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N-Glycosidase treatment with 18O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa

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Abstract

In prokaryote organisms, N-glycosylation of proteins is often correlated to cell–cell recognition and extracellular events. Those glycoproteins are potential targets for infection control. To date, many surface-glycosylated proteins from bacterial pathogens have been described. However, N-linked Pseudomonas surface-associated glycoproteins remain underexplored. We report a combined enrichment and labeling strategy to identify major glycoproteins on the outside of microorganisms. More precisely, bacteria were exposed to a mix of biotinylated lectins able to bind with glycoproteins. The latter were then recovered by avidin beads, digested with trypsin, and submitted to mass spectrometry. The targeted mixture of glycoproteins was additionally deglycosylated in the presence of H2 18O to incorporate 18O during PNGase F treatment and were also analyzed using mass spectrometry. This approach allowed us to identify a few tens of potential N-glycoproteins, among which flagellin FliC was the most abundant. To detect the possible sites of FliC modifications, a de novo sequencing step was also performed to discriminate between spontaneous deamidation and N-glycan loss. This approach led to the proposal of three potential N-glycosylated sites on the primary sequence of FliC: N26, N69, and N439, with two of these three asparagines belonging to an N-X-(S/T) consensus sequence. These observations suggest that flagellin FliC is a heterogeneous protein mixture containing both O- and N-glycoforms.

Analytical scenario developed for bacterial glycoprotein enrichment. This strategy includes three main steps: (1) exposure of Pseudomonas aeruginosa cells to a mixture of biotinylated lectins [wheat germ agglutinin (WGA) and concanavalin A (ConA)]; (2) enrichment of N-glycoproteins by elution with avidin beads; and (3) mass spectrometry (MS) identification and characterization of intact and deglycosylated peptides before and after H2 18O PNGase F enzymatic treatment, respectively

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Abbreviations

ConA:

Concanavalin A

HPLC:

High-performance liquid chromatography

MS:

Mass spectrometry

PTM:

Posttranslational modification

WGA:

Wheat germ agglutinin

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Acknowledgments

These investigations were made possible thanks to FEDER funding (program number 33267). A.K. is the recipient of a postdoctoral fellowship endorsed within an ANR program (ANR-BLANC-732-01 grant). The authors also thank F. Jarnier for help in implementation of de novo sequencing.

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Authors and Affiliations

  1. CNRS, UMR 6270, Laboratory “Polymères, Biopolymères, Surfaces”, 76820, Mont-Saint-Aignan, France

    Arbia Khemiri, Bertrand Naudin, Thierry Jouenne & Pascal Cosette

  2. Normandy University, University of Rouen, 76821, Mont Saint Aignan Cedex, France

    Arbia Khemiri, Bertrand Naudin, Xavier Franck, Philippe Chan Tchi Song, Thierry Jouenne & Pascal Cosette

  3. PISSARO Proteomic Facility, IRIB, 76820, Mont-Saint-Aignan, France

    Arbia Khemiri, Bertrand Naudin, Philippe Chan Tchi Song, Thierry Jouenne & Pascal Cosette

  4. CNRS, UMR6014, Laboratory COBRA, 76820, Mont-Saint-Aignan, France

    Xavier Franck

  5. UMR6270 CNRS - PISSARO Proteomics Platform, University of Rouen, 76821, Mont Saint Aignan Cedex, France

    Pascal Cosette

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  1. Arbia Khemiri
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Correspondence to Pascal Cosette.

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Khemiri, A., Naudin, B., Franck, X. et al. N-Glycosidase treatment with 18O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa . Anal Bioanal Chem 405, 9835–9842 (2013). https://doi.org/10.1007/s00216-013-7424-x

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