Fig. 5.

Analysis of observed and predicted C1s cleavage sites in PfEMP1. (A) SDS/PAGE showing C1s cleaves the full-length recombinant ectodomains of IT4var13 and IT4var20 PfEMP1 into two major bands (arrows, numbered 1–4). (B) Protein schematics of the IT4var13 and IT4var20 ectodomains showing the C1s cleavage products (black arrows, above the protein schematic from A). The boxed Insets show the interdomain sequences where C1s cleavage sites were identified in IT4var13 and IT4var20 by LS/MS and N-terminal protein sequencing analyses. C1s cleaves at one site in IT4var13 and three sites in IT4var20 (red arrows) at residues (large amino acid letters) resembling C1s-motifs. Of the four C1s-like sequence motifs found in IT4var20, three (red arrows) in B were experimentally confirmed and the fourth site (green arrow) in B may also have been cleaved but could not be validated due to its presence in a small peptide product lost in gel electrophoresis. (C) C1s cleavage sites in PfEMP1 resemble previously identified semiconserved sequence motifs, known as homology blocks HB49, HB134, and HB241 (11), found in the otherwise low sequence complexity interdomain regions of PfEMP1. (D) Reanalysis of conserved sequence motifs in PfEMP1 interdomains from 15 long-read sequenced P. falciparum genomes using MEME confirms the presence of C1s-like cleavage motifs (sequence LOGOs shown at Left). The right side shows 275 representative full-length PfEMP1 from 15 PacBio long-read sequenced P. falciparum genomes (44). The different PfEMP1 are stacked from Top to Bottom in three columns and aligned by their domain composition (not scaled to sequence length). Each domain type is color-coded, and white gaps between domains represent interdomain regions, which vary from 10 to 225 amino acids in length. Interdomain regions containing one or more predicted C1s-like cleavage site(s) (MEMEs 1 and 2) are indicated by red crosses. ATS, acidic terminal segment or cytoplasmic tail; NTS, N-terminal segment at the beginning of protein.