Antigen-antibody interactions: elucidation of the epitope and strain-specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K.
The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain-specific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain-specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.
Wong
W Y
WY
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Irvin
R T
RT
Paranchych
W
W
Hodges
R S
RS
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D001665:Binding Sites; D001667:Binding, Competitive; D004563:Electrochemistry; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000276:immunology; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D013045:Species Specificity; D014867:Water / Q000737:chemistry
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D014867:Water; D036781:Fimbriae Proteins