Functional Interaction Analysis of GM1-related Carbohydrates and Vibrio Cholerae Toxins Using Carbohydrate Microarray

Anal Chem. 2012 Aug 7;84(15):6884-90. doi: 10.1021/ac301511t. Epub 2012 Jul 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cholera Toxin / metabolism*
  • G(M2) Ganglioside / chemistry
  • G(M2) Ganglioside / metabolism
  • Microarray Analysis*
  • Oligosaccharides / chemistry
  • Oligosaccharides / metabolism*
  • Protein Binding
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Vibrio cholerae / metabolism*

Substances

  • GM1 pentasaccharide
  • Oligosaccharides
  • Protein Subunits
  • G(M2) Ganglioside
  • Cholera Toxin