Further studies on Pseudomonas aeruginosa LasA: analysis of specificity

Abstract

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.

Inhibitors and Specificity of Pseudomonas aeruginosa LasA

“…LasA has been reported to cleave ␤-casein at the Lys 29 -Ile 30 peptide bond (14). This cleavage seemed inconsistent with the apparent affinity of LasA for Gly-Gly peptide bonds.…”

“…␤-Casein Is Resistant to LasA-LasA has been reported to cleave ␤-casein into two distinct fragments by cleavage at the Lys 29 -Ile 30 peptide bond (14,17). Our LasA preparation, which eluted as fraction I on a DEAE-cellulose column (16), initially exhibited a similar activity toward ␤-casein (Fig.…”

“…and TLCK) had no effect on the staphylolytic activity of LasA, and 1,10-phenanthroline, an inhibitor of LasA staphylolytic activity, did not block ␤-casein degradation (17). Hence, LasA was proposed to be a "modified" serine protease (14,17).…”

“…Peters and Galloway (11) isolated a 22-kDa LasA fragment from culture filtrates of P. aeruginosa that enhanced elastolytic activity of Pseudomonas elastase as well as that of other proteases. Subsequently, elastase-negative mutants of P. aeruginosa were found to retain some elastolytic activity (12,13), and the purified LasA fragment was shown to possess basal elastolytic activity (14). The 40-kDa product of lasA was identified as a precursor of the extracellular LasA protein (15), and because of its action on elastin, the secreted 22-kDa form of LasA has been referred to as a second elastase (12)(13)(14).…”

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

“…Synthesized as a 42-kDa protein, LasA is processed to a 21-kDa active peptide which has both elastolytic and staphylolytic activities [5]. In addition, it substantially enhances elastolytic activities of several proteases, including pseudolysin and human leukocyte elastase [6]. LasA was demonstrated to hydrolyse pentaglycine into di-and triglycine [5].…”