Figure 1. Erythrocyte-binding assay on transfected COS7 cells expressing different regions of PfRH1 before and after enzymatic treatment.

(A) Chimeric constructs for the expression of different regions of PfRH1 in 3D7. 3D7 clone RH1 DNA sequence (Genbank accession no: AF533700) with intro spliced out includes signal sequence (SS, black) and Exon2 (grey) encoding a large extracellular domain and excludes transmembrane domain and cytoplastic tail. The extracellular domain was divided into eight regions (From I to VIII). Each region (black line with amino acids no.) is approximately 2 kb with 1 kb overlap between 2 consecutive regions except VIII, which is 1.3 kb. (B) Duffy-positive human erythrocytes bound to the surface of COS7 cells were visualized as rosettes at a magnification of ×200. (i) Two cells formed rosettes with Duffy-positive human erythrocytes on bright field. (ii) Cell-associated fluorescence as GFP (green) was found on the same field as in panel (i). (iii) Negative count of rosette on transfected COS7 cells on bright field. (iv) But two transfected cells (green) were shown on the same field as in panel (iii). Bar, 50um. (C) Comparison of Erythrocyte-binding assay with different regions of PfRH1 after enzymatic treatment. Rosettes were scored in 30 fields at 200× magnification. Data are present as percentage of binding activity (%) after the number of observed rosettes was normalized to 5% transfection efficiency in three independent experiments. The error bar denotes the SE. * p<0.001, indicating that the binding ability of RII is significantly different from those obtained from other regions. + p<0.001, indicating that after neuraminidase-treatment, the binding of RII was significantly decreased compared to untreated erythrocytes.