Inhibitors and specificity of Pseudomonas aeruginosa LasA.
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.
Kessler
E
E
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
Safrin
M
M
Abrams
W R
WR
Rosenbloom
J
J
Ohman
D E
DE
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
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