N-glycosidase treatment with 18O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa.
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(18)O to incorporate (18)O 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.
CNRS, UMR 6270, Laboratory "Polymères, Biopolymères, Surfaces", 76820, Mont-Saint-Aignan, France.
Arbia
A
Khemiri
Bertrand
B
Naudin
Xavier
X
Franck
Philippe Chan Tchi
PC
Song
Thierry
T
Jouenne
Pascal
P
Cosette
D000595:Amino Acid Sequence; D005408:Flagellin / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010104:Oxygen Radioisotopes; D010446:Peptide Fragments / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D011110:Polymorphism, Genetic / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
D006023:Glycoproteins; D010104:Oxygen Radioisotopes; D010446:Peptide Fragments; D005408:Flagellin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase