A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells
Fig 2
TvROM catalytic activity analyses.
The activity of T. vaginalis rhomboid proteases were tested by co-transfecting the proteases with known model rhomboid substrates in a heterologous cell cleavage assay using HEK293 cells. Proteases were HA tagged and substrates contained an N-terminal GFP tag to allow detection. (A, B, D and E) Whole cell lysates (WCL) and conditioned media (CM) collected from co-transfectants was analyzed by Western blot analyses [39]. Top panels: rhomboid protease detected in WCL using an anti-HA antibody; middle panels: full-length (FL, filled arrowheads) and cleaved substrates (open arrowheads) detected in WCL using an anti-GFP antibody; bottom panels: cleaved substrate fragments detected in CM using an anti-GFP antibody (open, red arrowheads). The substrates tested were (A) APP+7 residues of the Drosophila melanogaster Spitz protein encompassing the rhomboid protease cleavage site (APP+Spi7), (B) Plasmodium falciparum EBA-175, (D) human EphB3, and (E) Providencia stuartii TatA. The positive control HA-tagged rhomboid protease (lane 2) used for cleavage of Spitz (A), human EphB3 (D), and TatA (E) was DmRho1; the positive control protease for cleavage of EBA-175 (B) was HA-tagged PfROM4 (lane 2). Negative controls (lane 1) were transfected with only substrates. TvROM1/TvROM3 = wild type protease; TvROM1 mut/TvROM3 mut = protease with the catalytic histidine mutated to alanine. TvROM3 was found to cleave only the Spitz TM domain segment of APP+Spi7 (A middle panel, lane 5) whereas TvROM1 does not cleave the Spitz sequence (A bottom panel, lane 3) but does cleave the other 3 substrates (B, D, and E bottom panels, lane 3). (F) Summary of the cleavage data shown in panels A, B, D, and E. (C) The location of EBA-175 cleavage by PfROM4 (control; top panel) and TvROM1 (bottom panel) determined by subjecting the immunoprecipitated cleavage fragment to MALDI-TOF analysis. Red arrows indicate that both proteases cleave the substrate between the two small amino acids alanine (A) and glycine (G).