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, α-glucosidase I removes the outermost α1,2-glucose unit from the N-linked core Glc3Man9GlcNAc2. 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 α-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.
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Abbreviations
- CNP:
-
Castanospermin
- DEPC:
-
Diethyl pyrocarbonate
- DNJ:
-
1-Deoxynojirimycin
- ER:
-
Endoplasmic reticulum
- G3M9 :
-
Glc3Man9GlcNAc2
- MU:
-
4-Methylumbelliferone
- MUαGlc:
-
4-Methylumbelliferyl-α-d-glucopyranoside
- NEM:
-
N-Ethylmaleimide
- SDS-PAGE:
-
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
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Acknowledgments
This work was supported by Grants No. CONACyT-2002-CO1-39528/A-1 and 83414 from Consejo Nacional de Ciencia y Tecnología, México, and Convocatoria 2008 from Dirección de Investigación y Posgrado, Universidad de Guanajuato.
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Frade-Pérez, M.D., Hernández-Cervantes, A., Flores-Carreón, A. et al. Biochemical characterization of Candida albicans α-glucosidase I heterologously expressed in Escherichia coli . Antonie van Leeuwenhoek 98, 291–298 (2010). https://doi.org/10.1007/s10482-010-9437-1
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DOI: https://doi.org/10.1007/s10482-010-9437-1