Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity

Peters, J. E.; Galloway, Darrell R.

doi:10.1128/jb.172.5.2236-2240.1990

Cited by 78 publications

(57 citation statements)

References 27 publications

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“…Although purified LasA alone does not show high elastolytic activity, culture filtrates of lasA mutants (e.g. PAO-E64 and FRD2128) show much less elastolytic activity (30 -90%) when compared with their respective wild type strains (8,10,11,16). Peters and Galloway (11) examined this further with purified components and showed that the elastolytic activity of elastase (10 g input) increased 25-fold in the presence of purified LasA (10 g input), which is a LasA/ elastase molar ratio of ϳ1.7.…”

Section: Resultsmentioning

confidence: 96%

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Inhibitors and Specificity of Pseudomonas aeruginosa LasA

Kessler

Safrin

Abrams

et al. 1997

Journal of Biological Chemistry

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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.

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Section: Resultsmentioning

confidence: 96%

“…LasA Enhancement of Elastase Activity-LasA has been shown to enhance the elastolytic activity of Pseudomonas elastase in a dose-dependent manner (11). However, the effect has only been shown with high elastase input (10 g) and a relatively narrow range of LasA/elastase molar ratios (0.1 to 1.7, 0.5-10 g of LasA).…”

Section: Resultsmentioning

confidence: 99%

Inhibitors and Specificity of Pseudomonas aeruginosa LasA

Kessler

Safrin

Abrams

et al. 1997

Journal of Biological Chemistry

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“…It can also activate the host kinin cascade to cause inflammation (11). The elastolytic activity of elastase is enhanced by the product of the lasA gene (7,24,26) which has recently been demonstrated in and purified from culture supernatants of P. aeruginosa (24).…”

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confidence: 97%

Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase

1991

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The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasBi) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species.These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase.

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“…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).…”

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confidence: 99%

A Secreted Aminopeptidase of Pseudomonas aeruginosa

Cahan¹,

Axelrad²,

Safrin³

et al. 2001

Journal of Biological Chemistry

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Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was ϳ56 kDa; hence, it was designated AP 56 . Heating (70°C) of the partially purified aminopeptidase preparations led to the conversion of AP 56 to a ϳ28-kDa protein (AP 28 ) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP 28 was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine-or thiol-proteases, suggesting that AP 28 is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP 56 and AP 28 were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39 -58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, ϳ35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29 -32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.

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Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity

Cited by 78 publications

References 27 publications

Inhibitors and Specificity of Pseudomonas aeruginosa LasA

Inhibitors and Specificity of Pseudomonas aeruginosa LasA

Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase

A Secreted Aminopeptidase of Pseudomonas aeruginosa

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