Conversion of α1,2-mannosidase E-I from Candida albicans to α1,2-mannosidase E-II by limited proteolysis

Abstract

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble alpha1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide. Here, alpha1,2-mannosidase E-I from strain ATCC 26555 was purified by conventional methods of protein isolation and affinity chromatography in Concanavalin A-Sepharose 4B. Analytical electrophoresis of the purified enzyme revealed two polypeptides of 52 and 23 kDa, the former being responsible for enzyme activity as revealed by zymogram analysis. Time course proteolysis with an aspartyl protease from Aspergillus saitoi, converted alpha1,2-mannosidase E-I into an active polypeptide of 43 kDa which trimmed Man(9)GlcNAc(2), generating Man(8)GlcNAc(2) isomer B and mannose. Trimming was inhibited preferentially by 1-deoxymannojirimycin. Both, the molecular mass and the enzyme properties of the proteolytic product were identical to those described for alpha1,2-mannosidase E-II therefore supporting the notion that E-I is the precursor of E-II.

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

“…As the incubation continued, M 7 B and most probably M 6 and M 5 were detected after 24 h. This result is in agreement with the hydrolysis reported for α1,2-mannosidases beloging to S. cerevisiae, C. albicans and human cells, in which M 8 B can be processed to shorter oligosaccharides under both in vitro and in vivo conditions , Mora-Montes et al 2004, 2008a, Avezov et al 2008. The inability to trim Man 5 GlcNAc 2 -Asn was expected, as this oligosaccharide lacks α1,2-linked mannose residues.…”

“…Alpha-mannosidase assay -Enzyme activity was measured using MUαMan, p-nitrophenyl-α-Dmannopyranoside (p-NP-Man), M 9 or Man 5 GlcNAc 2 -Asn oligosaccharides as described previously (JelinekKelly et al 1985, Mora-Montes et al 2004. For assays using oligosaccharides as substrates, 30 µL reactions (containing 5 µg of partially purified enzyme and 2 µg of oligosaccharide resuspended in buffer A) were incubated at 37ºC for either 12 h or 24 h, then boiled in water for 10 min and an equal volume of deionised water was added.…”

“…Active fractions eluted with salt-free buffer were pooled and freeze-dried. For both columns, protein concentration was monitored by absorbance at 280 nm and enzyme activity was measured with the fluorogenic substrate 4-methylumbelliferyl-α-D-mannopyranoside (MUαMan) as described below.Alpha-mannosidase assay -Enzyme activity was measured using MUαMan, p-nitrophenyl-α-Dmannopyranoside (p-NP-Man), M 9 or Man 5 GlcNAc 2 -Asn oligosaccharides as described previously (JelinekKelly et al 1985, Mora-Montes et al 2004. For assays using oligosaccharides as substrates, 30 µL reactions (containing 5 µg of partially purified enzyme and 2 µg of oligosaccharide resuspended in buffer A) were incubated at 37ºC for either 12 h or 24 h, then boiled in water for 10 min and an equal volume of deionised water was added.…”

“…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).…”

Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

“…Montes et al 2004Montes et al , 2006. Previously, we have shown that the α1,2-mannosidase E-II enzyme is the product of limited proteolysis of either enzyme E-I or a membranebound α-mannosidase (Mora- Montes et al 2006Montes et al , 2008, suggesting that the three α-mannosidase isoforms are related each other. Indeed, this is supported by our recent report indicating that a C. albicans mns1∆ null mutant lacks both soluble and membrane-bound α-mannosidase activity (Mora-Montes et al 2007).…”