Extracellular Cysteine Protease Research Articles

Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection.

Streptococcal cysteine proteinase releases kinins: a virulence mechanism.

Previous work has indicated a crucial role for the extracellular cysteine proteinase of Streptococcus pyogenes in the pathogenicity and virulence of this important human pathogen. Here we find that the purified streptococcal cysteine proteinase releases biologically active kinins from their purified precursor protein, H-kininogen, in vitro, and from kininogens present in the human plasma, ex vivo. Kinin liberation in the plasma is due to the direct action of the streptococcal proteinase on the kininogens, and does not involve the previous activation of plasma prekallikrein, the physiological plasma kininogenase. Judged from the amount of released plasma kinins the bacterial proteinase is highly efficient in its action. This is also the case in vivo. Injection of the purified cysteine proteinase into the peritoneal cavity of mice resulted in a progressive cleavage of plasma kininogens and the concomitant release of kinins over a period of 5 h. No kininogen degradation was seen in mice when the cysteine proteinase was inactivated by the specific inhibitor, Z-Leu-Val-Gly-CHN2, before administration. Intraperitoneal administration into mice of living S. pyogenes bacteria producing the cysteine proteinase induced a rapid breakdown of endogenous plasma kininogens and release of kinins. Kinins are hypotensive, they increase vascular permeability, contract smooth muscle, and induce fever and pain. The release of kinins by the cysteine proteinase of S. pyogenes could therefore represent an important and previously unknown virulence mechanism in S. pyogenes infections.

Purification and characterization of an extracellular cysteine proteinase produced byMicrococcussp. INIA 528

Micrococcus sp. INIA 528, a micro‐organism isolated from raw ewe's milk Manchego cheese, produced an extracellular proteinase. This enzyme was purified to homogeneity from culture supernatant fluid in two chromatographic steps, with a 29‐fold increase of specific activity and a 28% recovery of proteinase activity. The homogeneous protein was characterized biochemically. The molecular weight of the enzyme was determined to be 19.4 kDa by mass spectrometry. The purified enzyme was inhibited by E‐64, PMSF and iodoacetamide and activated by cysteine, glutathione, dithiothreitol and β‐mercaptoethanol. These results suggest that the enzyme is a cysteine proteinase. Optimal conditions for activity on azocasein were 34°C and a pH of 7.0. The proteinase preferentially degraded β‐casein, while after a longer incubation period αs1‐casein was also extensively hydrolysed. The proteinase had a Km value of 6.12 g 1−1 for casein and 2.20 g 1−1 for azocasein.

Substitution of cysteine 192 in a highly conserved Streptococcus pyogenes extracellular cysteine protease (interleukin 1beta convertase) alters proteolytic activity and ablates zymogen processing.

Virtually all strains of the human pathogenic bacterium Streptococcus pyogenes express a highly conserved extracellular cysteine protease. The protein is made as an inactive zymogen of 40,000 Da and undergoes autocatalytic truncation to result in a 28,000-Da active protease. Numerous independent lines of investigation suggest that this enzyme participates in one or more phases of host-parasite interaction, such as inflammation and soft tissue invasion. Replacement of the single cysteine residue (C-192) with serine (C192S mutation) resulted in loss of detectable proteolytic activity against bovine casein, human fibronectin, and the low-molecular-weight synthetic substrate 7-amino-4-trifluoromethyl coumarin. The C192S mutant molecule does not undergo autocatalytic processing of zymogen to mature form. Taken together, these data support the hypothesis that C-192 participates in active-site formation and enzyme catalysis.

Streptococcal cysteine protease augments lung injury induced by products of group A streptococci

Streptococcus pyogenes infections in humans may be associated with severe clinical manifestations, including adult respiratory distress syndrome and a toxic shock-like syndrome. These observations have led to the investigation of products of group A streptococci that may contribute to increased virulence. Streptococcal pyrogenic exotoxin B is a highly conserved precursor of an extracellular cysteine protease that is secreted by S. pyogenes. We investigated the ability of this streptococcal cysteine protease (SCP) to act synergistically with either streptococcal cell wall antigen (SCW) or streptolysin-O (SLO) to augment lung injury in rats. Intratracheal administration of either SCW or SLO alone caused lung injury, as measured by pulmonary vascular leak. Bronchoalveolar lavage (BAL) fluid analysis showed that SCW induced neutrophil accumulation and appearance of interleukin-1beta and tumor necrosis factor alpha. In contrast, SLO induced neither neutrophil influx nor significant cytokine elevations in BAL fluids. Intratracheal administration of SCP with either SCW or SLO resulted in synergistic augmentation of lung vascular permeability and accumulation of BAL neutrophils. The synergy was reduced when SCP was either heat inactivated or coinstilled with a peptide inhibitor of the protease. SCP in the presence of SCW resulted in a significant increase in BAL fluid tumor necrosis factor alpha content but not in immunoreactive interleukin-1beta. Moreover, the copresence of SAP with SAW resulted in increased BAL fluid nitrite-nitrate levels, indicative of nitric oxide production. These data demonstrate that SCP acts synergistically with other S. pyogenes products (SCW or SLO) to increase tissue injury and provide additional evidence that SCP may function as an important virulence factor in group A streptococcal infections.

Characterization of Extracellular Proteinases of Tritrichomonas foetus

Proteinases released from Tritrichomonas foetus into a reducing buffer were characterized because we previously showed that they digested host proteins important in defense of the reproductive tract. These proteinases were shown to be extracellular because cell numbers did not decrease and trichomonads remained motile during their 3.5-hr incubation. Detectable proteinase activity was attributable to enzymes of the cysteine class by spectrophotometric and fluorometric automated assays for peptide substrate specificity. Proteinases from the trichomonad-conditioned buffer were partially purified by bacitracin affinity chromatography. Separation of the eluate on nondenaturing SDS-PAGE gels copolymerized with gelatin, revealed primarily low molecular weight proteinases. Bacitracin-purified T. foetus extracellular cysteine proteinase (TFECP) was separated in a denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, electroblotted, and a 31-kDa band cut out for amino acid sequencing. Four internal fragments were sequenced, 1 of which contained the highly conserved asparagine region of the cysteine proteinase active site. The combined sequences of these enzyme fragments were 66% and 55% identical to and the corresponding deduced amino acid sequences of 2 T. foetus cysteine proteinase genes (TFCP1 and TFCP2, respectively), which we previously cloned. These results indicate that this enzyme (TFECP) is a distinct cysteine proteinase. The extracellular release of TFECP from T. foetus suggests that it is a potential virulence factor in bovine trichomoniasis.

New concepts regarding the pathogenesis of amebiasis.

Our understanding of the pathogenesis of amebiasis has progressed significantly since Dr. Braude's description of the major clinical syndromes of amebic liver abscess. His hypothesis that invasive amebic strains must be resistant to complement-mediated lysis has been confirmed. We have also shown that Entamoeba histolytica activates complement by a unique mechanism, i.e., cleavage of C3 by an extracellular cysteine proteinase. Cysteine proteinases are important virulence factors encoded by at least three genes; one gene, acp1, is unique to invasive strains. The amebic cysteine proteinases are homologous to proteinases released by transformed cells and may represent a common mechanism of tissue invasion. The initial division of Entamoeba into invasive E. histolytica and noninvasive Entamoeba dispar by isoenzymes has been supported by genetic differences between amebae. Thus, a model of pathogenesis differentiating between two separate species of Entamoeba best explains the epidemiology, clinical syndromes, and pathology of amebiasis.

The extracellular neutral cysteine proteinase of Entamoeba histolytica degrades anaphylatoxins C3a and C5a.

We have shown previously that the extracellular cysteine proteinase of Entamoeba histolytica trophozoites activates the alternative pathway of complement by specifically cleaving C3. This unique mechanism of complement activation leads to passive lysis of nonpathogenic, but not of pathogenic strains. In an attempt to investigate the relationship between the cleavage of complement components C3 and C5 and the pathogenesis of amebiasis, we investigated the production of the anaphylatoxins C3a and C5a, which have diverse effects on the host immune response. The concentration of proteinase required to cleave purified C5 was at least 5 to 10 times that needed for C3 cleavage, but these levels are easily obtainable as demonstrated by cleavage of 125I-labeled C5 during incubation with purified trophozoites. When the C3a-like cleavage fragments were purified by gel filtration, they were found to be extensively degraded during a 1-h incubation of C3 with the proteinase. Subsequent evaluations of the C3a- and C5a-like cleavage products generated earlier in the reaction using immunoblots and cellulose acetate electrophoresis revealed rapid degradation, even during incubation periods as short as 5 min. Because C-terminal fragments as small as 20 amino acid residues can mimic the biologic functions of C3a or C5a, we tested cleavage products for activity. In sensitive bioassays, including guinea pig platelet aggregation for C3a activity and chemotaxis for C5a activity, we demonstrated that proteolysis renders these molecules inactive. These studies suggest that the extracellular cysteine proteinase of E. histolytica, which is capable of activating the complement system, may also provide a mechanism to circumvent normal host immunity by inactivating the proinflammatory factors C3a and C5a.

Streptococcal Cysteine Proteinase Releases Biologically Active Fragments of Streptococcal Surface Proteins

Streptococcus pyogenes are important pathogenic bacteria which produce an extracellular cysteine proteinase contributing to their virulence and pathogenicity. S. pyogenes also express surface molecules, M proteins, that are major virulence determinants due to their antiphagocytic property. In the present work live S. pyogenes bacteria of the M1 serotype were incubated with purified cysteine proteinase. Several peptides were solubilized, and analysis of their protein-binding properties and amino acid sequences revealed two internal fibrinogen-binding fragments of M1 protein (17 and 21 kDa, respectively), and a 36-kDa IgG-binding NH2-terminal fragment of protein H, an IgGFc-binding surface molecule. M protein also plays a role in streptococcal adherence, and removal of this and other surface proteins could promote bacterial dissemination, whereas the generation of soluble complexes between immunoglobulins and immunoglobulin-binding streptococcal surface proteins could be an etiological factor in the development of glomerulonephritis and rheumatic fever. Thus, in these serious complications to S. pyogenes infections immune complexes are found in affected organs. The cysteine proteinase also solubilized a 116-kDa internal fragment of C5a peptidase, another streptococcal surface protein. Activation of the complement system generates C5a, a peptide stimulating leukocyte chemotaxis. C5a-mediated granulocyte migration was blocked by the 116-kDa fragment. This mechanism, by which phagocytes could be prevented from reaching the site of infection, may also contribute to the pathogenicity and virulence of S. pyogenes.

Structural Characterization of Argingipain, a Novel Arginine-Specific Cysteine Proteinase as a Major Periodontal Pathogenic Factor from Porphyromonas gingivalis

Argingipain, so termed due to its peptide cleavage specificity at arginine residue, is a unique extracellular cysteine proteinase produced by the anaerobic rod Porphyromonas gingivalis, which is known as a major pathogenic factor of the progressive periodontal disease (T, Kadowaki, M. Yoneda, K. Okamoto, K. Maeda, and K, Yamamoto (1994) J. Biol. Chem. 269, 21371-21378), The catalytic specificity and functional importance of this enzyme prompted us to elucidate its structural features. A DNA fragment for argingipain was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the NH2-terminal amino acid sequence of the purified enzyme. Although the extracellular mature enzyme was shown to have an apparent molecular mass of 44 kDa in gels, the nucleotide sequence of the isolated gene revealed a single gene coding for a 109-kDa precursor of argingipain. The deduced amino acid sequence exhibited no significant similarity to the sequences of representative members of the cysteine protease family. The precursor contained four functional domains: the NH2-terminal signal peptide required for the inner membrane transport; the NH2-terminal prosequence, which is assumed to stabilize the precursor structure; the proteinase domain; and the COOH-terminal hemagglutinin domain, which appears to be essential for extracellular secretion of the proteinase domain. Experiments involving the addition of the argingipain inhibitors to the culture medium of P. gingivalis suggested that the maturation of argingipain occurs intracellularly via an autocatalytic cleavage of the pro-argingipain propeptide.

Vaccination with streptococcal extracellular cysteine protease (interleukin-1β convertase) protects mice against challenge with heterologous group A streptococci

Virtually all clinical isolates of group A streptococci secrete a highly conserved extracellular cysteine protease that cleaves human fibronectin and vitronectin, and converts IL-1β precursor to biologically active IL-1β. Based on the high degree of gene conservation within the species and its role in host pathogenicity, it was postulated that antibodies to the cysteine protease would confer protective immunity against S. pyogenes infection. To test this hypothesis, Swiss CD1 mice were intraperitoneally administered either saline, rabbit IgG, or IgG from rabbits immunized with the protease, and challenged with a highly virulent (minimum lethal dose ∼ 10 cfu) clinical isolate of S. pyogenes expressing a heterologous cysteine protease. The results indicate that mice administered IgG from rabbits immunized with purified cysteine protease had significantly enhanced survival when compared with mice given either non-specific rabbit IgG (log rank test; χ 2; p = 0.0195) or saline (log rank test; χ 2; p = 0.0002). Moreover, mice actively immunized with the cysteine protease had a significantly longer time to death than the control group (log rank test; χ 2; p = 0.0418). The results show that the cysteine protease elicits non-type-specific immunity to challenge with heterologous S. pyogenes.

Analysis of the extracellular proteases of Trichomonas vaginalis.

We have previously isolated two extracellular cysteine proteases (60 and 30 kDa, respectively) from the cell filtrate of an isolate of Trichomonas vaginalis. In this study the clinical presentation of 12 clinical isolates of T. vaginalis was correlated with their protease activity. All 12 isolates produced a 60-kDa protease as demonstrated by immunoblotting. However, only 5 of 12 isolates produced a 30-kDa protease in the extracellular filtrate. Protease activity did not differentiate between isolates obtained from symptomatic versus asymptomatic women. The 60-kDa protease, which breaks down into a 43- and 23-kDa subunit, was detected by immunoblotting with anti-23-kDa cross-reacting rabbit serum in the vaginal washes of 3/3 women with active T. vaginalis infection, 0/1 woman cured of T. vaginalis and 0/2 control women with vaginal candidiasis. These results suggest that the 60-kDa protease is found in all isolates testes in vitro, is present in active disease and may be important in the pathogenesis of T. vaginalis infection.

A conserved Streptococcus pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin

Streptococcus pyogenes secretes an extracellular cysteine protease that cleaves human interleukin 1β precursor to form biologically active IL 1β, a major cytokine mediating inflammation and shock. To further investigate the potential role of the cysteine protease in host-parasite interactions, the enzyme was purified to apparent homogeneity and tested for ability to degrade several human extracellular matrix proteins. Purified protease cleaved fibronectin, apparently at specific sites, and rapidly degraded vitronectin. In contrast, the protease did not have substantial activity against laminin. The cysteine protease also cleaved fibronectin from human umbilical vein endothelial cells grown in vitro. Allelic variation in the cysteine protease structural gene was studied in 67 strains expressing 39 M protein serotypes and five provisional M serologic types, and representing 50 phylogenetically distinct clones identified by multilocus enzyme electrophoresis. The gene is well conserved and allelic variation is due solely to accumulation of point mutations. Based on predicted amino acid sequences, one mature cysteine protease variant would be made by clones expressing serotypes M2, M3, M4, M5, M6, M9, M10, M11, M12, M14, M18, M22, M23, M25, M27, M41, M49, M56, M59, two provisional M types, and two clones non-typeable for M protein. Moreover, 33 of the 39 speB alleles identified encode one of three mature protease variants that differ from one another at only one or two amino acids clustered in a ten-amino acid region. All 39 alleles, and virtually all strains, encode a product that reacts with polyclonal antisera specific for purified cysteine protease. No compelling evidence was found for a primitive differentiation of the speB gene into two distinct classes, as has been proposed for M protein, opacity factor phenotype, and vir regulon architecture. The results demonstrate that the cysteine protease is well conserved in natural populations of S. pyogenes, provide additional evidence that this enzyme is involved in host-parasite interactions, and suggest that the protease plays a role in bacterial dissemination, colonization, and invasion, and inhibition of wound healing.

Lysis of complement-sensitive Entamoeba histolytica by activated terminal complement components. Initiation of complement activation by an extracellular neutral cysteine proteinase.

Activation of complement by Entamoeba histolytica may be initiated by the extracellular 56-kD neutral cysteine proteinase which cleaves the alpha chain of C3. To determine the relationship between the fluid-phase activation of complement and our observation that only strains isolated from patients with invasive disease are resistant to complement-mediated lysis, we investigated the fate of C3 with recent amebic isolates. When 125I-C3 was incubated with trophozoites in serum, C3 in the fluid phase was cleaved to C3b or C3bi, but the alpha chain of the C3 molecules on the cell surface appeared intact. Since the lysis of nonpathogenic strains takes place in the absence of bound C3b, we demonstrated that this reaction occurs by reactive lysis initiated in the fluid phase: (a) the killing of nonpathogenic strains was enhanced when alternative pathway activation was accelerated by the addition of cobra venom factor; (b) non-pathogenic strains were lysed by purified terminal components; and (c) sera incubated with pathogenic E. histolytica produced passive lysis of chicken erythrocytes. These results demonstrate for the first time that complement-sensitive E. histolytica are lysed by activation of the terminal complement components in the fluid phase where the 56-kD neutral cysteine proteinase cleaves C3, and not by the surface deposition of activated C3.

Production, characterization and use of monoclonal antibodies against the major extracellular human cysteine proteinase inhibitors cystatin C and kininogen.

Murine monoclonal antibodies against the major cysteine proteinase inhibitors of human biological fluids, cystatin C and kininogen, were produced. The cystatin C antibody, HCC3, with a Ka of 2 x 10(7) l/mol, increased the inhibition of papain by cystatin C and was suitable for use in immunoblotting, immunohistochemistry and in the construction of a sensitive sandwich enzyme immunoassay for quantification of cystatin C. It recognized not only free cystatin C but also cystatin C in complexes with cysteine proteinases. The kininogen antibody, HK4, was directed against the third, cysteine proteinase inhibitory domain of the heavy chain of kininogen (Ka = 1 X 10(7) l/mol), but did not influence the papain inhibitory activity of kininogen. It reacted with free kininogen as well as kininogen in complex with cysteine proteinases. Both antibodies could be used for the production of specific immunosorbents.