title: Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.
abstract: The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.
authors: Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.
authors: Chang Sup
authors: CS
authors: Kim
authors: Jeong Hyun
authors: JH
authors: Seo
authors: Hyung Joon
authors: HJ
authors: Cha
mesh_terms: D002772:Cholera Toxin / Q000378:metabolism*; D005678:G(M2) Ganglioside / Q000737:chemistry / Q000378:metabolism; D046228:Microarray Analysis; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014734:Vibrio cholerae / Q000378:metabolism*
chemical_list: C519498:GM1 pentasaccharide; D009844:Oligosaccharides; D021122:Protein Subunits; D005678:G(M2) Ganglioside; D002772:Cholera Toxin