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

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable hosts. It may produce various virulence factors including proteases. Among them, LasA possesses both elastolytic and staphylolytic (hydrolysis of pentaglycine cross-links in the cell wall peptidoglycan) activities. To understand if its elastolytic activity results from a preference for glycine-rich substrates, we studied its ability to hydrolyse the 65 pentapeptides of human tropoelastin containing at least three glycines. As demonstrated by capillary electrophoresis (CE), 22 of these peptides were hydrolysed by LasA, generally at a single peptide bond and the catalytic ratio kcat/KM was determined for most of them. The highest value was obtained for LGGGA, 59 +/- 9 min(-1) x mmol(-1) x L. The specificity of hydrolysis was elucidated by CE, liquid secondary ion mass spectrometry and, in some cases, collision activated dissociation-mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GG(cleavage site)A being especially sensitive to hydrolysis. Both positions P2 and P'2 must be occupied for hydrolysis and the presence of an amino acid in P3 (but not in P'3) significantly increases the catalytic ratio. Considering these results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin could be susceptible to LasA elastolysis.

Use of liquid chromatography-mass spectrometry coupling for monitoring the serralysin-catalyzed hydrolysis of a peptide library.

The use of a peptide library of limited size, is considered to be more appropriate for studying a protease with a complex specificity, but very sensitive and efficient analytical techniques must be used. We have designed and synthesized a 49-peptide library of the type Z-AlaXXAla(amide) (X=Ala, Leu, Val, Phe, Ser, Arg, Glu) for studying the Pseudomonas aeruginosa serralysin specificity. All compounds of the peptide library could be identified by a LC-MS procedure. After hydrolysis of the library by pseudomonal serralysin, the LC-MS procedure also allowed the identification of the hydrolysis products and the different cleavage sites of the substrates.

Lichenysins G, a novel family of lipopeptide biosurfactants from Bacillus licheniformis IM 1307: production, isolation and structural evaluation by NMR and mass spectrometry.

A series of 9 lactonic lipopeptide biosurfactants was isolated from Bacillus licheniformis IM 1307 as representatives of the lichenysin group and we propose to name them lichenysins G. They were recovered from the culture medium as complex mixtures of molecules having different peptide sequences and different structures of beta-hydroxy fatty acids. Their separation was achieved by a reversed-phase HPLC method leading to eight well-separated compounds. The complete structure of individual isoforms was proposed following the results of amino acid and fatty acid analysis, LSI-MS and 2D NMR spectroscopies. Compared to surfactin, lichenysins G are at least 10 fold more efficient biosurfactants.

Use of a 49-peptide library for a qualitative and quantitative determination of pseudomonal serralysin specificity.

The Pseudomonas aeruginosa serralysin (E.C. 3.4.24.40.), which is a zinc-dependent metalloprotease from the metzincin superfamily, has quite a broad specificity, which has not yet been clearly identified. We have studied it with an original approach, using a 49-peptide library of the type Z-AXXA (amide) (X = A, L, V, F, S, R, E). The library was analyzed by LC-MS before and after enzymatic hydrolysis. A great number of hydrolyzed peptides were screened and the preferential hydrolysis was the X-X peptide bond, even if in some cases, A-X and X-A bond could be hydrolyzed. No amino acids with a ionized side chain could be found in the P1' position. The results obtained suggest that the specificity in the P1' position, where an hydrophobic residue was preferentially found, seems more selective that in the Pn position. The P1 position was not very specific, but, on a quantitative point of view, the enzymatic activity was particularly increased when R, F or A were in this position. The results allow us to define the P1' and P1 residues for an optimal substrate of pseudomonal serralysin and usable for the design and the synthesis of a specific inhibitor.

Specificity of Pseudomonas aeruginosa serralysin revisited, using biologically active peptides as substrates.

The characterization of the specificity of alkaline protease from Pseudomonas aeruginosa has not yet been clearly defined. Some previous results suggested that its specificity was influenced more by amino acids far from the hydrolyzed peptide bond, than by amino acids in P1 or P'1 position. From other data, it was a C-(COOH)-type endoprotease where the preferential amino acid in P1 position was an arginine residue. We have studied the hydrolysis of several biologically active peptides. Many various sites of cleavage have been characterized but no arginine in P1 position was found, despite the presence of arginine in the peptide sequence. In fact P1 and P'1 position could be occupied by various amino acids. It seems unlikely that Pseudomonas alkaline protease may only be considered as a protease specific to arginine in P1 position. On the other hand, we have observed that increase of the peptide chain length led to an important increase of the hydrolysis rate, suggesting an extended number of subsites.

Capillary electrophoresis in the assay of the hydrolysis of glycine-containing peptides by a protease from Pseudomonas aeruginosa.

A rapid and simple capillary zone electrophoresis (CZE) method for measuring the activity of a minor protease from Pseudomonas aeruginosa is described. When glycine-containing oligopeptides were used as substrates, it was possible to separate and quantify substrate and products. Moreover oligopeptide hydrolysates were analysed by fast atom bombardment mass spectrometry providing the sites of splitting of the substrates. By comparison with CZE calibration curves constructed with reference peptides, the initial rates of hydrolysis were calculated. The method, validated for pentaglycine hydrolysis, was also used for the analysis of reaction mixtures and for monitoring the enzymic hydrolysis of various peptides in order to investigate enzyme specificity.

LasA, alkaline protease and elastase in clinical strains of Pseudomonas aeruginosa: quantification by immunochemical methods.

Thirty Pseudomonas aeruginosa strains were isolated from the sputa of cystic fibrosis patients. In each culture supernatant, the amount of three exoproteases (LasA, alkaline protease and elastase) was determined using immunochemical procedures. These assays used selected peptide-MAP (multiple antigen peptide) strategy as antigen for animal immunisation. The method appeared to be reproducible, simple, sensitive and specific without cross-reactivity between the antisera. The resulting values differed from one strain to another mostly for elastase production. Despite the fact that four genes (lasA, lasB, lasR and rhlR) were shown to be necessary for full elastolytic activity, it was obvious that if LasA was not secreted in a naturally non-elastase-producing strain, in return in an elastase-producing strain, there were no apparent relationships between LasA and elastase production and between LasA and alkaline protease secretion. Furthermore, in vitro, the secretion of the three exoproteases seemed to be independent of the mucoid or non-mucoid phenotype of the bacteria.

Biochemical properties of protease resistant prion protein PrPsc in natural sheep scrapie.

Prions are infectious agents involved in neurodegenerative diseases, such as scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cows and Creutzfeldt-Jakob disease (CJD) in humans. These pathogens are characterized by unusual properties, and, in particular, by their strong resistance to common procedures of disinfection used against conventional microorganisms. A major component of highly infectious fractions is a proteinase K-resistant prion protein PrPsc (PrP-res), the normal host prion protein PrPc being sensitive to PK (PrP-sen). We used a biochemical approach to further characterize PrPsc protein in natural sheep scrapie. Western blot analyses using rabbit antiserum that recognized both normal and pathologic sheep prion proteins, were undertaken to study the biochemical behaviour of PrPsc extracted from brains of sheep naturally infected with scrapie after protease digestion and under denaturing conditions. Increasing concentrations of urea (1-7 M) or GdnSCN (0.25-3 M) and different pH from 2 to 11 were tested for their effects on protease resistance of PrPsc. Alkaline pH (pH = 10) and high concentrations of urea (> 3 M) and GdnSCN (> 0.75 M) greatly decreased the protease resistance of the prion protein. Identical experiments carried out on three different sheep from the same flock gave similar results. The biochemical behaviour of PrPsc under denaturing conditions and in the presence of proteinase K could thus provide a biochemical means for further characterization of different natural scrapie isolates.

Diversity in the processing events at the N-terminus of type-V collagen.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)