Blastocystis Legumain Is Localized on the Cell Surface, and Specific Inhibition of Its Activity Implicates a Pro-survival Role for the Enzyme

Abstract

Programmed cell death (PCD) is crucial for cellular growth and development in multicellular organisms. Although distinct PCD features have been described for unicellular eukaryotes, homology searches have failed to reveal clear PCD-related orthologues among these organisms. Our previous studies revealed that a surface-reactive monoclonal antibody (mAb) 1D5 could induce multiple PCD pathways in the protozoan Blastocystis. In this study, we identified, by two-dimensional gel electrophoresis and mass spectrometry, the target of mAb 1D5 as a surface-localized legumain, an asparagine endopeptidase that is usually found in lysosomal/acidic compartments of other organisms. Recombinant Blastocystis legumain displayed biphasic pH optima in substrate assays, with peaks at pH 4 and 7.5. Activity of Blastocystis legumain was greatly inhibited by the legumain-specific inhibitor carbobenzyloxy-Ala-Ala-AAsn-epoxycarboxylate ethyl ester (APE-RR) (where AAsn is aza-asparagine) and moderately inhibited by mAb 1D5, cystatin, and caspase-1 inhibitor. Interestingly, inhibition of legumain activity induced PCD in Blastocystis, observed by increased externalization of phosphatidylserine residues and in situ DNA fragmentation. In contrast to plants, in which legumains have been shown to play a pro-death role, legumain appears to display a pro-survival role in Blastocystis.Programmed cell death in the unicellular protozoa is now accepted as a well established phenomenon. Several stereotypic apoptotic morphological markers similar to those observed in apoptotic metazoan cells have been described in human parasitic protozoa such as Leishmania amazonensis, Leishmania donovani, Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma rhodesiense, Plasmodium falciparum, and Blastocystis (1, 2). Despite a wealth of information on the organelles and cytochemical features involved in protozoan PCD, there is a scarcity of information on PCD 3 -related molecular mediators. Our earlier studies showed that Blastocystis undergoes programmed cell death when exposed to the surface-reactive monoclonal antibody mAb 1D5 with typical features of apoptotic cells (3, 4). mAb 1D5 was shown to target a 30-kDa protein found on the plasma membrane of Blastocystis (5-7). This protein is functionally important, but not all cells within a clonal population would be susceptible to the cytotoxic effects of mAb 1D5 (6). These results suggest that this protein may have multiple localizations and is potentially important for cell survival. In this study, an asparaginyl cysteine protease legumain was identified as the mAb 1D5 targeting protein. Legumain is a recently described lysosomal protease, well conserved and present in plants, mammals, helminth worms, and the protozoan Trichomonas vaginalis (8 -12). The active site of legumain contains the catalytic dyad His-Gly-spacer-Ala-Cys, a characteristic shared with caspases, aspartyl cysteine proteases important as molecular mediators of apoptosis cascades (13). Legumains have specificity for the hydrolysis of bonds on the carb...

A Rapid, High-Throughput Viability Assay forBlastocystisspp. Reveals Metronidazole Resistance and Extensive Subtype-Dependent Variations in Drug Susceptibilities

“…The significance of cysteine proteases in Blastocystis pathobiology is well reported (36,48,58,71). In this study, Inhibition of cysteine protease activity of the parasite by IA resulted in complete inhibition of all four isolates with similar IC 50 s, suggesting the importance of cysteine proteases in parasite survival.…”

“…Blastocystis cysteine proteases have been reported to cleave human secretory IgAs (58) and to induce upregulation of proinflammatory cytokines (48). A prosurvival role of legumain, a cysteine protease, has also been reported recently for Blastocystis (71). Accumulating data in recent years suggest the therapeutic potential of protease inhibitors in parasitic infections (1,42).…”

“…Concomitantly, to determine whether Blastocystis undergoes morphological changes after drug exposure, parasites were stained with propidium iodide and annexin V-FITC. Both PI and annexin V stain only dying parasites (71). PI binds to the parasite nuclear material (71).…”

“…Both PI and annexin V stain only dying parasites (71). PI binds to the parasite nuclear material (71). Annexin V binds with high affinity to phosphatidylserine (PS).…”

“…Annexin V binds with high affinity to phosphatidylserine (PS). PS is located at the cytosolic face of the cell membrane and has access to annexin V only when it becomes exposed at cell death (71). Healthy parasites are impermeable to both PI and annexin V (71).…”