Isolation and characterisation of putative adhesins from Helicobacter pylori with affinity for heparan sulphate proteoglycan

A pool of heparan sulphate-binding proteins (HSBPs) from Helicobacter pylor i culture supernates was obtained by sequential ammonium sulphate precipita tion and affinity chromatography on heparin-Sepharose, The chromatographic procedure yielded one major fraction that contained proteins with heparan s ulphate affinity as revealed by inhibition studies of heparan sulphate bind ing to H. pylori cells. Preparative iso-electric focusing, SDS-PACE and blo tting experiments, with peroxidase(POD)-labelled heparan sulphate as a prob e, indicated the presence of two major extracellular proteins with POD-hepa ran sulphate affinity. One protein had a molecular mass of 66.2 kDa and a p I of 5.4, whilst the second protein had a molecular mass of 71.5 kDa and a pI of 5.0. The N-terminal amino acid sequence of the 71.5-kDa HSBP did not show homology to any other heparin-binding protein, nor to known proteins o f H, pylori, whereas the 66.2-kDa HSBP showed a high homology to an Escheri chia coli chaperon protein and equine haemoglobin. A third HSBP was isolate d from an outer-membrane protein (OMP) fraction of H. pylori cells with a m olecular mass of 47.2 kDa, The amino acid sequence of an internal peptide o f the OMP-HSBP did not show homology to the extracellular HSBP of H, pylori , or to another microbial HSBP.