INHIBITORS AND SPECIFICITY OF PSEUDOMONAS-AERUGINOSA LASA

LasA is an extracellular protease of Pseudomonas aeruginosa that enhan ces the clastolytic activity of Pseudomonas elastase and other proteas es 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 d ithiothreitol. However, LasA was not inhibited by several serine or cy steine proteinase inhibitors including diisopropyl fluorophosphate, ph enylmethylsulfonyl fluoride, leupeptin, and N-ethylmaleimide. LasA sta phylolytic activity was also insensitive to N-alpha-p-tosyl-L-lysine c hloromethyl ketone or phosphoramidon. EDTA and EGTA were inhibitory on ly at concentrations greater than 20 mM. Without added inhibitors, Las A obtained by DEAE cellulose fractionation was active to ward beta-cas ein, but the same cleavage patterns were observed with column fraction s containing little or no LasA. The beta-casein cleaving activity was fully blocked in the presence of inhibitors that did not affect staphy lolytic activity. In the presence of such inhibitors, purified LasA wa s inactive toward acetyl-Ala(4) 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 d erived from soluble elastin indicated that both resulted from cleavage s of Gly-Ala peptide bonds located within similar sequences, Pro-Gly-V al-Gly-Gly-Ala-Xaa (where Xaa is Phe or Gly). In addition, Ala was ide ntified as the predominant N-terminal residue in fragments released by LasA from insoluble elastin. A dose-dependence study of elastase stim ulation by LasA indicated that a high molar ratio of LasA to elastase was required for significant enhancement of elastolysis. The present r esults suggest that LasA is a zinc metalloendopeptidase selective for Gly-Ala peptide bonds within Gly-Gly-Ala sequences in elastin. Substra tes that contain no Gly-Gly peptide bonds such as beta-casein appear t o be resistant to LasA.