Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation

Abstract

Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated in Neisseria meningitidis, Neisseria gonorrhoeae, and some strains of Pseudomonas aeruginosa. In this work, we characterized the P. aeruginosa 1244 and N. meningitidis MC58 O glycosylation systems in Escherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL in N. meningitidis and PilO in P. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of "O-OTases" that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized in E. coli.

FIG. 1.

Reconstitution of P. aeruginosa 1244 O glycosylation system in E. coli. (A) Western blot assay of whole-cell extracts containing unglycosylated and glycosylated P. aeruginosa (PA) 1244 pilin. Pilin was detected by using polyclonal anti-pilin antibody. Lanes: 1, P. aeruginosa 1244 expressing glycosylated pilin containing its endogenous trisaccharide; 2, P. aeruginosa 1244 pilO derivative expressing unglycosylated pilin; 3, E. coli CLM24 with pPAC46 (containing pilA-pilO); 4, E. coli CLM24 with pPAC46 and pACYCpglB_mut, which carries all the enzymes required for the assembly of the C. jejuni glycan except for PglB. WT, wild type. (B) Glycosylated P. aeruginosa pilin is recognized by the C. jejuni glycan-specific antiserum R12 (left panel) and the SBA lectin (right panel). SBA binds to GalNAc, the most abundant sugar of the C. jejuni glycan. Only lanes 3 and 4 of panel A show results of probes with antibody. (C) Whole-cell lysates of E. coli CLM24 transformed with pACYCpglB_mut, pAMF1 (expressing P. aeruginosa 1244 pilin), and pDOM3 (expressing PilO under an arabinose-dependent promoter) were analyzed by Western blotting (right panels). Extracts of cells carrying pMLBAD (empty vector) instead of pDOM3 were included as a negative control (left panels). PilA was detected by using anti-pilin polyclonal antibody (upper panels), and glycosylated pilin was detected by R12 antibody (lower panels). The arabinose concentrations (conc.) used were 0, 0.0002, 0.002, 0.02, and 0.2%. Plasmids are described in Table 1.

FIG. 2.

MS/MS analysis shows glycosylation of recombinant P. aeruginosa pilin with the C. jejuni glycan at Ser148 in E. coli. MS/MS analysis of two glycopeptides matching the predicted doubly charged peptide ¹⁴⁴NCPKS¹⁴⁸ modified with acrylamide and containing the glycan DATDH(HexNAc)₅Hex (m/z 1,012.8²⁺) (upper panel) or (HexNAc)₆Hex (m/z 1,000.3²⁺) (lower panel). The fragmentation patterns of the glycopeptides are shown in the insets. The assignment of the hexoses is based on the known structure of the C. jejuni glycan (47). DATDH is 2,4-diacetimido-2,4,6-trideoxyhexopyranose. C^a represents acrylamide attached to Cys.

FIG. 3.

Reconstitution of N. meningitidis (MC) MC58 O glycosylation system in E. coli. (A) Western blot assay of whole-cell E. coli CLM24 extracts producing unglycosylated and glycosylated MC pilin. Pilin was detected by the SM1 anti-pilin monoclonal antibody (upper panel) or the C. jejuni glycan antiserum R12 (lower panel). Lanes: 1, pAMF3 (expressing MC pilin) and pAMF5 (expressing PglL); 2, pAMF3, pACYCpglB_mut, and pEXT22 (cloning vector); 3, pAMF3, pACYCpglB_mut, and pAMF5. (B) Results of analysis similar to that for panel A, showing the effects of mutations S63A and T62A on pilin glycosylation. Plasmid details are presented in Table 1. NM, N. meningitidis.

FIG. 4.

MS/MS analysis shows glycosylation of recombinant MC pilin with the C. jejuni glycan in E. coli. (A) MS/MS spectrum of doubly charged ion at m/z 1,430.0²⁺, corresponding to the glycopeptide ⁵⁹LNHGEWPGNNTSAG⁷² modified with DATDH(HexNAc)₅Hex resulted from thermolysin digestion. (B) MS/MS spectrum of a doublely charged glycopeptide ion at m/z 905.8²⁺, corresponding to DATDH(HexNAc)5Hex attached to peptide ⁶³SAGVA⁶⁷ resulted from proteinase K digestion of MC pilin. The fragmentation patterns of the glycopeptides are shown in the insets. As shown, the common peptide fragment ions (y and b) are observed in addition to the sugar fragments and the peptide with sugar fragments. The assignment of the hexoses is based on the known structure of the C. jejuni glycan (47).

FIG. 5.

Translocation of lipid-linked oligosaccharides to the periplasm is a prerequisite for glycosylation. Glycosylation of MC and P. aeruginosa pilins occurs only in the presence (+) of a functional flippase, either Wzx (lanes 1), a pgl-encoded PglK (lanes 3), or a PglK encoded in trans (lanes 4). Cell extracts were analyzed by Western blotting by using antibodies directed against P. aeruginosa and MC pilins (upper panels, left and right, respectively) and the glycan specific antiserum R12 (lower panels). Left panels, CLM24 containing pPAC46 and pACYCpglB_mut (lanes 1); SCM7 transformed with plasmid pPAC46 and pACYCpglK_mut (lanes 2); SCM7 containing pPAC46 and pACYCpgl (lanes 3); SCM7 transformed with pPAC46, pACYCpglK_mut, and pCW27, expressing PglK in trans (lanes 4), and SCM7 transformed with pACYCpglK_mut and pMLBAD (cloning vector) (lanes 5). Right panels, same as left panels, except for plasmids pAMF5 and pAMF6 expressing MC pilin and PglL, respectively, instead of pPAC46. Details of the strains and plasmids are presented in Table 1. The arrows indicate the presence of LPS containing the C. jejuni oligosaccharide in the strains where a functional WaaL (ligase) and a flippase are present. −, absence of active protein.

FIG. 6.

PglL, but not PilO, transfers polysaccharides to pilin. (A) Lectin blot analysis of three different forms of E. coli O7 LPS produced in E. coli SΦ874. A lectin specific for rhamnose, one of the sugars of the O7 antigen, has been used: wild type (WT) (lane 1); wzy (polymerase) mutant (lane 2), and wzz (chain length regulator) mutant (lane 3). The numbers on the right indicate the numbers of O7-repeating units attached to lipid A-core. (B) The ability of PglL (left panel) and PilO (right panel) to transfer O7 antigen of different lengths to their respective pilins in the E. coli SCM3 strain (ligase-deficient derivative of SΦ874) was analyzed by Western blotting. MC pilins containing the three O antigen versions shown in panel A were detected by using the anti-MC pilin monoclonal antibody. PglL was able to transfer fully polymerized O7 antigen to MC pilin (lane 2). P. aeruginosa pilin containing O antigen of up to only two repeating units was detected in the wzz mutant strain (lane 8), despite the observation that O antigen containing two or more repeating units is equally abundant (panel A, lane 3). Lanes 1 to 4: pAMF5 (expressing PglL) and pAMF6 (expressing MC pilin). Additionally, lane 2 contains pJHCV32 (wild-type O7 antigen), lane 3 contains pJHCV32-134 (O7 wzy mutant), and lane 4 contains pJHCV32-136 (O7 wzz mutant). Lanes 5 to 8, pPAC46 (expressing P. aeruginosa pilin and PilO). Additionally, lane 6 contains pJHCV32, lane 7 contains pJHCV32-134, and lane 8 contains pJHCV32-136. (C) O7 antigen from the wzz mutant strain is not transferred to the S63A variant of MC pilin (lane 2). Glycosylation is not affected in the mutant T62A (lane 1). Unglycosylated (lane 3) and wild-type pilin glycosylated with the O7 antigen (lane 4) are included for comparison.