Role of cysteines in Plasmodium falciparum circumsporozoite protein: Interactions with heparin can rejuvenate inactive protein mutants

Various pathogenic bacteria, viruses, and protozoan bind to glycosaminoglyc an-based receptors on host cells and initiate an infection. Sporozoites of Plasmodium predominantly express circumsporozoite (CS) protein on their sur face, which binds to heparan sulfate proteoglycans on liver cell surface th at subsequently leads to malaria. Here we show that the interaction of free heparin with this parasite ligand has the potential to be a critical compo nent of invasion. CS protein of P. falciparum contains four cysteines at po sitions 361. 365, 396. and 401. In this study, all four cysteine residues w ere mutagenized to alanine both individually and in different combinations. Conversion of cysteine 396 to alanine (protein CS3) led to a 10-fold incre ase in the binding activity of the protein to HepG2 cells. Replacement of c ysteines at positions 361, 365. and 401 either alone or in different combin ations led to a near total loss of binding. Surprisingly, activity in these inactive mutants could be effectively restored in the presence of submolar concentrations of heparin. Heparin also up-regulated binding of CS3 at sub molar concentrations with respect to the protein but downregulated binding when present in excess. Given the significantly different concentrations of heparin in different organs of the host and the in vitro results described here one can consider in vivo ramifications of this phenomenon for pathoge n targeting of specific organs and for the functional effects of antigenic variation on receptor ligand interaction.