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. 2012 Jan;80(1):22-30.
doi: 10.1128/IAI.05920-11. Epub 2011 Nov 14.

Novel protein substrates of the phospho-form modification system in Neisseria gonorrhoeae and their connection to O-linked protein glycosylation

Jan Haug Anonsen  1 , Wolfgang Egge-Jacobsen, Finn Erik Aas, Bente Børud, Michael Koomey, Ashild Vik
Affiliations

Novel protein substrates of the phospho-form modification system in Neisseria gonorrhoeae and their connection to O-linked protein glycosylation

Jan Haug Anonsen et al. Infect Immun. 2012 Jan.

Abstract

The zwitterionic phospho-form moieties phosphoethanolamine (PE) and phosphocholine (PC) are important components of bacterial membranes and cell surfaces. The major type IV pilus subunit protein of Neisseria gonorrhoeae, PilE, undergoes posttranslational modifications with these moieties via the activity of the pilin phospho-form transferase PptA. A number of observations relating to colocalization of phospho-form and O-linked glycan attachment sites in PilE suggested that these modifications might be either functionally or mechanistically linked or interact directly or indirectly. Moreover, it was unknown whether the phenomenon of phospho-form modification was solely dedicated to PilE or if other neisserial protein targets might exist. In light of these concerns, we screened for evidence of phospho-form modification on other membrane glycoproteins targeted by the broad-spectrum O-linked glycosylation system. In this way, two periplasmic lipoproteins, NGO1043 and NGO1237, were identified as substrates for PE addition. As seen previously for PilE, sites of PE modifications were clustered with those of glycan attachment. In the case of NGO1043, evidence for at least six serine phospho-form attachment sites was found, and further analyses revealed that at least two of these serines were also attachment sites for glycan. Finally, mutations altering glycosylation status led to the presence of pptA-dependent PC modifications on both proteins. Together, these results reinforce the associations established in PilE and provide evidence for dynamic interplay between phospho-form modification and O-linked glycosylation. The observations also suggest that phospho-form modifications likely contribute biologically at both intracellular and extracellular levels.

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Figures

Fig 1
Fig 1
Identification of glycan and phospho-form modifications of NGO1043. (A) The MS2 HCD spectrum of precursor peptide at m/z 1,117.98 [M + 2H]2+ confirmed that peptide 38DTAASAAESAASAVEEAK55 was modified with diNAcBac-acetylhexose and PE. The reporter oxonium ion of diNAcBac-acetylhexose at m/z 433.18 was detected. Moreover, the mass difference between the ions at m/z 1,801.77 [M + H]+ and m/z 1,660.75 [M + 1H]1+ represented a neutral loss of 141.02 Da, which corresponded to the loss of PE. MS analyses were performed on an LTQ OrbitrapXL mass spectrometer. (B) Immunoblots of purified NGO1043 from wild-type (wt), glycosylation-negative mutant (pglC), phospho-form transferase mutant (pptA), double mutant deficient for both PptA and PglC (pptA pglC), oligosaccharide transferase mutant (pglO), and minor pilin-like protein mutant (pilV) strains. Purified proteins were checked for reactivity against the TEPC-15 MAb (recognizing PC) and the npg-2 (recognizing the disaccharide glycan) and tetra-His antibodies.
Fig 2
Fig 2
Identification of PE modification sites in NGO1043 by MS2. MS analyses were performed on an LTQ OrbitrapXL mass spectrometer. (A) MS2 spectrum of the PE-modified peptide 27EAAQAVESDVK37 at m/z 635.29 [M + 2H]2+. A dehydroalanine (DHA) was detected at position y4 (m/z 430.23), identified by amino acid sequencing using the y-ion series from y2 to y7. Peptide 27EAAQAVESDVK37 was modified with a PE at serine 34. (B) MS2 spectrum of glycan- and PE-modified peptide 27EAAQAVESDVKDTAASAAESAASAVEEAK55 at m/z 1,107.50 [M + 3H]3+. A dehydroalanine (DHA) was detected at position y10 (m/z 944.46), by amino acid sequencing using the y-ion series from y7 to y10. Reporter ions for diNAcBac and diNAcBac-hexose were detected at m/z 229.12 and at m/z 391.17, respectively, confirming that peptide 27EAAQAVESDVKDTAASAAESAASAVEEAK55 was modified with a PE at serine 49 and a glycan. (C) Phospho-form occupancy sites on NGO1043. Phospho-form-modified peptides (orange rectangles) and modification sites (in red) are shown. Underlining of sites denotes serines modified with a PC in a pglC background. Residues are numbered according to the unprocessed protein. Asterisks denote phospho-form-modified peptides shown to be simultanously modified by a glycan (two asterisks) or phospho-form-modified serines shown to be alternatively modified by a glycan (one asterisk). The blue line denotes the lipoprotein processing (proc.) site.
Fig 3
Fig 3
Effect of glycan length on the PC modification status of NGO1043. Shown are affinity-purified NGO1043-6His from a glycosylation-negative mutant (pglC), a double mutant deficient for both phospho-form transferase PptA and PglC (pptA pglC), a mutant producing monosaccharide (pglA), a double mutant producing monosaccharide and deficient for PptA (pptA pglA), a wild-type (wt) background, a phospho-form transferase mutant (pptA), a mutant producing trisaccharide (pglEon), and a double pptA pglEon mutant immunoblotted with antibodies against tetra-His, npg-1 (recognizing the monosaccharide glycan), npg-2 (recognizing the disaccharide glycan), and npg-3 (recognizing the trisaccharide glycan), as well as the TEPC-15 MAb (recognizing PC).
Fig 4
Fig 4
Identification of phospho-form modification of NGO1237-6His. (A) NGO1237 was purified from a wild-type (wt) background, a glycosylation-negative mutant (pglC), a phospho-form transferase mutant (pptA), a double mutant deficient for both PptA and PglC (pptA pglC), an oligosaccharide transferase mutant (pglO), and a pilin-like protein mutant (pilV). All purified proteins were immunoblotted with the TEPC-15 MAb (recognizing PC) and antibodies against npg-2 (recognizing the disaccharide glycan) and tetra-His. (B) MS2 CID spectrum of the precursor peptide at m/z 1,063.3 [M + 3H]3+ confirmed that peptide 25DNSAAQAASSSASAPAAENAAKPQTRGT52 was modified with diNAcBac-acetylhexose and PE. The reporter oxonium ion of diNAcBac-acetylhexose disaccharide at m/z 433.0 could be detected. The charge-corrected difference between the ions at m/z 1,377.6 [M + 2H]2+ and m/z 1,306.9 [M + 2H]2+ represented the 141.02-Da neutral loss, indicative of a loss of PE. MS analyses were performed on an Agilent 1100 LC/MSD Trap XCT Ultra mass spectrometer. (C) Phospho-form modification site on NGO1237. The phospho-form-modified peptide was identified by MS2, and the location is indicated by an orange rectangle. Residues are numbered from unprocessed protein. Two asterisks denote that this is a phospho-form-modified peptide shown to be modified simultanously by a glycan.
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