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Entamoeba histolytica: purification of cathepsin B
Abstract:
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.
Insights
A cytotoxic cysteine proteinase, amoebal cathepsin B, was isolated from Entamoeba histolytica. Higher levels of this enzyme correlate with increased virulence, suggesting its role in amoebiasis pathogenesis.
Area of Science:
- Biochemistry
- Parasitology
- Molecular Biology
Background:
- Entamoeba histolytica is a protozoan parasite responsible for amoebiasis.
- Virulence factors of E. histolytica are crucial for understanding disease pathogenesis.
- Cysteine proteinases are implicated in parasitic diseases.
Purpose of the Study:
- To isolate and characterize a cytotoxic cysteine proteinase from E. histolytica.
- To investigate the role of this enzyme in E. histolytica virulence.
- To determine if the enzyme's activity correlates with parasite strain virulence.
Main Methods:
- Purification of the enzyme using ion-exchange, affinity, and size-exclusion chromatography.
- Enzyme activity assays using azocasein, hemoglobin, and a cathepsin B-specific substrate (Z-arg-arg-AFC).
- Assessment of enzyme stability and inhibition/activation by various agents.
- Quantification of enzyme activity in different E. histolytica strains of varying virulence.
Main Results:
- A 16,000 MW cytotoxic cysteine proteinase, identified as amoebal cathepsin B, was purified.
- The enzyme exhibited proteinase activity and its function was dependent on free sulfhydryl groups.
- Both proteinase and cytotoxic activities were inhibited by leupeptin and serum, and activated by sulfhydryl groups.
- Virulent E. histolytica strains showed significantly higher cathepsin B activity compared to less virulent strains.
Conclusions:
- The purified amoebal cathepsin B possesses both proteinase and cytotoxic activities.
- These activities are characteristic of amoebal cathepsin B and are linked to its function.
- Elevated cathepsin B activity in virulent E. histolytica strains suggests a role in the pathogenesis of amoebiasis.
