Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

doi: 10.1073/pnas.2104166118

Figure Lengend Snippet: Fig. 1. Serum fractions B11 and C3 inhibit IT4var19 IE binding to brain endothelial cells. (A) Chromatographic fractionation of serum by size exclusion chromatography. IgG-depleted serum was run on a Superdex 200 Increase 10/300 GL column. Protein fractions were eluted using a linear gradient and the absorbance (mAbs) monitored at 280 nm. The arrows show the binding inhibitory fractions B11 and C3. (B) Binding of IT4var19 IEs to HBEC-5i in binding medium with 10% human serum, IgG-depleted human serum, or serum fractions (A6-C6). A no-serum control was included. Each data point is from two independent experiments done in duplicate, and the mean and SEM are shown for each fraction. Fractions B11 and C3 inhibited the binding the most. These fractions were run on MS/MS, and the components were run to determine their effect on binding as shown in C to identify potential inhibitors for binding. (C) Binding of IT4var19 IEs to HBEC-5i in binding medium with proteins identified by MS/MS in the serum inhibitory B11 and C3 fractions (attractin, C1s, C4b, factor H, IgD, ITIH1, ITIH2, and kappa light chain). Data are mean ± SEM, and each data point is from an independent experiment. **P < 0.01; ***P < 0.001.

Article Snippet: For binding inhibition assays, serum and serum fractions (at 10%), recombinant human attractin (7238-AT-050; Novus Biologicals), C1s (A104; Complement Technology; or 2060-SE; R&D Systems), human complement C4b (H00000721-Q01; Novus Biologicals), human complement factor H (H00003075-P03; Novus Biologicals), IgD (NB100-62667; Novus Biologicals), human kappa light chain (H00003514-P01; Novus Biologicals), human inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) (H00003697-P01; Novus Biologicals), and H2 (ITIH2) (NBP2-31750PEP; Novus Biologicals) (at physiological concentrations in serum, ∼10% of the physiological concentrations or an estimated concentration where serum levels could not be found; see SI Appendix, Table S1) were added to the parasites in DMEM binding medium immediately before coincubation with HBEC-5i.

Techniques: Binding Assay, Fractionation, Size-exclusion Chromatography, Control, Tandem Mass Spectroscopy