Characterization of the Structurally Diverse N-Linked Glycans of Campylobacter Species

Abstract

cThe Gram-negative bacterium Campylobacter jejuni encodes an extensively characterized N-linked protein glycosylation system that modifies many surface proteins with a heptasaccharide glycan. In C. jejuni, the genes that encode the enzymes required for glycan biosynthesis and transfer to protein are located at a single pgl gene locus. Similar loci are also present in the genome sequences of all other Campylobacter species, although variations in gene content and organization are evident. In this study, we have demonstrated that only Campylobacter species closely related to C. jejuni produce glycoproteins that interact with both a C. jejuni N-linked-glycan-specific antiserum and a lectin known to bind to the C. jejuni N-linked glycan. In order to further investigate the structure of Campylobacter N-linked glycans, we employed an in vitro peptide glycosylation assay combined with mass spectrometry to demonstrate that Campylobacter species produce a range of structurally distinct N-linked glycans with variations in the number of sugar residues (penta-, hexa-, and heptasaccharides), the presence of branching sugars, and monosaccharide content. These data considerably expand our knowledge of bacterial N-linked glycan structure and provide a framework for investigating the role of glycosyltransferases and sugar biosynthesis enzymes in glycoprotein biosynthesis with practical implications for synthetic biology and glycoengineering.

Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation

“…Derivatives of the same structure with reduced complexity exist in lower eukaryotes, such as protozoan species (3,4). In contrast, the glycan structures in archaea and eubacteria show a far greater variety of monosaccharides and their linkages (1,(5)(6)(7). This is probably because the N-glycosylation is not essential for survival in archaea and eubacteria, and the structures have been optimized for their special purposes.…”

Bacterial Protein N-Glycosylation: New Perspectives and Applications

“…1) (30,32). In all species examined, the first two to three reducing end sugars (which include diNAcBac) were conserved, and all of the variation occurred at the nonreducing end.…”

“…The non-thermotolerant Campylobacter species (Group II) express different N-glycan chain lengths from hexasaccharides to octasaccharides and, in some cases, produce from two to four structural variants. The presence of monosaccharides with unusual masses of 217 Da (as observed in H. pullorum (31), Hilicobacter winghamensis (31), Campylobacter rectus, Campylobacter showae, Campylobacter curvus, Campylobacter mucosalis (32), and Campylobacter concisus (30) and predicted to be 2-acetamido-2-deoxy-D-galacturonic acid (32)), 234 Da (identified as glucolactilic acid in C. concisus but also present in other species (32)), and 245 Da (present in Campylobacter hominis (32) and predicted to be an O-acetylated HexNAc (32)) suggests that some Campylobacter species have acquired these unique sugars independently (32).…”

“…Helicobacter pullorum produces a linear pentasaccharide with unknown sugar residues with masses of 216 and 217 Da (31). Wolinella succinogenes produces a hexasaccharide containing three 216-Da mon-osaccharides and an unusual 232-Da residue at the nonreducing end and is the only species so far that has a sugar branching off the second sugar from the reducing end (30).…”

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…It seems possible that archaeal enzymes, in particular AglB, might be useful for glycoengineering of vaccines. A limitation of the use of PglB in protein glycan coupling technology is the requirement of this OST for a linking sugar presenting an acetamido group in the C-2 position (231,232). The employment of AglB in these efforts may help overcome this requirement, since Archaea use a broader variety of linking sugars in their N-glycans, including those lacking this C-2 modification.…”