👤 James H McKerrow

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence. Show less

Extracellular neutral cysteine proteinases are an important virulence factor of E. histolytica. Experimental evidence supporting its role in invasion includes the ability to degrade components of the extracellular matrix and activate complement by specifically cleaving C3. We had previously reported the isolation of fragments encoding cysteine proteinase genes from HM-1 (ACP1) and a nonpathogenic strain (REF291, ACP2) by PCR using consensus sequences based on conserved structural motifs of eukaryotic cysteine proteinases. Using similar techniques, we have now identified a third gene encoding a cysteine proteinase which is present in both pathogenic and nonpathogenic strains and have correlated cysteine proteinase specific-mRNA levels with enhanced proteolytic activity and cytopathic effect on a fibroblast cell monolayer, a quantitative assay of virulence. Show less