Differential glycosylation of polar and lateral flagellins in Aeromonas hydrophila AH-3

Abstract

Polar and lateral flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be glycosylated with different carbohydrate moieties. The lateral flagellin was modified at three sites in O-linkage, with a single monosaccharide of 376 Da, which we show to be a pseudaminic acid derivative. The polar flagellin was modified with a heterogeneous glycan, comprised of a heptasaccharide, linked through the same 376-Da sugar to the protein backbone, also in O-linkage. In-frame deletion mutants of pseudaminic acid biosynthetic genes pseB and pseF homologues resulted in abolition of polar and lateral flagellar formation by posttranscriptional regulation of the flagellins, which was restored by complementation with wild type pseB or F homologues or Campylobacter pseB and F.

FIGURE 1.

SDS-PAGE of purified flagellins. 1, polar flagellins. 2, lateral flagellin. St, size standard.

FIGURE 2.

MS/MS analysis of modified tryptic peptides from lateral flagellar protein of Aeromonas hydrophila AH-3. a, nLC-MS/MS spectrum of the doubly protonated glycopeptide ion at m/z 870.4 from lateral flagellin. Visible were peptide fragment ions of low intensity, giving the peptide sequence ¹⁷⁰TVTSVNTAISTASAAA¹⁸⁴. Also observed was a daughter ion corresponding to the singly charged unmodified peptide ion (m/z 1363.7). The observed mass excess was 376 Da, with a corresponding intense oxonium ion visible at m/z 377, with ion corresponding to likely loss of water at m/z 359. b, protein sequence coverage from mass spectrometry analyses of flagellin protein is shown. Indicated in boldface type are unmodified peptides, boldface type and underlined letters indicate peptides modified with glycan. Of note, de novo sequencing of flagellin peptides showed the amino acid at position 80 to be serine, not glycine as predicted from the gene sequence. c, doubly charged glycopeptide ion at m/z 870.2 was targeted for MS/MS with alternating collision energies of 20 and 24 V in CID mode. The combined spectrum results in a typical y and b ion series as well as y and b ions harboring the intact 376-Da glycan. The singly charged ion at m/z 954.3 (y7 + glycan) and the singly charged ion at m/z 1249.3 (b9 + glycan) suggests that the glycan is attached in O-linkage and that serine 178 is the modified residue. , 376-Da sugar.

FIGURE 3.

MS/MS analysis of modified tryptic peptides from polar flagellar protein of A. hydrophila AH-3. a, nLC-MS/MS spectrum of the triply protonated glycopeptide ion at m/z 1060.7 from polar flagellin. Visible were peptide fragment ions of low intensity, giving the peptide sequence ¹⁶⁰TMTSAFTISGIASSTK¹⁷⁴. Also observed was a daughter ion corresponding to the singly charged unmodified peptide ion (m/z 1501.1). The observed mass excess was 1678 Da, and a putative glycan oxonium ion was observed at m/z 1679. Indicated in the MS/MS spectrum are a series of neural losses from the glycan oxonium ion, giving a monosaccharide sequence on 376-162-162-203-297-377-102. It is likely that masses of 203 and 162 Da correspond to N-acetylhexosamine and hexose, respectively. Dominating the low mass region of the peptide MS/MS spectrum were glycan-related ions, at m/z 377 and 359. In addition, a doubly charged ion series was also observed, corresponding to glycopeptide fragments. b, protein sequence coverage from mass spectrometry analyses of flagellin proteins FlaA and FlaB are shown. Indicated in boldface type are unmodified peptides; boldface and underlined type indicates peptide modified with glycan. c, the triply charged glycopeptide ion at m/z 1060.7 was targeted for ETD with a reaction time of 300 s. In the resulting spectrum, the singly charged ion at m/z 1928.2 (c2 + glycan) along with ions corresponding to c3, c5, c6, c7, c8, c10, c11, c12, c14 all with the glycan still attached suggest that the peptide is modified at the threonine in position 161 of the protein sequence in O-linkage. , 376-Da sugar; ●, hexose; ■, HexNAc; ▴, 102-Da unknown moiety; •, phosphorylation; ■, methylation.

FIGURE 4.

Polar flagellin glycopeptide heterogeneity. nLC-MS spectrum of the flagellin tryptic digest at 12.8–13.4 min of elution, showing clusters of multiply charged ions. Further targeted analyses showed these to be flagellin glycopeptide ions as annotated. The glycan chains range from a single 376-Da modification to a 1772-Da heptasaccharide chain, including variably phosphorylated and methylated HexNAc residues. , 376-Da sugar; ●, hexose; ■, HexNAc; ▴, 102-Da unknown moiety; •, phosphorylation; ■, methylation.

FIGURE 5.

Electrospray mass spectrometry of intact lateral flagellin protein from Aeromonas hydrophila AH-3. a, electrospray mass spectrum of intact lateral flagellin protein, showing an envelope of multiply charged protein ions. b, the reconstructed molecular mass profile of lateral flagellin, showing masses at 29,703, 30,424, 30,819, and 30,899 Da. This corresponds in total to three sites of modification with a 376-Da glycan moiety. c, tandem mass spectrometry analysis of the multiply charged protein ion at m/z 1186. A peptide type y ion fragmentation series was observed, the sequence of which corresponded to the C-terminal peptide of the lateral flagellin LSNSNQMTGMVTGLLR. In addition, several ions were observed that did not correspond to peptide fragment ions, observed at m/z 377, with likely daughter ion at 359 due to loss of water. , 376-Da sugar.

FIGURE 6.

Electrospray mass spectrometry of intact polar flagellin protein from Aeromonas hydrophila AH-3. a, electrospray mass spectrum of intact polar flagellin protein showing a complex envelope of multiply charged protein ions. b, the reconstructed molecular mass profile of the polar flagellin is not as well resolved as the lateral flagellin due to the increased complexity, but does clearly show masses at 35,019, 36,685, 38,376, 40,149, and 41,712 Da. This seems to correspond to FlaB, modified with two, three, four, five, and six heptasaccharide glycan chains, ranging in mass from 1563 to 1773 Da. Similar glycan heterogeneity is also observed in the nLC-MS/MS analysis of tryptic digests. c, feCID of polar flagellin, generated by increasing the cone voltage of the QTOF2 mass spectrometer to 85 V. Clear glycan related oxonium ions are present in the low m/z region, resulting from nonspecific fragmentation of the intact flagellin glycoprotein. , 376-Da sugar; ■ = HexNAc.

FIGURE 7.

Glycan structural characterization by mass spectrometry. Tandem mass spectrum of singly charged ion at m/z 377 following feCID of intact flagellin protein showed consecutive losses of water and an acetyl group from the parent ion giving rise to an ion at m/z 317. Subsequent losses from this ion gave rise to a fragmentation pattern similar to that observed with nonulosonic acid sugars. Daughters ions marked with an asterisk denote those fragment ions found in MS/MS spectra of pseudaminic acid. , 376-Da sugar; *, fragment ions common to pseudaminic acid; Ac, acetyl group.

FIGURE 8.

Western blots using specific antiserum against purified polar flagellins. Molecular weights are indicated. a, cytoplasmic fractions of strains: 1, A. hydrophila AH-3 (wild type); 2, AH-3ΔPseB mutant; 3, AH-3ΔPseF mutant; 4, AH-3ΔPseB mutant complemented with pBAD-PseB; and 5, AH-3ΔPseB mutant complemented with pBAD-PseB_Cj. b, A. hydrophila AH-3 (wild type). C, cytoplasmic fraction; M, whole membrane fraction. The fractions were separated as described under “Experimental Procedures.”