Identification of the Pseudomonas aeruginosa 1244 pilin glycosylation site.
Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.
Comer
Jason E
JE
Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282, USA.
Marshall
Mark A
MA
Blanch
Vincent J
VJ
Deal
Carolyn D
CD
Castric
Peter
P
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology; D036781:Fimbriae Proteins; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D018933:Pili, Sex / Q000276:immunology*; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000276:immunology*; D014312:Trisaccharides / Q000276:immunology*
D001425:Bacterial Outer Membrane Proteins; D018985:Epitopes, B-Lymphocyte; D008565:Membrane Proteins; D011498:Protein Precursors; D014312:Trisaccharides; D036781:Fimbriae Proteins