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

Abstract

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...

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis

“…The genomes of S. schenckii and S. brasiliensis contained the orthologs involved in elaboration of the N -linked glycan core, its transference to proteins and in early trimming. These genes are also known to be involved in glycoprotein endoplasmic reticulum-associated degradation, a quality control system for proteins synthesized within the secretory pathway [90–92] (Additional file 2: Table S11). Furthermore, the genomes contain the putative orthologs encoding Golgi-resident glycosidases and glycosyltransferases that further modify N -linked glycans, generating both hybrid and complex N -linked glycans.…”

Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil

“…Mendes et al (2011) reported a frequency of 36.1% of LAM among 93 isolates from the metropolitan São Paulo area, and Gomes et al (2011), who provided the first insight into the population structure of TB isolates in Brazil, reported that the lowest percentage of the LAM lineage (35.0%) had been observed among 180 isolates from São Paulo state. Noguti et al (2010) showed the lowest percentage (34.7%) of this lineage in Maringá (93 isolates), and Miranda et al (2011) showed the highest percentage (55.3%) of this lineage in Minas Gerais (114 isolates).…”

Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

“…Cells were washed twice with deionized water and mechanical disrupted in a Braun homogenizer, with 16 cycles of 30 s and cooling on ice in-between (Mora-Montes et al, 2010b). Cell walls were pelleted by centrifuging and cleaned with sequential incubations with hot 2% (v/v) SDS (BioRad), 0.3 M β-mercaptoethanol (Sigma), and 1 M NaCl (Sigma), as described (Mora-Montes et al, 2007).…”