Biochemical characterization of Candida albicans α-glucosidase I heterologously expressed in Escherichia coli

Abstract

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.

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.…”

Glycoside Hydrolase MoGls2 Controls Asexual/Sexual Development, Cell Wall Integrity and Infectious Growth in the Rice Blast Fungus

“…The transferred oligosaccharide is then processed in the ER by the sequential action of specialized trimming enzymes. Glucosidase I (Gls1) removes the outermost 1,2-linked glucose residue to yield the G2 form of the glycan (Glc2Man9GlcNAc2) [8], then glucosidase II (Gls2) removes the middle and innermost 1,3-linked glucose residues, yielding the G1 and M9 forms (Glc1Man9GlcNAc2; Man9GlcNAc2), respectively. Finally, mannosidase I selectively removes a specific mannose residue, yielding the M8 form (Man8GlcNAc2).…”

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

“…Further evidence of the type of a-glucosidase purified in this study was obtained by testing the effect of selective inhibitors of N-glycan trimming glycosidases. These included the glucose analog 1-deoxynojirimycin (DNJ) and the plant alkaloids castanospermine (CNP) and australine (AUST) which have been used to demonstrate the function of a-glucosidases I and II in glycoprotein processing (Elbein 1991;Moremen et al 1994;Herscovics 1999b;Frade-Pérez et al 2010). DNJ is a more specific inhibitor of a-glucosidase II (Saunier et al 1982;Kaushal et al 1990;Herscovics 1999b), CNP preferentially inhibits a-glucosidase I (Szumilo et al 1986;Zeng and Elbein 1998) and AUST appears to be more active on a-glucosidase I than on a-glucosidase II (Elbein 1991).…”