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Infection and Immunity, June 2002, p. 2837-2845, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.2837-2845.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site

Jason E. Comer,¹ Mark A. Marshall,¹ Vincent J. Blanch,¹ Carolyn D. Deal,^2, and Peter Castric^1*

Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282,¹ Department of Bacterial Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910²

Received 12 November 2001/ Returned for modification 14 January 2002/ Accepted 18 March 2002

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.

Editor: V. J. DiRita

Present address: Division of Bacterial Products, CBER/FDA, Rockville, MD 20892.

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