Identification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni

It was demonstrated recently that there is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To characterize such glycoproteins, we identified a lectin, Soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie-stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28 kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34 kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein extracts from mutant and wild-type strains were analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA-reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA-reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid-glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of alpha-linked N-acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni Furthermore, we have identified two such glycoproteins, the first non-flagellin campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain alpha-linked N-acetylgalactosamine residues.