Entamoeba histolytica: Purification of cathepsin B

Lushbaugh, William B.; Hofbauer, Ann; Pittman, Fred E.

doi:10.1016/0014-4894(85)90088-8

Cited by 71 publications

(36 citation statements)

References 29 publications

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“…Cell fractionation studies indicate that although present in the cytosol, they are enriched in the plasma and internal membranes. In addition, secreted protease activities of 16, 26, and 56 kDa have been reported (64,80,81). A total of six distinct genes (EhCP1 through EhCP6) encoding prepro forms of cysteine proteinases have been identified.…”

Section: Early Invasive Lesions With Superficial Ulcerationmentioning

confidence: 99%

Pathogenesis of Intestinal Amebiasis: From Molecules to Disease

Espinosa‐Cantellano¹,

Martı́nez-Palomo²

2000

Clinical Microbiology Reviews

191

115

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Section: Early Invasive Lesions With Superficial Ulcerationmentioning

confidence: 99%

Pathogenesis of Intestinal Amebiasis: From Molecules to Disease

Espinosa‐Cantellano¹,

Martı́nez-Palomo²

2000

Clinical Microbiology Reviews

191

115

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“…All of the ameba cp genes encode mature cysteine proteases with calculated molecular masses between 24 and 35 kDa. However, earlier studies using substrate gel electrophoresis suggested that E. histolytica contains a considerable number of cysteine proteases ranging from 16 to more than 100 kDa, some of which were considered to be associated with the ameba membrane (3,19,24,27,30,35). This may imply that only a subset of E. histolytica cysteine proteases has been identified so far.…”

mentioning

confidence: 99%

The Intestinal Protozoan ParasiteEntamoeba histolyticaContains 20 Cysteine Protease Genes, of Which Only a Small Subset Is Expressed during In Vitro Cultivation

Loftus²,

et al. 2003

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Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.Cysteine proteases (EC 3.4.22) of the papain superfamily occur in a wide range of organisms including bacteria, plants, invertebrates, and vertebrates (7). In mammals, they are well documented as intracellular enzymes involved in protein turnover within lysosomes. In addition, extracellular cysteine proteases have been implicated in various physiological and pathophysiological processes, including tumor invasion and metastasis (33,43). Cysteine proteases are important virulence factors of various infectious agents and the main proteolytic enzymes in many protozoon parasites (22,29). The protozoan Entamoeba histolytica, the causative agent of human amoebiasis, is characterized by its great capacity to destroy host tissue, leading to potentially life-threatening diseases such as ulcerative colitis or liver abscess. Convincing evidence exists that cysteine proteases are essential for E. histolytica-induced pathology. Treatment of the ameba with sublethal doses of a specific cysteine protease inhibitor or the addition of laminin, which blocks the substrate-binding pocket of cysteine proteases, greatly reduces its ability to produce liver abscesses in laboratory animals (18, 37). Likewise, liver abscess formation was totally blocked and significantly less gut inflammation and damage to the intestinal permeability barrier were obser...

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“…Nevertheless, when such bands were examined with SDS-PAGE, they revealed several bands with different molecular masses and proteolytic activities, which reverted to a single protein band of apparent 30 kDa when the sample was boiled in the presence of EDTA, DTT and iodoacetamide and reexamined by SDS-PAGE. Since all genes so far cloned that encode proteases of Entamoeba histolytica have been found to encode proteins of approximately 30 kDa (Bruchhaus et al 1996), the existence of CPs derived from the same parasite with molecular masses varying over 16-96 kDa (Avila and Caldero´n 1993;Keene et al 1986;Luaces and Barrett 1988;Lushbaugh et al 1985;McLaughlin and Faubert 1976;Montfort et al 1994;Navarro-Garcı´a et al 1995) should have a posttranslational explanation. It was suggested (Lo´pez-Revilla et al 1993) that, during SDS-PAGE, the lower molecular mass CPs are derived from a higher molecular mass precursor of CPs by autoproteolysis.…”

Section: Discussionmentioning

confidence: 96%

“…Purified amebic CPs have a cytopathic effect on monolayers of HeLa cells (Lushbaugh et al 1985), BHK cells (Keene et al 1986) and human fibroblasts (Luaces and Barrett 1988), without cytotoxic effect.…”

Section: In Vitro Experimentsmentioning

confidence: 99%

Amebic cysteine proteinase 2 (EhCP2) plays either a minor or no role in tissue damage in acute experimental amebic liver abscess in hamsters

Olivos-García¹,

González-Canto²,

López-Vancell³

et al. 2003

Parasitol Res

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Amebic cysteine protease 2 (EhCP2) was purified from ethyl ether extracts of axenically grown trophozoites of Entamoeba histolytica strain HM1-IMSS. The purification procedure involved molecular filtration and electroelution. Sequence analysis of the purified product revealed EhCP2 and ubiquitin(s). Electrophoretic migration patterns, isoelectric point determination and Western blot studies failed to reveal other EhCP molecules. Polyclonal antibodies against the purified EhCP2 prepared in rabbits either stabilized or enhanced the enzyme activity in a dose-response manner. Purified EhCP2 was enclosed within inert resin microspheres (22-44 microm in diameter) and injected into the portal vein of normal hamsters. In the liver, the microspheres caused mild acute inflammation and occasional minimal necrosis of short duration. Sections of the liver were immunohistochemically stained with the anti-EhCP2 antibody and the microspheres were positive for only a very short period (1 h) after injection. Sections of experimental acute (1 day, 5 days) amebic liver abscess produced in hamsters were also stained with the anti-EhCP2 antibody; and amebas were intensely positive but no staining was observed at any time in the surrounding necrotic structures. It is suggested that EhCP2 plays either a minor or no role in the causation of tissue damage in experimental acute liver amebiasis.

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Entamoeba histolytica: Purification of cathepsin B

Cited by 71 publications

References 29 publications

Pathogenesis of Intestinal Amebiasis: From Molecules to Disease

Pathogenesis of Intestinal Amebiasis: From Molecules to Disease

The Intestinal Protozoan ParasiteEntamoeba histolyticaContains 20 Cysteine Protease Genes, of Which Only a Small Subset Is Expressed during In Vitro Cultivation

Amebic cysteine proteinase 2 (EhCP2) plays either a minor or no role in tissue damage in acute experimental amebic liver abscess in hamsters

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