The genes involved in the synthesis and transfer of the heptasaccharide are found clustered together in the pgl (for protein glycosylation) locus (Fig. ) and can be functionally transferred into Escherichia coli (). The locus is conserved in gene content and organization throughout the Campylobacter genus (), with the exception of wlaJ, which is present in approximately half of all C. jejuni strains. Recent analyses of the gene content from the genome-sequenced strains of Wolinella succinogenes and Desulfovibrio desulfuricans have identified genes with significant identity to those in the campylobacter pgl locus, including the gene encoding the oligosaccharide transferase, PglB, required for N-linked protein glycosylation. PglB, the homolog of the eukaryotic Stt3p, contains the WWDYG catalytic domain that is conserved in all three domains of life and is essential for N-linked protein glycosylation (, ). Thus, although it has not yet been confirmed structurally, it is likely these organisms also synthesize N-linked glycans.
Although the HR-MAS NMR spectrum of the pglI mutant showed anomeric resonances that corresponded to those of the N-linked glycan, they exhibited altered stoichiometry compared to the wild type. MS analysis of Peb3 fractions from this mutant did not detect glycosylation, yet pglI-negative protein extracts reacted with SBA at a level similar to that of the wild type. To resolve these observations and to determine whether Peb3 was inefficiently glycosylated in this mutant, total glycoproteins were extracted from the pglI mutant by SBA affinity chromatography and separated by SDS-PAGE, and the glycoprotein bands were analyzed by in-gel tryptic digestion and nanoLC-MS/MS as described. The MS/MS spectra of glycopeptides derived from one of these glycoproteins, the secreted transglycolase (Cj0843c) from wild-type C. jejuni NCTC 11168 and the pglI mutant are presented in Fig. . The identity of Cj0843c was confirmed by the observation of weak peptide fragment ions in these spectra (not highlighted in Fig. ) and by the presence of other peptides derived from this protein in the gel band digest extracts. The MS/MS spectrum of the doubly charged wild-type glycopeptide ion at m/z 1325.6 was dominated by the glycan oxonium ions in the low-mass region of the spectrum (m/z 204.1, HexNAc; m/z 366.1, HexNAc plus Hex; m/z 407.2, 2× HexNAc) and by the sequential loss of monosaccharides in the upper regions and was entirely consistent with the structure described previously for this glycan (). On the other hand, the MS/MS spectrum of the corresponding doubly protonated pglI-negative glycopeptide ion at m/z 1244.5 lacked the HexNAc plus Hex oxonium ion at m/z 366.1 and any fragment ions in the higher m/z regions indicating the presence of a hexose. This evidence, plus the difference in mass between the two glycopeptides, indicates that the Cj0843c glycan from the pglI mutant lacked the hexose moiety representative of Glc. Similar analyses were carried out with the pglD, pglH, and pglK mutants (not shown). As expected, there were no detectable glycopeptides for pglH and pglK mutants; however, there was evidence of very low levels of glycosylation for the pglD mutant. Unfortunately, the levels were too low to analyze further.
Pathways of O-glycan biosynthesis in cancer cells
The GalNAc -Ser/Thr linkage has been considered a hallmark of mucins where it occurs in clusters. However, this linkage has also been found in a wide variety of other proteins (). No primary amino acid consensus sequence has emerged for mucin-type O-linked glycosylation. In general, glycosylation of Thr is preferred over Ser () and the linkage is found in clusters of Ser/Thr residues with a turn near Pro and at a distance from charged amino acids ().
The biosynthesis of branched O-glycans | Request PDF
In individual studies, some proteins have been demonstrated to contain N-linked oligosaccharides. , a member of the /Notch family of receptor proteins essential for distal tip cell (DTC) control of germline proliferation () is a glycoprotein ().
Glycan Biosynthesis and MetabolismRelease 2
When examined by HR-MAS NMR, the majority of double pgl/kpsM C. jejuni mutants were missing N-glycan resonances (Fig. ). These strains included pglB/kpsM and gne/kpsM, as was predicted from previous experiments; pglD/kpsM, pglE/kpsM, and pglF/kpsM (Bac biosynthesis mutants); pglA/kpsM, pglH/kpsM, and pglJkpsM (GalNAc transferase mutants); and pglK/kpsM (putative inner-membrane transport mutant). In contrast, the glycan produced by pglG/kpsM appeared to be identical to the wild type. This gene flanks the pgl locus (Fig. ), but its function is currently unknown.
Glycan Biosynthesis and Metabolism
Recently developed methods have allowed the identification of 304 proteins containing N-glycans and the simultaneous determination of the glycosylation sites (; ; ). These studies employed lectin affinity chromatography to isolate glycopeptides generated by tryptic digestion of protein fractions, followed by a variety of mass spectrometry approaches. All but four of the N-glycosylation sites identified correspond to the conventional Asn-X-Ser/Thr. Three peptides contain Asn-X-Cys (; ). The identified N-glycoproteins comprise soluble and hydrophobic proteins, and many of them are extracellular matrix components which have been implicated in cell adhesion or are components of basement membranes. Among them, , (the two integrin subunits), (the integrin subunit), , , , (the four laminin subunit gene products), (nidogen) and (dystroglycan) contain at least one N-linked glycan [see ; for complete lists].