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J Saulnier

Publications and source records attributed to J Saulnier.

At least 19 recordsLinked to original sources
PubMed2006

Human leukocyte elastase hydrolysis of peptides derived from human elastin exon 24.

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a beta-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S(5) to S'(3)). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.

Amino Acid Sequence↗
PubMed2005

Proteinase 3 hydrolysis of peptides derived from human elastin exon 24.

In normal and pathological tissues, elastin-derived peptides proceed of elastin degradation by polymorphonuclear leukocyte proteases: elastase, cathepsin G and proteinase 3. They were demonstrated to have a chemotactic activity, to promote cell proliferation and protease release, . . .. To be biologically active, their structures, which reflect elastase specificity, must adopt a beta-turn conformation which accommodate to the cell surface-located elastin binding protein. In this study, we establish that human elastin exon 24-derived peptides containing at least two repeated VGVAPG sequences are hydrolyzed by the proteinase 3 (Pr3). As shown by mass spectrometry analyses, the demonstrated cleavage sites are in agreement with previously reported Pr3 substrate specificity and its lengthy substrate binding site. The characterization of the Pr3-generated products indicate that they contain at least one GXXPG sequence known to stimulate cellular effects after binding to the elastin receptor.

Elastin↗
PubMed2005

Recent trends in protease-catalyzed peptide synthesis.

Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions: 1-Search for proteases with high and/or new P'1 and P1 specificities. 2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity. 3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide. 4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization. 5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures. This review will illustrate these new orientations with examples described in the recent literature.

Animals↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed1999

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.

Catalysis↗
PubMed1997

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.

Electrophoresis, Capillary↗
PubMed1996

Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P. aeruginosa elastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.

Amino Acid Sequence↗
PubMed1995

Effects of synthesized elastin peptides on human leukocytes.

It is well demonstrated that various peptides derived from elastin are biologically active. The hexapeptide (Val-Gly-Val-Ala-Pro-Gly; VI) as well as elastin peptides were demonstrated to be chemotactic for fibroblasts, while kappa-elastin had marked biological effects on human PMNLs. The aim of our present work was to synthesize various elastin peptides and compare their action to that of kappa-elastin and this hexapeptide. The results indicate that the hexapeptide (Val-Gly-Val-Ala-Pro-Gly) and the two other synthesized hexapeptides (Pro-Gly-Val-Gly-Val-Ala; III and Val-Gly-Val-Gly-Val-Ala; IV) had very similar and specific effects on intracellular free calcium metabolism, on superoxide anion production and elastase release. The other peptides had no effects on these parameters, except a tripeptide (Val-Gly-Val; V) on superoxide anion production. Moreover, the effect of the hexapeptides (III and VI) could be abolished by Pertussis toxin preincubation. All peptides had very similar stimulating effects on H2O2 production and myeloperoxidase release. We conclude that most probably the peptide size and conformation, as well as peptide composition play a role in the biological effects of these peptides, through specific receptors on PMNLs surface.

Amino Acid Sequence↗
PubMed1994

Evaluation of desmopressin for dental extractions in patients with hemostatic disorders.

We evaluated the effectiveness of desmopressin to control bleeding of patients with coagulation defects during dental surgery. Thirty-five patients, mainly with moderate and mild hemophilia and Willebrand disease, were undergoing dental extractions (over 80 extractions in total). Bleeding was successfully prevented in 28 patients with the use of a combined treatment incorporating IV desmopressin, an antifibrinolytic agent (tranexamic acid), and local methods (surgical glue and compression techniques). Seven patients had a bleeding episode after dental extraction, which was controlled in two cases by repeated injection of desmopressin and in another two by local methods; Factor VIII substitutive treatment was needed in only three patients. Desmopressin offers an alternative to blood products to control bleeding risk in patients with moderate and mild coagulation defects. Our experience tends to specify the mode of administration of both desmopressin and the associated treatments. Our findings suggest that desmopressin can be used in conjunction with other treatments to prevent bleeding in patients with coagulation defects who undergo dental surgery. This work highlights the concept of multifactorial medical care of these patients in which desmopressin plays a major role.

Adolescent↗
PubMed1993

Peptide synthesis catalysed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used for peptide-bond synthesis with benzyloxycarbonylalanine and amino acid amides as nucleophilic substrates. Dipeptide-bond synthesis was observed only for hydrophobic amino acid amides. The rate of peptide synthesis, measured by h.p.l.c., was in the decreasing order: Phe > Leu > Tyr > Val, Ile > Ala, which is consistent with the decreasing order of hydrolysis rates of the corresponding tetrapeptides Ala-Ala-Xaa-Ala. In contrast with thermolysin, Ps. aeruginosa elastase permits the synthesis of tyrosine-containing peptides with tyrosine in the P'1 position. Furthermore, the rates of synthesis for other hydrophobic amino acid amides are higher with elastase than with thermolysin.

Amino Acid Sequence↗
PubMed1992

Comparison of four procedures for measuring elastase production by Pseudomonas aeruginosa strains from cystic fibrosis patients.

Forty-five Pseudomonas aeruginosa strains were isolated from the sputa of cystic fibrosis patients. The elastase production of each strain was assayed in the culture supernatant using four different procedures, i.e. two immunological assays (RIA and ELISA), and two enzymatic assays, the latter employing either elastin or tetraalanine as substrate, with conductometric measurement of substrate hydrolysis. Elastase concentrations were determined from standard curves prepared with the same purified elastase, and expressed in mg of elastase per litre of supernatant. The resulting values were in the range reported in the literature, and differed greatly from one strain to another (0-230 mg/l). Linear relationships were found when assays were compared in pairs. Significant correlation coefficients were obtained (r greater than 0.76, p less than 0.001) but the values were quite different for different assays. Thus, ELISA measurements were always from three to five times higher, and RIA results were from two to five times lower, than those from the other assays. Enzymatic assays with elastin gave higher values than those using tetraalanine. Most P. aeruginosa strains produce two other proteinases, alkaline proteinase and Las A protein. Both enzymes have limited elastolytic and peptidasic activities. The presence of alkaline proteinase does not result in falsely elevated elastase values, but an increase of elastase activity was observed when Las A was preincubated with elastin. Since this increase was not observed when tetraalanine was used as the substrate, the presence of Las A in the supernatants could explain the differences observed between the enzymatic assays. The assay with the synthetic substrate is therefore preferred.

Cystic Fibrosis↗
PubMed1991

Hydrolysis of alanine oligomers and of elastin by P. aeruginosa proteinases and thermolysin.

The hydrolysis of alanine oligomers by P. aeruginosa proteinases, thermolysin and porcine pancreatic elastase was studied. The concentrations of substrates and cleavage products were determined using reverse phase high pressure liquid chromatography. Tetraalanine was the shortest oligomer for which we could demonstrate hydrolysis by all the proteinases, except for porcine pancreatic elastase which only significantly hydrolyzed peptides longer than hexaalanine. Porcine pancreatic elastase hydrolyzes hexaalanine at a single site, whereas the other enzymes may split it either into two trialanine molecules, or into di- and tetraalanine, the latter being further cleavable to dialanine. A kinetic model based on first-order kinetic rate constants is proposed and the individual constants determined. Although P. aeruginosa elastase and thermolysin are closely similar in structure, they have shown a marked difference in their hydrolysis of either elastin or tetraalanine. Elastolytic activity of thermolysin was higher than that of elastase but tetraalanine was hydrolyzed more slowly by thermolysin.

Alanine↗
PubMed1990

[Mechanism of action of proteinases of Pseudomonas aeruginosa].

Of the two proteases produced by Pseudomonas aeruginosa, one whose optimal pH is neutral, exhibits elastolytic activity. This elastase is produced as a prepropeptide, subsequently modified by proteolysis, and finally secreted as an active enzyme. Both proteases act mainly on hydrophobic aminoacids. The most important site for the elastase seems to be the P'1 site where Phe, Tyr and Leu residues enhance hydrolysis. The alkaline protease is less specific of this site. At least four aminoacids are needed to obtain measurable rates of hydrolysis. A synthetic substrate, Ala-Ala-Phe-Ala, is proposed for conductimetric measurements of Pseudomonas aeruginosa elastase activities in the nanomolar range. A review of recent studies shows that a very wide range of proteins can be hydrolyzed by the two Pseudomonas aeruginosa proteases, a fact which may explain why these enzymes are major determinants of the bacteria's infectivity.

Conductometry↗
PubMed1990

Elastolysis of kappa-elastin sub-fractions by pancreatic and leukocyte elastases.

Kappa-elastin peptides, obtained from insoluble elastin by organo-alkaline hydrolysis, were fractioned by gel filtration on Biogel agarose. Rates of hydrolysis by pancreatic and leukocyte elastases of the fractions were measured using a conductometric method. Kinetics obey to Michaelis-Menten model for both substrates and enzymes. KM and Vmax values derived from Lineweaver-Burk plots indicate that, if KM remains quite constant, differences were observed in catalytic rates. The kcat values decreased with molecular-weight, the high-molecular-weight kappa-elastin peptides being hydrolyzed 3 to 5 times faster than the low-molecular-weight ones. Apparent differences between potentiometric (pH-Stat) and conductometric results were discussed, in relation with buffer capacity of soluble and insoluble elastins.

Animals↗
PubMed1988

Conductimetry: a new tool for studying inhibition of elastolysis.

The conductimetric method was applied to the measurement of human leukocyte elastase activity, using insoluble elastin as a substrate. From conductance changes, initial rates of elastolysis were derived. A linear relationship of enzyme activity with enzyme concentration was demonstrated up to 400nM of enzyme, for three different substrates. In this concentration range, inhibition of elastolysis by eglin c was studied for different concentrations of eglin c. A 50% inhibitory concentrations of 0.13-0.15 microM of eglin c was derived from our results, corresponding to an inhibitor/enzyme ratio of about 0.5, indicating a strong inhibition, as previously demonstrated by authors using synthetic substrates.

Animals↗