Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase in vitro

Human plasma prekallikrein, precursor of the bradykinin-generating enzyme, was activated in a purified system under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase which is a tissue-destructive metalloproteinase. Compared with that, Pseudomonas aeruginosa alkaline proteinase poorly activated it with a rate as low as less than one-twentieth of that of elastase. The activation by elastase was blocked with a specific inhibitor of elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2 (10 microM). Generation of kallikrein-like amidolytic activity was also observed in plasma deficient in Hageman factor by treatment with elastase, but was not in plasma deficient in prekallikrein. The kallikrein-like activity generated in Hageman factor deficient plasma as well as the generation process itself was indeed inhibited by anti-human prekallikrein goat antibody. These results suggest that the pathological activation of the kallikrein-kinin system might occur under certain clinical conditions in pseudomonal infections.     

Activation of an elastase precursor by the lasA gene product of Pseudomonas aeruginosa.

To study the role of the lasA gene product in the secretion of enzymatically active elastase by Pseudomonas aeruginosa, we constructed mutants by gene replacement with in vitro-derived insertion and deletion mutations in the cloned lasA gene. lasA mutants were deficient in the production of elastolytic activity. A membrane-associated, higher-molecular-weight (approximately 47,000) precursor of elastase was observed in both the wild-type and the lasA mutants. Unlike the wild-type strain, the lasA mutant accumulated the 47,000-molecular weight elastase species in the soluble fraction of the cell, suggesting that the lasA gene product has a role in elastase secretion. Although lasA mutants were deficient in elastolytic activity, they produced a proelastase with a mature molecular weight (approximately 37,000) that still retained general proteolytic activity. Final yields of elastase-related material were approximately the same in both the wild-type strain and lasA mutant supernatants. The lasA gene was expressed in Escherichia coli, and the approximate molecular weight of the lasA gene product was 31,000. Extracts of E. coli containing the lasA gene product were shown in vitro to activate the proelastase produced by P. aeruginosa lasA mutants to an enzyme with elastolytic activity. Thus the lasA gene product has a direct effect on broadening the substrate specificity of secreted proelastase, as well as a second role (direct or indirect) in the secretion of elastase.     

Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces

Human blood coagulation factor XI was activated by either autoactivation or thrombin. These reactions occurred only in the presence of negatively charged materials, such as dextran sulfate (approximately Mr 500,000), sulfatide, and heparin. During the activation, factor XI was cleaved at a single Arg-Ile bond by thrombin or factor XIa to produce an amino-terminal 50-kDa heavy chain and a carboxyl-terminal 35-kDa light chain. This activation pattern is identical to that produced by factor XIIa. The addition of a small amount of thrombin and sulfatide to factor XII-deficient plasma produced shorter clotting times than when these agents were added to factor XI/factor XII combined-deficient plasma. These results suggest that the activation of factor XI by thrombin and possibly the autoactivation of factor XI proceed in plasma to lead fibrin clot formation. These reactions may have a role on an appropriate negatively charged surface in normal hemostasis.     

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.     

Degradation of IgA proteins by Pseudomonas aeruginosa elastase

Human colostral IgA and myeloma proteins of both IgA1 and IgA2 subclasses were susceptible to cleavage by Pseudomonas aeruginosa elastase. Detailed analysis of the cleavage products of IgA myeloma proteins revealed complete degradation of Fab with no evidence of intact Fab fragments as intermediate cleavage products. In contrast, both IgA1 and IgA2 proteins were resistant to cleavage by alkaline protease from P. aeruginosa. The susceptibility of human IgA proteins to elastase suggests a mechanism by which P. aeruginosa might evade the potentially protective function of IgA by producing this enzyme.     

Activation of the chick oviduct progesterone receptor by heparin in the presence or absence of hormone.

Activation (transformation) of the chick oviduct progesterone receptor was found to be induced at 0 degrees C by heparin free in solution as well as by chromatography on a column of heparin linked to acrylamide/agarose. The transformed molecule displayed properties of the activated form of [3H]progesterone-receptor complex obtained by heat treatment or by high ionic strength: smaller size (s20,w = 3.9 S, Stokes radius = 5.2 nm), lower rate of dissociation (t 1/2 approx. 50 h at 0 degrees C compared with approx. 20 h for the 'native' form) and increased binding to phosphocellulose. In all cases, molybdate was an effective inhibitor of transformation and stabilized a large 'native' form (s20,w = 7.9 S, Stokes radius = 7.6 nm). Transformation by neither KCl nor heparin depended on the presence of ligand bound to the receptor, and the properties of the receptor molecule produced by treatment of ligand-free receptor with high ionic strength or with heparin were identical with those of the activated progesterone-receptor complex, demonstrating that receptor activation can be obtained experimentally in the absence of hormone. Our data are compatible with a model in which activation implies separation of the 4 S units, which compose the approx. 8 S 'native' form.     

Maturation of Pseudomonas aeruginosa elastase. Formation of the disulfide bonds

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.     

Thermotropic properties of mixtures of negatively charged phospholipids with cholesterol in the presence and absence of Li+ or Ca2+ ions

Mixtures of cholesterol with dipalmitoylphosphatidylserine or phosphatidic acid were investigated by differential scanning calorimetry. As in mixtures of natural phosphatidylserine with cholesterol (Bach, D. (1984) Chem. Phys. Lipids 35, 385-392), also here phase separation of cholesterol at molar ratios of 2:1 (phospholipid:cholesterol) and below is observed. The limited solubility of cholesterol in negatively charged phospholipids is found to be independent of the nature of the acyl chain residues, and independent of whether the negative charge resides on both COO- and PO- groups (as in phosphatidylserine) or on PO- only (as in phosphatidic acid). The separate cholesterol phase is also seen by DSC in mixtures of natural phosphatidylserine or phosphatidic acid with cholesterol in the presence of Ca2+; and in phosphatidylserine/cholesterol mixtures in the presence of Li+, by DSC and X-ray diffraction.     

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase in vitro

Human plasma prekallikrein, precursor of the bradykinin-generating enzyme, was activated in a purified system under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37°C) by Pseudomonas aeruginosa elastase which is a tissue-destructive metalloproteinase. Compared with that, Pseudomonas aeruginosa alkaline proteinase poorly activated it with a rate as low as less than one-twentieth of that of elastate. The activation by elastase was blocked with a specific inhibitor of elastase, HONHCOCH(CH₂C₆H₅)CO-Ala-Gly-NH₂ (10 μM). Generation of kallikrein-like amidolytic activity was also observed in plasma deficient in Hageman factor by treatment with elastase, but was not in plasma deficient in prekallikrein. The kallikrein-like activity generated in Hageman factor deficient plasma as well as the generation process itself was indeed inhibited by antihuman prekallikrein goat antibody. These results suggest that the pathological activation of the kallikrein-kinin system might occur under certain clinical conditions in pseudomonal infections.     

Cloning and characterization of elastase genes from Pseudomonas aeruginosa.

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.     

Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.     

Antigenic specificities of Pseudomonas aeruginosa alkaline protease and elastase defined by human T cell clones

Virulent strains of Pseudomonas aeruginosa derive their pathogenicity, in part, from their secretion of two proteolytic enzymes, alkaline protease (AP) and elastase (E). Human T lymphocytes specific for AP and E can be detected in the blood of immune donors and have afforded the opportunity to characterize the antigenicity of these proteins. To accomplish this goal, we have recently selected 68 human T cell clones from five different Pseudomonas-immune donors and determined their fine specificities. Fifty-five (81%) were found to be protease specific, demonstrating the immunogenicity of the exoenzymes in humans. These clones defined five AP and three E specificities and suggested the existence of at least five allomorphic determinants expressed on the proteases of various Pseudomonas strains. Limiting dilution analysis confirmed a number of antigenic relationships suggested by the long-term T cell clones and revealed that T cells specific for allomorphic protease determinants were at least as frequent in the blood of immune donors as were T cells committed to conserved determinants. Thus, both primary and long-term human T cell clones showed specificity patterns that distinguished proteases from different Pseudomonas strains. These observations describe a heretofore unknown antigenic system of Pseudomonas aeruginosa that should assist in defining the nature and specificity of Pseudomonas immunity in humans.     

Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids.

The hydroxamic acid HONHCOCH2CH(i-Bu)CO-L-Trp-NHMe, isomer 6A (GM 6001), inhibits human skin fibroblast collagenase with Ki of 0.4 nM using the synthetic thiol ester substrate Ac-Pro-Leu-Gly-SCH(i-Bu)CO-Leu-Gly-OEt at pH 6.5. The other isomer, 6B, which has the opposite configuration at the CH2CH(i-Bu)CO alpha-carbon atom, has a Ki of 200 nM for this enzyme. GM 6001 is one of the most potent inhibitors of human skin fibroblast collagenase yet reported. GM 6001 has a Ki of 20 nM against thermolysin and Pseudomonas aeruginosa elastase. Isomer 6B has a Ki of 7 nM against thermolysin and 2 nM against the elastase. 6A and 6B are the most potent hydroxamate inhibitors reported for these bacterial enzymes. The pattern of inhibition for all three enzymes suggests that isomer 6A is the (R,S) compound, stereochemically analogous to the L,L-dipeptide, and isomer 6B is the (S,S) compound, analogous to the DL-dipeptide. The tolerance of the D configuration by thermolysin and the elastase allows these inhibitors to discriminate between the human and bacterial enzymes simply by inversion of configuration at the CH2CH(i-Bu)CO alpha-carbon atom. Substitution of the potential metal liganding groups carboxylate and hydrazide for the hydroxamate group yields much weaker inhibitors for all three enzymes.     

Synthesis, processing, and transport of Pseudomonas aeruginosa elastase.

Three cell-associated elastase precursors with approximate molecular weights of 60,000 (P), 56,000 (Pro I), and 36,000 (Pro II) were identified in Pseudomonas aeruginosa cells by pulse-labeling with [35S]methionine and immunoprecipitation. In the absence of inhibitors, cells of a wild-type strain as well as those of the secretion-defective mutant PAKS 18 accumulated Pro II as the only elastase-related radioactive protein. EDTA but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] inhibited the formation of Pro II, and this inhibition was accompanied by the accumulation of Pro I. P accumulated in cells labeled in the presence of ethanol (with or without EDTA), dinitrophenol plus EDTA, or carbonyl cyanide m-chlorophenyl hydrazone plus EDTA. Pro I and Pro II were localized to the periplasm, and as evident from pulse-chase experiments, Pro I was converted to the mature extracellular enzyme with Pro II as an intermediate of the reaction. P was located to the membrane fraction. Pro I but not Pro II was immunoprecipitated by antibodies specific to a protein of about 20,000 molecular weight (P20), which, as we showed before (Kessler and Safrin, J. Bacteriol. 170:1215-1219, 1988), forms a complex with an inactive periplasmic elastase precursor of about 36,000 molecular weight. Our results suggest that the elastase is made by the cells as a preproenzyme (P), containing a signal sequence of about 4,000 molecular weight and a "pro" sequence of about 20,000 molecular weight. Processing and export of the preproenzyme involve the formation of two periplasmic proenzyme species: proelastase I (56 kilodaltons [kDa]) and proelastase II (36 kDa). The former is short-lived, whereas proelastase II accumulates temporarily in the periplasm, most likely as a complex with the 20-kDa propeptide released from proelastase I upon conversion to proelastase II. The final step in elastase secretion seems to required both the proteolytic removal of a small peptide from proelastase II and dissociation of the latter from P20.     

Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids

The endothelial cell-type plasminogen activator inhibitor (PAI-1) may exist in an inactive, latent form that can be converted into an active form upon treatment of the protein with denaturants, such as sodium dodecyl sulfate, guanidine HCl, or urea. The present paper demonstrates that latent PAI-1 can be activated by lipid vesicles containing the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol. The presence of a net negative charge on the phospholipid headgroup is essential for activation, since lipid vesicles consisting exclusively of zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, do not activate PAI-1. In the presence of PS vesicles, PAI-1 inhibited tissue-type plasminogen activator 50-fold more effectively than in the absence of phospholipids, whereas sodium dodecyl sulfate enhanced PAI-1 activity by 25-fold. In mixed phospholipid vesicles containing PS and phosphatidylcholine in various molar ratios, the extent of PAI-1 activation was directly related to the PS content of the phospholipid membrane. Ca2+ ions interfered with the inhibitory activity of PS-activated PAI-1, suggesting that Ca2+ ions may regulate PAI-1 activity in the presence of negatively charged phospholipids. An important consequence of these findings is that, as in blood coagulation, negatively charged phospholipids may play an important regulatory role in controlling the fibrinolytic system by activating an inhibitor of tissue-type plasminogen activator.     

Activation mechanism of human Hageman factor-plasma kallikrein-kinin system by Vibrio vulnificus metalloprotease.

Vivrio vulnificus, an opportunistic human pathogen, secretes a metalloprotease (VVP). The VVP inoculated into a guinea pig is known to generate bradykinin through activation of the Hageman factor-plasma kallikrein-kinin system. VVP was shown to possess the ability to activate the human system through the same mechanism as that clarified in the guinea pig system, namely, VVP converted both human zymogens (Hageman factor and plasma prekallikrein) to active enzymes (activated Hageman factor and plasma kallikrein), and the then generated kallikrein liberated bradykinin from high-molecular-weight kininogen. However, in the presence of plasma alpha 2-macroglobulin (alpha 2M), the VVP action was drastically decreased. This finding suggests that the human system might be activated only at the interstitial-tissue space which contains negligible amounts of alpha 2M or in the bloodstream of the individuals whose plasma alpha 2M level is extremely reduced.