Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA)

The Plasmodium falciparum serine repeat antigen (SERA), a malaria vaccine candidate, is processed into several fragments (P73, P47, P56, P50, and P18) at the late schizont stage prior to schizont rupture in the erythrocytic cycle of the parasite. We have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not an autocatalytic event. The processing activities appeared at late schizont stage. The proteases are membrane associated, correlating with the secretion and accumulation of SERA within the parasitophorous vacuole membrane (PVM). The activity responsible for the primary processing step of SERA to P47 and P73 was inhibited by serine protease inhibitor DFP. In contrast, the activity responsible for the conversion of P56 into P50 was inhibited by each of the cysteine protease inhibitors E-64, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing is mediated by at least three distinct proteases.