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N-glycosylation of proteins is a common post-translational modification in eukaryotic cells. Following the translocation of nascent proteins into the endoplasmic reticulum (ER), the Glc 3 Man 9 GlcNAc 2 oligosaccharide is transferred to asparagine residues and sequentially processed by ER α-glucosidases, which remove the three glucose residues and by α-mannosidases that trim at least one mannose residue (Kornfeld & Kornfeld 1985, Herscovics 1999a.Alpha-mannosidases are grouped within families 38 and 47 of the glycosyl hydrolase classification (Henrissat 1991, Henrissat & Davies 1997. Members of family 38 are less specific than enzymes from family 47, removing α1,2, α1,3 and α1,6-linked mannose units (Daniel et al. 1994, Herscovics 1999a. Enzymes from family 47 are α1,2-mannosidases that participate in the trimming of N-glycans and in ER-associated degradation (Herscovics 1999a, b, 2001, Helenius & Aebi 2004. Two subgroups of enzymes comprise family 47: the ER α1,2-mannosidases that trim only one mannose residue from Man 9 GlcNAc 2 (M 9 ) generating Man 8 GlcNAc isomer B (M 8 B) (Ziegler & Trimble 1991, Tremblay & Herscovics 1999, Mora-Montes et al. 2004, Movsichoff et al. 2005; and Golgi α1,2-mannosidases from mammalian cells and filamentous fungi, which act after the ER enzyme to remove three α1,2-linked mannose units, generating Man 5 GlcNAc 2 , an intermediate required for the biosynthesis of complex and hybrid N-glycans (Yoshida et al. 1993, Lal et al. 1998, Ichishima et al. 1999, Eades & Hintz 2000, Tremblay & Herscovics 2000, Lobsanov et al. 2002, Akao et al. 2006. Lower eukaryotes, such as Saccharomyces cerevisiae, lack Golgi α1,2-mannosidases and synthesise only high-mannose N-glycans (Herscovics 1999b).Sporothrix schenckii, the etiological agent of sporotrichosis, is a dimorphic fungus that grows as filamentous and yeast cells during saprophytic and parasitic phases, respectively. This organism is closely related to Ophiostoma stenoceras, a non-pathogenic fungus that grows on sapwood from coniferous and hardwood trees (de Beer et al. 2003). Sporotrichosis is a subcutaneous mycosis caused by traumatic inoculation of colonised materials (soil, wood, decomposed vegetable matter) or inhalation of the conidia (Ramos-e-Silva et al. 2007). The disease has been reported as an emerging mycosis in HIV-infected humans (Durden & Elewski 1997). The cell wall of S. schenckii is being actively researched, as this structure has an important role in adhesion to host tissues and is the main source of antigens that can be exploited in the immuno-diagnosis of the disease (LopesBezerra et al. 2006). The cell wall of S. schenckii contains β1,3, β1,6 and β1,4-glucans (Lopes-Bezerra et al. In order to gain insight into the processing steps of Nglycan biosynthesis, a membrane-bound α-mannosidase from S. schenckii was targeted for purification and characterisation. Biochemical characterisation and intracellular localisation studies indicated that this enzyme is an ER α1,2-mannosidase and therefore a new member of glycosyl hydrol...

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.