2-mannosidases is similar to that present in lower eukaryotes.

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

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, á1,2-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 á1,2-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 á-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-á1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This á1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised á1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi á1,2-mannosidases and therefore, the processing of N-glycans by á1,2-mannosidases is similar to that present in lower eukaryotes.