Figure 4.

Plasmepsin V activity. a) Substrate cleavage. PM V was isolated from parental (3D7) and PM V-GFP fusion (DC6) clones using anti-GFP (yellow and red, respectively) or anti-PM V (blue and green, respectively). A similar isolation without Ab (protein A) served as a control for non-specific binding (purple and cyan, respectively). Purified enzyme was incubated at pH 6.5 for 35 min with fluorogenic peptide (Anaspec) corresponding to the PEXEL motif for HRPII (DABCYL-LNKRLLHETQ-EDANS). Error bars indicate standard deviation of three activity curves. b) PM V specificity for PEXEL motif. Processing of fluorogenic peptides containing the PEXEL motifs of HRPII (blue) or PfEMP2 (red, DABCYL-RYVRILSETE-EDANS) are shown. Left - wild-type peptide (WT); middle - L to A mutant peptide; right - R to A mutant peptide. Inset: peptide sequences with mutated residues in black. Error bars as in a. c) pH dependence in Tris-Malate buffer. Two separate experiments are shown in different colors. Error bars as in a. d) Activity of active-site mutant PM V compared to wild type. PMV-GFP was isolated from 3D7 (untransfected mock isolation), wild-type- or mutant-PM V-GFP-transfected parasites using anti-GFP for purification. Activity on HRPII-PEXEL peptide is normalized to PM V protein content. Error bars as in a. e) Cleavage of pro-HRPII by isolated enzyme from d. p: pro-form; m: mature protein.