Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin

Flagellins from three strains of Campylobacter jejuni and one strain of Cam pylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the prot ein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray m ass spectrometry with a high/low orifice stepping to identify peptide segme nts of aberrant masses together with their corresponding glycosyl appendage s. These modified peptides were further characterized by tandem mass spectr ometry and preparative high performance liquid chromatography followed by n ano-NMR spectroscopy to identify the nature and precise site of glycosylati on. These analyses have shown that there are 19 modified Ser/Thr residues i n C. jejuni 81-176 flagellin. The predominant modification found on C. jeju ni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-ma nno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heteroge neity conferred by substitution of the acetamido groups with acetamidino an d hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesi s and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appe ars unrelated to a consensus peptide sequence but is rather based on surfac e accessibility of Ser/Thr residues in the folded protein.