Epitope specific antibodies to N- and C cytoplasmic domains of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) differentiate native and post-translationally modified variant

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser⁴¹¹ and Thr⁴¹⁶, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser⁴¹¹ and Thr⁴¹⁶. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined.