Blastocystis legumain is localized on the cell surface, and specific inhibition of its activity implicates a pro-survival role for the enzyme.
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.
Wu
Binhui
B
Department of Microbiology, Yong Loo Lin School of Medicine, Life Sciences Institute, National University of Singapore, Singapore 117597.
Yin
Jing
J
Texier
Catherine
C
Roussel
Michaël
M
Tan
Kevin Shyong-Wei
KS
D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology; D016844:Blastocystis / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D016923:Cell Death; D002470:Cell Survival; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D053938:DNA Fragmentation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020287:In Situ Nick-End Labeling; D051379:Mice; D008969:Molecular Sequence Data; D010718:Phosphatidylserines / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D021381:Protein Transport; D051381:Rats; D013379:Substrate Specificity; D000099240:Asparaginyl Endopeptidase
D017304:Annexin A5; D000911:Antibodies, Monoclonal; D003546:Cysteine Endopeptidases; D010718:Phosphatidylserines; D011480:Protease Inhibitors; D000099240:Asparaginyl Endopeptidase