Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae

Abstract

Protein glycosylation is an important element of biologic systems because of its significant effects on protein properties and functions. Although prominent within all domains of life, O-linked glycosylation systems modifying serine and threonine residues within bacteria and eukaryotes differ substantially in target protein selectivity. In particular, well-characterized bacterial systems have been invariably dedicated to modification of individual proteins or related subsets thereof. Here we characterize a general O-linked glycosylation system that targets structurally and functionally diverse groups of membrane-associated proteins in the gram-negative bacterium Neisseria gonorrhoeae, the etiologic agent of the human disease gonorrhea. The 11 glycoproteins identified here are implicated in activities as varied as protein folding, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration. Along with their common trafficking within the periplasmic compartment, the protein substrates share quasi-related domains bearing signatures of low complexity that were demonstrated to encompass sites of glycan occupancy. Thus, as in eukaryotes, the broad scope of this system is dictated by the relaxed specificity of the glycan transferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus sequence. Together, these findings reveal previously unrecognized commonalities linking O-linked protein glycosylation in distantly related life forms.

Fig. 1.

Evidence for multiple Ngo glycoproteins revealed via detection of glycan-associated epitopes. Immunoblotting of whole-cell lysates with rabbit serum (termed antiglycan) raised against purified PilE bearing the O-AcHexDATDH glycan reacts with multiple proteins in a pgl pathway–dependent fashion (see Fig. S1 for an overview of the pgl pathway). Note that the lack of immunoreactivity observed in the pglA and pglE_on backgrounds is not associated with a lack of glycosylation but rather the presence of monosaccharide DATDH and trisaccharide O-AcHexHexDATDH glycans, respectively.

Fig. 2.

Affinity purification confirms the identities of candidate Ngo glycoproteins. (A–D) C-terminally 6xHis-tagged candidate proteins were affinity purified from wild-type (wt) and pgl backgrounds and tested for glycan-associated antigenicity by immunoblotting. (B) For Ng1371, a mutant form of the protein carrying a glycine substitution at Ser-337 was also purified and tested, whereas for Ng1717 (C), the Ng1548 paralogue (Fig. S3) was examined in parallel. (D) Ng1225 was identified as a potential glycoprotein on the basis of its lipoprotein processing site and proximal ASP-rich LCR (Table S1).

Fig. 3.

Identification of glycopeptides derived from endoproteolytic cleavage of affinity-purified proteins using tandem MS. CID experiments of the glycopeptide-related molecular ions yield mainly cleavage at glycosidic bonds, revealing characteristic oxonium ions at m/z 433 and m/z 229 (corresponding to O-AcHexDATDH and DATDH glycans, respectively). Diamonds denote the m/z of the precursor ion fragmented in the spectrum. Superscript numbering appended to ORF designations indicates peptides bearing 1 (¹) or 2 (²) glycans. The asterisk marks the unmodified peptide after loss of the glycan moiety resulting from CID. (A) MS/MS spectrum of [M + 2H] ²⁺ at m/z 733.2 derived from Ng1328 by AspN cleavage. (B) MS/MS spectrum of [M + 3H] ³⁺ at m/z 1134.2 derived from Ng1371 by tryptic cleavage. (C) MS/MS spectrum of [M + 4H] ⁴⁺ at m/z 911.6 derived from Ng1276 by tryptic cleavage. (D) MS/MS spectrum of [M + 4H] ⁴⁺ at m/z 1022.1 derived from Ng1237 by AspN cleavage. (E) MS/MS spectrum of [M + 3H] ³⁺ at m/z 1038.9 derived from Ng2139 by tryptic cleavage. (F) MS/MS spectrum of [M + 2H] ²⁺ at m/z 824.6 derived from Ng1717 by proteinase K cleavage. (G) MS/MS spectra of [M + 2H] ²⁺ at m/z 619.5 derived from Ng1043 by tryptic cleavage. (H) MS/MS spectra of [M + 2H] ²⁺ at m/z 1272.7 derived from Ng1043 by tryptic cleavage.

Fig. 4.

Identification of Ser-173 of Ng1328 as a glycan acceptor site using ETD. Fragmentation of the doubly charged peptide at m/z 733.2 shows the site of O-AcHexDATDH modification by detection of fragment ions c₉⁺ and c₁₁⁺ at m/z 1224.6 and 1392.7, respectively. These represent the only c-ions with a mass increase of 432 Da, which corresponds to the glycan moiety.

Fig. 5.

Domain organization and structural architecture of Ngo glycoproteins. (A) Glycoproteins for which glycopeptides have been identified by MS/MS. Regions encompassed by glycopeptides are indicated by yellow rectangles, and specific residues are numbered according to those of unprocessed polypeptides. Serines identified as acceptor sites (via ETD, substitution mutation, or by virtue of being the sole hydroxyl-bearing residue) are labeled in red. Asterisks denote glycopeptides bearing multiple glycan moieties. (B) Glycoproteins for which glycopeptides have yet to be identified. Sequences of the LCRs shown for these proteins are found in Table S1. Further details and specific Pfam family identifiers are found in Table 1.