Purification and partial biochemical characterization of a membrane-bound type II-like α-glucosidase from the yeast morphotype of Sporothrix schenckii

Abstract

The early steps of glycoprotein biosynthesis involve processing of the N-glycan core by endoplasmic reticulum α-glucosidases I and II which sequentially trim the outermost α1,2-linked and the two more internal α1,3-linked glucose units, respectively. We have demonstrated the presence of some components of the enzymic machinery required for glycoprotein synthesis in Sporothrix schenckii, the etiological agent of human and animal sporotrichosis. However, information on this process is still very limited. Here, a distribution analysis of α-glucosidase revealed that 38 and 50% of total enzyme activity were present in a soluble and in a mixed membrane fraction, respectively. From the latter, the enzyme was solubilized, purified to apparent homogeneity and biochemically characterized. Analysis of the enzyme by denaturing electrophoresis and size exclusion chromatography revealed molecular masses of 75.4 and 152.7 kDa, respectively, suggesting a homodimeric structure. Purified α-glucosidase cleaved the fluorogenic substrate 4-methylumbelliferyl-α-D: -glucopyranoside with high affinity as judged from K(m) and V(max) values of 0.3 μM and 250 nmol of MU/min/mg protein, respectively. Analysis of linkage specificity using a number of glucose α-disaccharides as substrates demonstrated a clear preference of the enzyme for nigerose, an α1,3-linked disaccharide, over other substrates such as kojibiose (α1,2), trehalose (α1,1) and isomaltose (α1,6). Use of selective inhibitors of processing α-glucosidases such as 1-deoxynojirimycin, castanospermine and australine provided further evidence of the possible type of α-glucosidase. Accordingly, 1-deoxynojirimycin, a more specific inhibitor of α-glucosidase II than I, was a stronger inhibitor of hydrolysis of 4-methylumbelliferyl-α-D: -glucopyranoside and nigerose than castanospermine, a preferential inhibitor of α-glucosidase I. Inhibition of hydrolysis of kojibiose and maltose by 1-deoxynojirimycin and castanoespermine was significantly lower than that of nigerose. Taken together, these properties are consistent with a type II-like α-glucosidase probably involved in N-glycan processing. To our knowledge, this is the first report of such an activity in a truly dimorphic fungus.

Insight into broad substrate specificity and synergistic contribution of a fungal α-glucosidase in Chinese Nong-flavor daqu

“…In addition, NFAg31A was a mesophilic α-glucosidase, being stable up to 41.3 ℃, which is litter higher than 38 ℃ of Lactobacillus johnsonii AG [ 26 ] and 40 ℃ of Schizosaccharomyces pombe AGII [ 34 ], and lower than 55 ℃ of A.cerana AGII [ 32 ]. Whereas, NFAg31A showed an optimal pH at ~ 7.0, which was similar as pH 6.5 to pH 7.5 for most of homologous AGIIs [ 16 , 18 , 29 , 30 , 41 ], AGs [ 13 , 42 ], and human MGAM and SI [ 22 , 43 , 44 ], different with pH 3.5 to 5.5 for A.cerana AGII and archaebacterial AGs. Moreover, NFAg31A was a neutral α-glucosidase, exhibiting well stability over a broad pH range of pH 6.0 to pH 10.0, which was similarly detected as pH 6.2 to pH 9.1 for S.pombe AGII and pH 5.0 to pH 8.0 for L.johnsonii AG.…”

Fungal Glycosidases in Sporothrix Species and Candida albicans

“…A distribution analysis of α-glucosidase activity in the yeast morphotype of S. schenckii was carried out by a fluorometric method using 4-methylumbelliferyl-α-D-glucopyranoside (MUαGlc) as a substrate [ 114 ]. The results revealed that 38% and 50% of total enzyme activity were present in a soluble and a mixed membrane fraction (MMF), respectively.…”

“…Taken together, these properties are consistent with a type II-like α-glucosidase that was probably involved in N -glycan processing. To our knowledge, this was the first report of this activity in a truly dimorphic fungus [ 114 ].…”

Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization

“…Homologs of these three Am HBGase isoforms (I, II and type II-like HBGases) have also been reported in the yeast Sporothrix schenckii , where they were found to be located in the endoplasmic reticulum and to be involved in processing of the N -glycan core for glycoprotein biosynthesis [12]. However, these three yeast homologs differ in their molecular mass and biochemical characters.…”