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Glycobiology. 2014 Dec;24(12):1312-22. doi: 10.1093/glycob/cwu075. Epub 2014 Jul 21.

Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity.

Author information

1
Department of Bioengineering Sciences, Structural Biology Research Center (SBRC), Vrije Universiteit Brussel, Brussels 1050, Belgium.
2
Department of Biochemical and Microbial Technology, Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Ghent 9000, Belgium.
3
Department of Bioengineering Sciences, Structural Biology Research Center (SBRC), Vrije Universiteit Brussel, Brussels 1050, Belgium ronnie.willaert@vub.ac.be.

Abstract

The N-terminal domain of the Epa1p adhesin from Candida glabrata (N-Epa1p) is a calcium-dependent lectin, which confers the opportunistic yeast the ability to adhere to human epithelial cells. This lectin domain is able to interact with galactosides and, more precisely, with glycan molecules containing the Galβ-1,3-GalNAc disaccharide, also known as the T-antigen. Based on the crystallographic structure of the N-Epa1p domain and the role of the variable loop CBL2 in glycan binding, saturation mutagenesis on some residues of the CBL2 loop was used to increase the binding affinity of N-Epa1p for fibronectin, which was selected as a model of a human glycoprotein. Two adhesin mutants, E227A and Y228W, with improved binding features were obtained. More importantly, a glycan array screening revealed that single-point mutations in the CBL2 could produce significant changes in the carbohydrate specificity of the protein. In particular, lectin molecules were generated with a high affinity for sulfated glycans, which may find an application as molecular probes for the identification of 6-sulfogalactose containing glycans and glycoconjugates.

KEYWORDS:

Candida glabrata; Epa1; epithelial adhesin; glycan array; saturation mutagenesis

PMID:
25049238
DOI:
10.1093/glycob/cwu075
[Indexed for MEDLINE]

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