Glycosylated Proteins of the Gastric Pathogen Helicobacter pylori

The aim of this study was to detect the glycosylated proteins of Helicobacter pylori (H. pylori) separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two dimensional gel electrophoresis (2-DE). Protein analysis of the H. pylori strains was performed using denaturing 8% SDS-PAGE. H. pylori 26695 cell proteins were also separated by 2-DE into hundreds of spots. Separated proteins of H. pylori strains by SDS-PAGE and 2-DE were transferred onto the Polyvinylidene Fluoride (PVDF) membrane by semi-dry blotting. Detection of glycosylated proteins of the protein bands or spots on blotted membranes were determined by overlay reactions with Digoxigenin (DIG)-Glycan Detection and Differentation kits (Roche Diagnostics, Germany). The Roche DIG-Glycan Differentiation Kit including peanut agglutinin (PNA) lectin was utilized to further characterize the glycosidic modifications. Analysis of protein bands on a blotted membrane with DIG Glycan Detection kit after SDS-PAGE analysis gave the general pattern of glycosylated proteins of H. pylori; interestingly PA4, PR20 and P12 strains of H. pylori gave the different patterns of protein glycosylation on 8% polyacrilamide gels. Moreover, a glycosylated protein band (similar to 54 kDa) was also detected dominantly on the outer membrane part of H. pylori. 2-DE analysis of H. pylori proteins showed about twelve clear spots indicating the O-glycosidically linked carbohydrate chains (galactose-beta(1-3)-N-acetylgalactosamine) determined by PNA lectin staining of the blotted membrane. Obtained results suggest the presence of some potentially glycosylated proteins in H. pylori.