title: Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity.
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.
authors: Department of Bioengineering Sciences, Structural Biology Research Center (SBRC), Vrije Universiteit Brussel, Brussels 1050, Belgium.
authors: Francesco S
authors: FS
authors: Ielasi
authors: Department of Biochemical and Microbial Technology, Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Ghent 9000, Belgium.
authors: Tom
authors: T
authors: Verhaeghe
authors: Department of Biochemical and Microbial Technology, Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Ghent 9000, Belgium.
authors: Tom
authors: T
authors: Desmet
authors: Department of Bioengineering Sciences, Structural Biology Research Center (SBRC), Vrije Universiteit Brussel, Brussels 1050, Belgium ronnie.willaert@vub.ac.be.
authors: Ronnie G
authors: RG
authors: Willaert
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002241:Carbohydrates / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D037102:Lectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D005656:Fungal Proteins; D037102:Lectins; C120505:epithelial adhesin 1, Candida glabrata
keywords: Candida glabrata; Epa1; epithelial adhesin; glycan array; saturation mutagenesis