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.2015 Nov 23;10(11):e0143362.
doi: 10.1371/journal.pone.0143362. eCollection 2015.

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide

Affiliations

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide

James M Daubenspeck et al. PLoS One..

Abstract

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The effect of substrate on glycoconjugate synthesis byM.pulmonis andM.pneumoniae.
Panels A and B show the relative abundance of glucose or xylose, respectively, linked to protein as determined by GC. The values represent averages of the areas under the curves from 3 replicates of gas chromatograms that were converted to μg of sugar per mg of protein. The colors of the bars representing the different substrates used to supplement the medium are shown on the right. Plus or minus standard error bars are shown and the asterisks indicate a significant difference between a sample and control.
Fig 2
Fig 2. SDS PAGE of TX-114 fractions ofM.pulmonis and M.pneumoniae.
Panel B shows a glycostained gel, and panel A is a Coomassie-stained gel run in parallel.M.pulmonis bands excised for further analysis by HR-MS are indicated by arrows with the designation of the genes coding for the identified proteins. The unlabeled top band was analyzed by HR-MS and while there were several spectra that suggested glycosylation, none met our criteria for publication.
Fig 3
Fig 3. Hexosylation of the peptide GTKDFLPIELQSLEVSK of MYPU_3230.
Orbitrap MS showing the doubly and triply charged ions. The 81.0262 shift forz = 2 between the non-glycosylated and glycosylated peptides equates to a mass shift of 162.0524 Da, which corresponds to the addition of hexose (162.0528 Da) with a mass accuracy of 0.0004 Da. The 54.0169 shift forz = 3 between non-glycosylated and glycosylated forms equates to a mass shift of 162.0507 Da, which corresponds to hexosylation with a mass accuracy of 0.0021 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.
Fig 4
Fig 4. LC MS/MS-CID showing hexosylation at Thr64 of the peptide Gt64KDFLPIELQSLEVSK of MYPU_3230.
The assigned b and y ions are shown in blue and red, respectively. Glycosylation of Q and S glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 87. The charge state of the parental ion wasz = 3.
Fig 5
Fig 5. Hexosylation of the peptide STLEYTINNSQELQNILKQTYEEFTK of MYPU_3200.
Orbitrap MS showing the doubly and triply charged ions. The monoisotopic mass of the doubly charged species at 1648.8077 is consistent with the hexosylated peptide atz = 2 with a mass accuracy of 0.0012 Da. The 54.0175 shift forz = 3 between non-glycosylated and hexose forms equates to a mass shift of 162.0525 Da with a mass accuracy of 0.0003 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.
Fig 6
Fig 6. LC MS/MS-CID showing hexosylation at Asn335 of the peptide STLEYTINNSQELQn335ILKQTYEEFTK of MYPU_3200.
The assigned b and y ions are shown in blue and red, respectively. Glycosylation of N, Q, T, S, and Y glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 60. The charge state of the parental ion wasz = 3.
Fig 7
Fig 7. Hexosylation of the peptide ITDLLSQKEVTETQK of MYPU_3460.
Orbitrap MS showing the doubly and triply charged ions. The 81.027 shift forz = 2 between the non-glycosylated and glycosylated peptides equates to a mass shift of 162.054 Da, which corresponds to the addition of hexose (162.0528 Da) with a mass accuracy of 0.0012 Da. The 54.0177 shift forz = 3 between non-glycosylated and glycosylated forms equates to a mass shift of 162.0531 Da, which corresponds to hexosylation with a mass accuracy of 0.0003 Da. The calculated theoretical and experimental values form/z are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.
Fig 8
Fig 8. LC MS/MS-CID showing hexosylation at Gln49 of the peptide ITDLLSq49KEVTETQK of MYPU_3460.
The assigned b and y ions are shown in blue and red, respectively. Glycosylation of Q, S, and T glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 74. The charge state of the parental ion wasz = 3.
See this image and copyright information in PMC

References

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