Proteolytic processing of the merozoite surface protein 1 from Plasmodium falciparum: Implications on its structure and function during invasion

Abstract

The most virulent form of malaria is caused by the apicomplexan parasite Plasmodium falciparum, which invades and replicates within erythrocytes. The most abundant surface protein of merozoites and a prime vaccine candidate is the merozoite surface protein 1 (MSP-1). In the course of merozoite maturation and invasion, MSP-1 is proteolytically processed several times. The parasite’s subtilisin-like protease PfSUB1 converts the MSP-1 precursor into MSP-183, MSP 130, MSP-138 and MSP-142. This primary processing may be essential for priming the malaria parasites for erythrocyte invasion and thus merozoite infectivity. The aim of this study was to unravel the biological significance of primary processing for MSP-1 function using a number of approaches. PfSUB1 cleavage sites within MSP 1 were identified in silico. Recombinant proteins with mutations in the respective sites were generated to investigate their in vitro cleavage susceptibility by exposure to recombinant PfSUB1. Some cleavage sites, in particular between MSP-138 and MSP-142, revealed redundant PfSUB1 recognition sequences, suggesting an important role of processed MSP-1. Mutation of all PfSUB1 cleavage sites rendered the protein entirely refractory to processing by the protease. To study the direct effect of primary processing on invasion, the binding of the MSP-1 precursor as well as its processed fragments to erythrocytes was investigated. Only PfSUB1-cleaved MSP-1 bound to fresh erythrocytes via the MSP-183 and MSP-138 fragments. This attachment was inhibited by MSP-1 specific antibodies but not by heparin or enzymatic treatment of erythrocytes, indicating MSP-1 to be a common receptor for invasion. In another approach, the effect of non-cleavable MSP-1 on parasite viability was investigated. Various cleavage-deficient versions of full length msp-1f (FCB1 strain) were expressed in P. falciparum 3D7 parasites which were analysed for their growth and MSP-1 expression. Dose-dependent regulation of the latter was accomplished via the blasticidin co-selection system. Indeed, transgenic MSP-1F correctly localised to the merozoite surface. However, primary processing of MSP 1 was only observed for wildtype MSP-1F, while the mutant versions remained as refractory to cleavage in vivo as previously found in vitro. Although only low levels of non-cleavable MSP-1F were tolerated in transgenic parasites, they displayed a significant growth defect. In summary, this study shows that PfSUB1 cleavage of MSP-1 is vital for merozoite maturation and infectivity and thus for parasite viability. A key function of processed MSP-1 is the direct binding to erythrocytes, thereby providing a prerequisite for merozoite invasion and replication.