Specificity of a cysteine proteinase of Entamoeba histolytica towards the α1-CB2 peptide of bovine collagen type I

Schulte, Werner; Scholze, Henning; Werries, Eckhard

doi:10.1016/0166-6851(87)90016-8

Cited by 17 publications

(5 citation statements)

References 14 publications

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“…Autoradiograms of gels clearly show three areas of radiolabeled bands, three of them at molecular sizes of 73, 68, and 56 kDa, others at molecular sizes of 40, 39, and 35 kDa, and the smaller molecular species at molecular sizes of 29 and 27 kDa. The soluble cytoplasmic proteins remaining after sedimentation by high-speed centrifugation contained only the lower-molecular-size bands (molecular sizes of 27 FIG. 3.…”

Section: Resultsmentioning

confidence: 99%

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica

Meester¹,

Shaw²,

Scholze³

et al. 1990

Infect Immun

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Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 ,ug/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN2). lodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 ,ug/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-,-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of 1251_ labeled protease inhibitor (10 ,ug/ml) showed as many as eight radioactive bands by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons). Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.

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Section: Resultsmentioning

confidence: 99%

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica

Meester¹,

Shaw²,

Scholze³

et al. 1990

Infect Immun

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“…In these cases reversed-phase high performance liquid chromatography (RP-HPLC) with its capability of separating the product from the substrate of the reaction could provide an invaluable method of monitoring such activities [16-191. In some cases, however, although the sensitivity of the assay is increased, HPLC is less suitable than spectrophotometry for routine analyses [16][17][18][19]. Thus, due to the low amount of sample and the short analysis times required, the potential of high performance capillary electrophoresis (HPCE) could be an attractive alternative to current methods.…”

Section: Introductionmentioning

confidence: 99%

Micellar electrokinetic chromatography: A convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity

et al. 1998

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High performance capillary electrophoresis (HPCE) has been exploited as an analytical method alternative to current procedures for the determination of proteolytic activity of elastases from different sources. Due to some drawbacks with capillary zone electrophoresis (CZE), the mode of operation employed for the assay of elastolytic activity was micellar electrokinetic chromatography (MEKC). Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM SDS and 15% v/v methanol, separation of intact peptide substrate from products of proteolytic reaction was easily achieved in a fused-silica capillary of 50 cm effective length x 75 microm ID. This allowed us to determine the rate of hydrolysis of substrates and to calculate the kinetic parameters Km and k(cat) of the proteases investigated. A comparison of these data with those obtained from high performance liquid chromatography (HPLC)-based experiments showed that MEKC is a convenient technique for studying protease kinetics.

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“…The analysis of E. histolytica extracts has shown several proteinases in the range of 16 kDa to ~150 kDa [ 59 , 60 ], but, in particular, four to six bands of proteinase activity are usually identified on gelatin-polyacrylamide gels. The first similar, but distinct, cysteine proteinase activities purified and characterized from E. histolytica were amebapain [ 61 ] and histolysin [ 62 ], now known as EhCP1 and EhCP2, respectively. Together with EhCP5, which was identified as the main surface-localized CP of the parasite [ 63 ], these three proteases were found to be highly expressed in trophozoites of E. histolytica to such an extent that they represent 90% of CP-encoding transcripts and virtually all of the CP activity identified in the amoeba [ 64 ].…”

Section: Entamoeba Histolytica Cysteine Proteases: Repertoir...mentioning

confidence: 99%

Extracellular Cysteine Proteases of Key Intestinal Protozoan Pathogens—Factors Linked to Virulence and Pathogenicity

Argüello-García

Carrero

Ortega-Pierres

2023

IJMS

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Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens’ virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CPs, some of which have been characterized. CPs of Giardia duodenalis (e.g., Giardipain-1) and Entamoeba histolytica (EhCPs 1-6 and EhCP112) degrade mucin and villin, cause damage to intercellular junction proteins, induce apoptosis in epithelial cells and degrade immunoglobulins, cytokines and defensins. In Cryptosporidium, five Cryptopains are encoded in its genome, but only Cryptopains 4 and 5 are likely secreted. In Blastocystis sp., a legumain-activated CP, called Blastopain-1, and legumain itself have been detected in the extracellular medium, and the former has similar adverse effects on epithelial integrity and enterocyte survival. Due to their different functions, these enzymes could represent novel drug targets. Indeed, some promising results with CP inhibitors, such as vinyl sulfones (K11777 and WRR605), the garlic derivative, allicin, and purified amoebic CPs have been obtained in experimental models, suggesting that these enzymes might be useful drug targets.

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Specificity of a cysteine proteinase of Entamoeba histolytica towards the α1-CB2 peptide of bovine collagen type I

Cited by 17 publications

References 14 publications

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica

Micellar electrokinetic chromatography: A convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity

Extracellular Cysteine Proteases of Key Intestinal Protozoan Pathogens—Factors Linked to Virulence and Pathogenicity

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