Fig. 1: Mutation of PMX cleavage site in PfRh5 reveals alternative processing.

a Construction of P. falciparum parasites expressing mutant PfRh5 at the PMX cleavage recognition sequence. Wild-type cleavage is shown with the mutant forms listed in the grey box. Cas9 cut site in pfrh5 is shown. Pfrh5 genes containing wt or mutant PMX cleavage sites were HA-tagged. Right-pointing arrow is the endogenous pfrh5 promoter. Black stalk depicts endogenous pfrh5 terminator. White stalk depicts P. berghei dhfr-ts terminator. Selection for WR99210-resistant parasites encoded by human dhfr. The five transfected genes integrated into the genome (Y = Yes). b Schematic of PfRh5 cleavage site residues⁵¹ and updated sequence logo (weblogo.berkeley.edu) for PMX cleavage sites. c Mutation of PfRh5 cleavage site identifies alternative processing sites. Immunoblots were probed with anti-HA mAb and anti-hsp70 for protein quantitation. Right panels show schematics of predicted unprocessed protein (p64), predicted PMX-cleaved product (p50) and observed cleavage fragments in the mutant parasites (p54 and p53). Cleavage at NFLQ site generating p50 highlighted in blue. The mean values for unprocessed/processed band intensities are shown. Band intensities for PfRh5 unprocessed/processed were calculated from the five parasite lines in three independent experiments. d Effect of mutations in endogenous NFLQ motif on cleavage by PMX. Mutation of either the P1’ or P1 residue block NFLQ cleavage and generation of the p50 product. e Processing Inhibition Assays (PIA) with Rh5-NFAA parasites, using PMX-specific inhibitor WM4 and the PMIX/PMX dual inhibitor WM382 probed with anti-HA mAb. Lower panel shows schematic of observed products. Band intensities for PfRh5 unprocessed/processed were calculated from Control and WM4 merozoites for three independent experiments. Error bars represent standard deviation. f First 160 amino acids of PfRh5 showing putative alternative cleavage sites and mutated NFAA sequence. Lower-case letters are the signal sequence. g Construction of P. falciparum parasites with C-terminally HA-tagged prodomain (Rh5PD-CHA). HA-tag was placed N-terminal to the ‘NFLQ’ PMX cleavage site. h PIA with Rh5PD-CHA parasites using Compound 1 (C1) and probed with anti-HA mAb. Schematic shows expected and observed proteins. Putative non-preferred (alternative) cleavage sites (VFNQ & LLNE) and the known PMX site (NFLQ) are shown. The p17 product is the HA-tagged prodomain (PD), p7 product the HA-tagged ‘VFNQ-to-NFLQ’ protein and p6 the ‘LLNE-to-NFLQ’ protein. Source data are provided as a source data file.