Purification and biochemical characterization of a novel cysteine protease of Entamoeba histolytica.
Cysteine proteases are important virulence factors of Entamoeba histolytica, the causative agent of amoebiasis. A novel cysteine protease from parasite extracts was purified 15-fold by a procedure including concanavalin A-Sepharose, hydroxylapatite and DEAE-Sepharose chromatography. The purification resulted in the obtainment of an homogeneous protein with a molecular mass of 66 kDa on native PAGE. In 10% SDS/PAGE, three bands of 60, 54 and 50 kDa were evident. Each of the three specific mouse antisera raised against these proteins showed cross-reactivity with the three bands obtained from the purified eluate. The N-terminal sequencing of the first 10 amino acids from the three proteins showed 100% identity. These results support the hypothesis of a common precursor for the 60, 54 and 50-kDa proteins. Protease activity of the purified enzyme was demonstrated by electrophoresis in a gelatine-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate such as N-carbobenzyloxy-arginyl-arginyl-7-amido-4-methylcoumarin. The optimum pH for the protease activity was 6.5; however, enzymatic activity was observed between pH 5 and pH 7.5. Typical of cysteine proteases, the enzyme was inhibited by 4-[(2S, 3S)-carboxyoxiran-2-ylcarbonyl-L-leucylamido]butylg uanidine and iodoacetamide, and activated by free sulfhydryl groups. The cellular location of the enzyme was examined on trophozoites before and after contact with red blood cells using indirect immunofluorescence and cellular fractionation. The 60-kDa cysteine protease translocated to the amoebic surface upon the interaction of trophozoites with red blood cells. This result provided evidence for participation of the 60-kDa protease in erythrophagocytosis.
Spinella
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Biologie et Contrôle des Parasites, Faculté de Pharmacie, Chatenay-Malabry, France.
Levavasseur
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Petek
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Rigothier
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D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000276:immunology / Q000472:pathogenicity; D004749:Entamoebiasis / Q000097:blood / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D015800:Protozoan Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014774:Virulence
D000913:Antibodies, Protozoan; D008565:Membrane Proteins; D011498:Protein Precursors; D015800:Protozoan Proteins; D013438:Sulfhydryl Compounds; C028326:concanavalin A-sepharose; D012685:Sepharose; D003546:Cysteine Endopeptidases