title: Inhibitors and specificity of Pseudomonas aeruginosa LasA.
abstract: LasA is an extracellular protease of Pseudomonas aeruginosa that enhances the elastolytic activity of Pseudomonas elastase and other proteases by cleaving elastin at unknown sites. LasA is also a staphylolytic protease, an enzyme that lyses Staphylococcus aureus cells by cleaving the peptidoglycan pentaglycine interpeptides. Here we showed that the staphylolytic activity of LasA is inhibited by tetraethylenepentamine and 1,10-phenanthroline (zinc chelators) as well as excess Zn2+ and dithiothreitol. However, LasA was not inhibited by several serine or cysteine proteinase inhibitors including diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, leupeptin, and N-ethylmaleimide. LasA staphylolytic activity was also insensitive to Nalpha-p-tosyl-L-lysine chloromethyl ketone or phosphoramidon. EDTA and EGTA were inhibitory only at concentrations greater than 20 mM. Without added inhibitors, LasA obtained by DEAE-cellulose fractionation was active toward beta-casein, but the same cleavage patterns were observed with column fractions containing little or no LasA. The beta-casein cleaving activity was fully blocked in the presence of inhibitors that did not affect staphylolytic activity. In the presence of such inhibitors, purified LasA was inactive toward acetyl-Ala4 and benzyloxycarbonyl-Gly-Pro-Gly-Gly-Pro-Ala, but it degraded soluble recombinant human elastin as well as insoluble elastin. N-terminal amino acid sequencing of two fragments derived from soluble elastin indicated that both resulted from cleavages of Gly-Ala peptide bonds located within similar sequences, Pro-Gly-Val-Gly-Gly-Ala-Xaa (where Xaa is Phe or Gly). In addition, Ala was identified as the predominant N-terminal residue in fragments released by LasA from insoluble elastin. A dose-dependence study of elastase stimulation by LasA indicated that a high molar ratio of LasA to elastase was required for significant enhancement of elastolysis. The present results suggest that LasA is a zinc metalloendopeptidase selective for Gly-Ala peptide bonds within Gly-Gly-Ala sequences in elastin. Substrates that contain no Gly-Gly peptide bonds such as beta-casein appear to be resistant to LasA.
authors.affiliation: Maurice and Gabriela Goldschleger Eye Research Institute, Tel-Aviv University Sackler Faculty of Medicine, Sheba Medical Center, Tel-Hashomer 52621, Israel. ekessler@ccsg.tau.ac.il
authors.forename: E
authors.initials: E
authors.lastname: Kessler
authors.forename: M
authors.initials: M
authors.lastname: Safrin
authors.forename: W R
authors.initials: WR
authors.lastname: Abrams
authors.forename: J
authors.initials: J
authors.lastname: Rosenbloom
authors.forename: D E
authors.initials: DE
authors.lastname: Ohman
mesh_terms: D001426:Bacterial Proteins; D002364:Caseins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004549:Elastin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005029:Ethylenediamines / Q000494:pharmacology; D005998:Glycine; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D002614:Chelating Agents; D005029:Ethylenediamines; D010446:Peptide Fragments; D010618:Phenanthrolines; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D004549:Elastin; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C066371:staphylolytic protease; D015032:Zinc; D004229:Dithiothreitol; D005998:Glycine; C025205:1,10-phenanthroline; C034269:tetraethylenepentamine