Abstract
The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a phospho-protein with three identified phosphorylation sites (Ser(33), Ser(411) and Thr(416)) at its cytosolic N- and C-termini. In this study, we report on the characterization of PfCRT anti-serum and show the presence of three epitope-specific immunoglobulin (IgG) pools (i.e., IgG-P1, P2, and P3), each recognizing a different epitope in PfCRT cytoplasmic C-terminal. IgG-P2 bound the heptapeptide sequence ((408)NEDSEGE(414)), including Ser(411). The effect of Ser(411) phosphorylation on the binding specificity of IgG-P2 was confirmed using heptapeptides and full-length PfCRT with substitutions of Ser(411) with aspartic acid (phospho-serine mimic) and alanine residues. Moreover, using purified IgG-P2, we show the presence of PfCRT homodimer that has unphosphorylated Ser(411) and migrates with an apparent molecular mass of 90 kDa on SDS-PAGE. In addition, parasite lysates showed PfCRT to be more phosphorylated at Ser(411) in CQ-sensitive (3D7) than CQ-resistant (Dd2-H) strains of P. falciparum. Taken together, the findings of this study suggest a role for Ser(411) phosphorylation in PfCRT structure-function. (C) 2021 Elsevier Inc. All rights reserved.