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 Zn 2؉ 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 N ␣ -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 ␤-casein, but the same cleavage patterns were observed with column fractions containing little or no LasA. The ␤-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-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 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 ␤-casein appear to be resistant to LasA.

Peptidoglycan Amidase MepA Is a LAS Metallopeptidase

“…This turned out to be the case. Zn 2ϩ at 10 mM concentration was also inhibitory to the enzyme, consistent with previous reports about the inhibition of zinc-dependent metallopeptidases by unphysiologically high concentrations of Zn 2ϩ (9,25) (Fig. 1B).…”

Hydrolysis of glycine‐containing elastin pentapeptides by LasA, a metalloelastase from Pseudomonas aeruginosa

“…All four possess an internal GGX sequence (X being alanine or glycine) and are hydrolysed between glycine and X. This result is in agreement with the previous conclusions of Kessler et al [7]. We must also mention that for these four peptides, the catalytic ratio is higher than for pentaglycine, the reference substrate for staphylolytic activity (k cat / K M 6.5^0.4 min 21´m mol 21´L21 ).…”

A Secreted Aminopeptidase of Pseudomonas aeruginosa

“…Protein Sequencing-Proteins were separated on 10% SDS-polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp.) as previously described (9). After staining with Coomassie Blue, protein bands of interest were excised from the membrane, and N-terminal sequences were determined by automated Edman degradation on an Applied Biosystems 490 protein sequencing system.…”

“…LasA protease (staphylolysin) (7) has high staphylolytic activity that results from cleavages of the pentaglycine cross-linkages within the peptidoglycan of Staphylococcus aureus cells (8). LasA protease can also nick elastin at certain Gly-Gly sequences (9,10), an activity that increases the susceptibility of elastin to other proteases and contributes to the elastinolytic potential of P. aeruginosa (9,11,12). The fourth endopeptidase secreted by P. aeruginosa (lysine-specific endopeptidase; protease IV) cleaves peptide bonds on the carboxyl side of lysine residues in peptides and proteins and can act on a number of host proteins including complement components, IgG, and fibrinogen (13)(14)(15).…”