Purification, refolding and autoactivation of the recombinant cysteine proteinase EhCP112 from Entamoeba histolytica

The cysteine proteinase EhCP112 and the adhesin EhADH112 assemble to form the EhCPADH complex involved in Entamoeba histolytica virulence. To further characterize this cysteine proteinase, the recombinant full-length EhCP112 enzyme was expressed and purified under denaturing conditions. After a refolding step under reductive conditions, the inactive precursor (ppEhCP112) was processed to a 35.5 kDa mature and active enzyme (EhCP112). The thiol specific inhibitor E-64, but not serine or aspartic proteinase inhibitors arrested this activation process. The activation step of the proenzyme followed by the mature enzyme suggests an autocatalytic process during EhCP112 maturation. The experimentally determined processing sites observed during EhCP112 activation lie close to processing sites of other cysteine proteinases from parasites. The kinetic parameters of the mature EhCP112 were determined using hemoglobin and azocasein as substrates. The proteinase activity of EhCP112 was completely inhibited by thiol inhibitors, E-64, TLCK, and chymostatin, but not by general proteinase inhibitors. Since EhCP112 is a proteinase involved in the virulence of E. histolytica, a reliable source of active EhCP112 is a key step for its biochemical characterization and to carry out future protein structure-function studies.     

EhCP112 is an Entamoeba histolytica secreted cysteine protease that may be involved in the parasite-virulence

EhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhCPADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.     

Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils

To study the role of cysteine proteinases in the pathogenicity of Entamoeba histolytica , we have attempted to overexpress the three main cysteine proteinases (EhCP1, EhCP2, EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic Entamoeba dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 and EhCP5, respectively, but could substantially induce expression of EhCP2 in both amoeba species by sevenfold, leading to a threefold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not influence expression of other cysteine proteinases and could be attributed to an increase of a single 35 kDa activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba cysteine proteinases are involved in erythrophagocytosis and liver abscess formation, cells overexpressing EhCP2 showed no difference in erythrophagocytosis or liver abscess formation compared with respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.     

The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites

The Entamoeba histolytica EhCPADH complex, formed by a cysteine proteinase (EhCP112) and an adhesin (EhADH112), is involved in adherence, phagocytosis and cytolysis. This makes this complex an attractive candidate as a vaccine against amoebiasis. Here, we produced the recombinant polypeptide EhADH243, which includes the adherence epitope detected by a monoclonal antibody against the EhCPADH complex. EhADH243 was purified, and the effect of the polypeptide on in vitro and in vivo virulence was studied. Antibodies against EhADH243 reacted with the EhCPADH complex and with the recombinant polypeptide. EhADH243 and antibodies against this polypeptide inhibited adherence, phagocytosis and destruction of cell monolayers by live trophozoites, but had little effect on cell monolayer destruction by trophozoite extracts. EhADH243 recognized a 97 kDa protein in the MDCK membrane fraction that could be a putative receptor for E. histolytica trophozoites. Hamsters immunized with EhADH243 developed humoral response against EhCPADH, and animals were partially protected from amoebic liver abscess.     

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.     

Entamoeba histolytica: purification of cathepsin B

A cytotoxic cysteine proteinase with a molecular weight of 16,000 was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified from frozen-thawed strain HM-1 by ion-exchange chromatography on DEAE-cellulose, organomercurial agarose affinity chromatography, and size-exclusion chromatography. The purified enzyme had proteinase activity that could be demonstrated on azocasein (pH 5), hemoglobin (pH 5), or carbobenzoxy-L-arginyl--L-arginyl-7-amino-4-trifluoromethylcoumarin++ + (Z-arg-arg-AFC), a substrate specific for cathepsin B. Enzyme activity was stable to high pH, but not to 40 C for 1 hr or 56 C for 0.5 hr. As typical of cysteine proteinases, inhibition of activity on Z-arg-arg-AFC by p-chloromercuribenzoate or mercury was reversed by free sulfhydryl groups. Both the proteinase and cytotoxic activities of the purified amoebal cathepsin B were inhibited by leupeptin and serum and activated by free sulfhydryl groups, supporting the hypothesis that both activities are characteristics of amoebal cathepsin B. Virulent strains of E. histolytica (HM-1 and Rahman) had significantly more cathepsin B activity per milligram protein than less virulent strains (HK-9, Laredo, and Huff). The correlation between higher levels of cathepsin B activity in strains with greater virulence could indicate a role for amoebal cathepsin B in the pathogenesis of amoebiasis.     

Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica

Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact-dependent killing of host cells via membrane-active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N-terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica , which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three-dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non-pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica .     

Evaluation of the efficacy of a recombinant Entamoeba histolytica cysteine proteinase (EhCP112) antigen in minipig

Cysteine proteinases 112 (EhCP112) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP112 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig-E. histolytica model. There was a 46.29% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP112 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). This is a first report demonstrating that a recombinant form of EhCP112 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP112 for human in the future.     

Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: functional convergence of a common protein fold

Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight β-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.     

Action of the major protease from Entamoeba histolytica on proteins of the extracellular matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica, a cysteine protease of Mr 27,000-29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated alpha 2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Action of the Major Protease from Entamoeba histolytica on Proteins of the Extracellular Matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica , a cysteine protease of M, 27,000–29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated α2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Human placental chorionic renin: production, purification and characterization

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50-200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43,000; the active enzyme 40,000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.     

Cloning, expression and evaluation of the efficacy of a recombinant Entamoeba histolytica cysteine proteinase (EhCP4) antigen in minipig

Cysteine proteinases 4 (EhCP4) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP4 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig-E. histolytica model. There was a 53.16% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP4 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). This is a first report demonstrating that a recombinant form of EhCP4 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP4 for human in the future.     

Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression.

A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.     

Activation of Intracellular Proteinases of Yeast

The existence of the inactive precursors of yeast proteinases B and C was confirmed in the autolysate of baker’s yeast and they were named as pro-proteinases B and C, respectively. The active and inactive forms of proteinase C were two distinct proteins, separable by chromatographical procedures. The two precursors were markedly activated by incubation at pH 5 or by treatment with denaturing agents, e.g. urea, dioxane, acetone and certain alcohols.

These activations were also observed with extracts from acetone-dried cells and from mechanically destructed cells, but the activation of proteinase A was not demonstrated under any conditions tested. Therefore, it was assumed that most of proteinases B and C exist in vivo as inactive precursors, whereas proteinase A originally exists in an active form.

Pro-proteinase C, the latent form of yeast proteinase C, was partially purified from the autolysate of baker’s yeast. It was strongly activated by incubation at pH 5 or by treatment with urea or dioxane. The former activation was prevented by treatment to inactivate yeast proteinase A, which co-existed with the pro-enzyme in the present preparation, but was promoted by addition of purified proteinase A. Thus, it was confirmed that A could activate pro-proteinase C. Furthermore, it was found that activation could be caused by extremes in pH or by heating to 55~60°C, accompanied by the simultaneous destruction of the enzyme produced. Pro-proteinase C was stable over a range of pH 5 to 8 after 60 min incubation at 50°C.     

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.     

Adherens junctions and desmosomes are damaged by Entamoeba histolytica: Participation of EhCPADH complex and EhCP112 protease

Entamoeba histolytica trophozoites adhere to epithelium at the cell–cell contact and perturb tight junctions disturbing the transepithelial electrical resistance. Behind tight junctions are the adherens junctions (AJs) that reinforce them and the desmosomes (DSMs) that maintain the epithelium integrity. The damage produced to AJs and DMSs by this parasite is unknown. Here, we studied the effect of the trophozoites, the EhCPADH complex, and the EhCP112 recombinant enzyme (rEhCP112) on AJ and DSM proteins. We found that trophozoites degraded β‐cat, E‐cad, Dsp l/ll, and Dsg‐2 with the participation of EhCPADH and EhCP112. After contact of epithelial cells with trophozoites, immunofluorescence and transmission electron microscopy assays revealed EhCPADH and rEhCP112 at the intercellular space where they colocalised with β‐cat, E‐cad, Dsp l/ll, and Dsg‐2. Moreover, our results suggested that rEhCP112 could be internalised by caveolae and clathrin‐coated vesicles. Immunoprecipitation assays showed the interaction of EhCPADH with β‐cat and Dsp l/ll. Besides, in vivo assays demonstrated that rEhCP112 concentrates at the cellular borders of the mouse intestine degrading E‐cad and Dsp I/II. Our research gives the first clues on the trophozoite attack to AJs and DSMs and point out the role of the EhCPADH and EhCP112 in the multifactorial event of trophozoites virulence.