Identification of neutralizing epitopes on Pseudomonas aeruginosa elastase and effects of cross-reactions on other thermolysin-like proteases.
Monoclonal antibodies (MAbs) to a Burkholderia (Pseudomonas) cepacia 36-kDa protease (PSCP) which neutralize PSCP and Pseudomonas aeruginosa elastase but not P. aeruginosa alkaline protease have been isolated (C. Kooi et al., Infect. Immun. 62:2811-2817, 1994). These MAbs, designated 36-6-6 and 36-6-8, react with N-chlorosuccinimide cleavage products of P. aeruginosa elastase, consistent with the recognition of a 13.9-kDa fragment which contains the active site. Overlapping 9-mer peptides that span this region were synthesized. Neutralizing MAbs to PSCP reacted strongly with two peptides (341HGFTEQNSG349 and 395RYM DQPSRD403). Peptide 341HGFTEQNSG349 overlaps the motif 337HEXXH341, which has been found in many zinc-dependent endopeptidases. Peptide 395RYMDQPSRD403 lies between E361, which binds a zinc atom, and H420, which acts as a proton donor at the active site. Polyclonal rabbit sera raised against these peptides reacted with elastase on Western immunoblots and by enzyme-linked immunosorbent assay. With hide powder azure as the substrate, antisera to either HGFTEQNG and RYMDQPSRD completely neutralized the activities of elastase, thermolysin, Vibrio cholerae hemagglutinin/protease, and PSCP but had no effect on P. aeruginosa alkaline protease or the Serratia marcescens major protease. These results suggest that the MAbs recognize two different epitopes on P. aeruginosa elastase and that antibodies raised against synthetic peptides corresponding to either of these epitopes neutralize proteolytic activity.
Kooi
C
C
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Alberta, Canada.
Hodges
R S
RS
Sokol
P A
PA
D000911:Antibodies, Monoclonal / Q000737:chemistry; D003429:Cross Reactions; D004789:Enzyme Activation / Q000276:immunology; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology*; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000276:immunology*; D013388:Succinimides / Q000494:pharmacology; D013820:Thermolysin / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D013388:Succinimides; C015752:N-chlorosuccinimide; D010196:Pancreatic Elastase; D013820:Thermolysin