Biochemical Characterization of the O-Linked Glycosylation Pathway in Neisseria gonorrhoeae Responsible for Biosynthesis of Protein Glycans Containing N,N′-Diacetylbacillosamine

Abstract

The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains (1, 2), but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH) and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-D-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically and the stereochemistry was assigned as uridine diphosphate N’-diacetylbacillosamine (UDP-diNAcBac) by NMR characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases and oligosaccharyltransferase (OTase) were analyzed in vitro and in most cases, these enzymes showed strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae or C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

“…Although the identities of these residues cannot be confirmed from the mass alone the convergence of similar residues, irrespective of their stereochemistry, is an observation of significant note. A similar concept of the utilization of a conserved carbohydrate repertoire has been noted within Neisseria and Campylobacter species where both systems utilize diBacNAc yet the enzymes responsible for the generation of diBacNAc represent two phylogenetically distinct clades (62)(63)(64). Widespread use of these unique carbohydrates by multiple bacterial glycosylation systems suggests preference for these sugars in protein modification, although the exact advantages of these residues are unknown.…”

O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry

“…After membrane translocation, the glycan portion of the polyprenyl-phosphate-glycan intermediate is transferred en bloc to the protein by an oligosaccharyltransferase active in the periplasm. Such mechanism has been demonstrated for N-glycosylation in Campylobacter jejuni (4 -6) and O-glycosylation in Neisseria gonorrheae (7)(8)(9). These pathways are responsible for the glycosylation of more than 65 proteins in C. jejuni (10) and 12 different proteins in N.…”

Diversity, Abundance and Distribution of O-linked Glycosylation Pathway Enzymes in Prokaryotes-A Comparative Genomics Study

“…The variation in the number of homologs belonging to different categories in case of many organisms reflects the diversity of the O-glycosylation pathway as has been demonstrated in Neisseria gonorrhoea [5,15]. These variations can be attributed to the horizontal gene transfer and selective loss of genetic material [46][47][48].…”

“…These are the O-glycosylation pathways of Neisseria [1][2][3][4][5], Helicobacter pylori [6], Pseudomonas aeruginosa [7], Bacteroides fragilis [8] and Acinetobacter baumannii [9], and the N-glycosylation pathways of Campylobacter jejuni [10][11][12], Haloferax volcanii [13] and Methanococcus voltae [14]. In the genus Neisseria, the O-glycosylation pathway ( Figure S1) has been delineated in the species gonorrhoeae [1,5], lactamica [15] and meningitidis [2,3]. The enzymes involved in these pathways have been characterized to various extents [1][2][3][4][15][16][17][18][19].…”

“…The enzymes involved in these pathways have been characterized to various extents [1][2][3][4][15][16][17][18][19]. For example, in Neisseria meningitidis, PglE has been shown to be a β1,4-GalT and pglE has been shown to be responsible for phase variation between tri-and disaccharide structures [5]. In Neisseria gonorrhoeae, enzyme activities, substrate specificities and steady state kinetics parameters have been determined [3].…”