RESUMO
Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1∆ null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.
Assuntos
Genes Fúngicos , Pirofosfatases/genética , Pirofosfatases/metabolismo , Sporothrix/enzimologia , Sporothrix/genética , Sequência de Aminoácidos , Parede Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Glicosilação , Complexo de Golgi/metabolismo , Dados de Sequência Molecular , Pirofosfatases/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Manosiltransferases/genética , Manosiltransferases/metabolismo , Sporothrix/enzimologia , Candida albicans/enzimologia , Candida albicans/genética , Cátions Bivalentes/metabolismo , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Ativadores de Enzimas/metabolismo , Deleção de Genes , Expressão Gênica , Teste de Complementação Genética , Magnésio/metabolismo , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sporothrix/genética , Sporothrix/metabolismo , Especificidade por SubstratoRESUMO
The cell surface of Candida albicans is enriched with highly glycosylated mannoproteins that are involved in the interaction with host tissues. N- and O-glycosylation are post-translational modifications that initiate in the endoplasmic reticulum, and finalize in the Golgi. The KRE2/MNT1 family encode a set of multifunctional mannosyltransferases that participate in O-, N- and phosphomannosylation. In order to gain insights into the substrate specificities of these enzymes, recombinant forms of Mnt1, Mnt2, and Mnt5 were expressed in Pichia pastoris and the enzyme activities characterized. Mnt1 and Mnt2 showed a high specificity for α-methylmannoside and α1,2-mannobiose as acceptor substrates. Notably, they also used Saccharomyces cerevisiaeO-mannans as acceptors and generated products with more than three mannose residues, suggesting than Mnt1 and Mnt2 could be the mannosyltransferases adding the fourth and fifth mannose residue to the O-mannans in C. albicans. Mnt5 only recognized α-methylmannoside as acceptor, suggesting that participates in the addition of the second mannose residues to the N-glycan outer chain.
Assuntos
Candida albicans/enzimologia , Proteínas Fúngicas/química , Mananas/biossíntese , Manosiltransferases/química , Proteínas Fúngicas/genética , Manosiltransferases/genética , Modelos Químicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, alpha-glucosidase I removes the outermost alpha1,2-glucose unit from the N-linked core Glc(3)Man(9)GlcNAc(2). We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3'-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed alpha-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.