Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity
- Author
- Francesco S Ielasi, Tom Verhaeghe (UGent) , Tom Desmet (UGent) and Ronnie G Willaert
- Organization
- 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 Gal beta-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
- Epa1, Candida glabrata, epithelial adhesin, glycan array, saturation mutagenesis, KERATAN SULFATE, SATURATION MUTAGENESIS, DIRECTED EVOLUTION, STRUCTURAL BASIS, FORCE-FIELD, SIALIC-ACID, LECTIN, CELLS, YEAST, PROTEIN
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-5796573
- MLA
- Ielasi, Francesco S., et al. “Engineering the Carbohydrate-Binding Site of Epa1p from Candida Glabrata: Generation of Adhesin Mutants with Different Carbohydrate Specificity.” GLYCOBIOLOGY, vol. 24, no. 12, 2014, pp. 1312–22, doi:10.1093/glycob/cwu075.
- APA
- Ielasi, F. S., Verhaeghe, T., Desmet, T., & Willaert, R. G. (2014). Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity. GLYCOBIOLOGY, 24(12), 1312–1322. https://doi.org/10.1093/glycob/cwu075
- Chicago author-date
- Ielasi, Francesco S, Tom Verhaeghe, Tom Desmet, and Ronnie G Willaert. 2014. “Engineering the Carbohydrate-Binding Site of Epa1p from Candida Glabrata: Generation of Adhesin Mutants with Different Carbohydrate Specificity.” GLYCOBIOLOGY 24 (12): 1312–22. https://doi.org/10.1093/glycob/cwu075.
- Chicago author-date (all authors)
- Ielasi, Francesco S, Tom Verhaeghe, Tom Desmet, and Ronnie G Willaert. 2014. “Engineering the Carbohydrate-Binding Site of Epa1p from Candida Glabrata: Generation of Adhesin Mutants with Different Carbohydrate Specificity.” GLYCOBIOLOGY 24 (12): 1312–1322. doi:10.1093/glycob/cwu075.
- Vancouver
- 1.Ielasi FS, Verhaeghe T, Desmet T, Willaert RG. Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity. GLYCOBIOLOGY. 2014;24(12):1312–22.
- IEEE
- [1]F. S. Ielasi, T. Verhaeghe, T. Desmet, and R. G. Willaert, “Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity,” GLYCOBIOLOGY, vol. 24, no. 12, pp. 1312–1322, 2014.
@article{5796573, 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 Gal beta-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.}}, author = {{Ielasi, Francesco S and Verhaeghe, Tom and Desmet, Tom and Willaert, Ronnie G}}, issn = {{0959-6658}}, journal = {{GLYCOBIOLOGY}}, keywords = {{Epa1,Candida glabrata,epithelial adhesin,glycan array,saturation mutagenesis,KERATAN SULFATE,SATURATION MUTAGENESIS,DIRECTED EVOLUTION,STRUCTURAL BASIS,FORCE-FIELD,SIALIC-ACID,LECTIN,CELLS,YEAST,PROTEIN}}, language = {{eng}}, number = {{12}}, pages = {{1312--1322}}, title = {{Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity}}, url = {{http://doi.org/10.1093/glycob/cwu075}}, volume = {{24}}, year = {{2014}}, }
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