Abstract
Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies. Refolding conditions were optimized and zymogen was processed into a mature form via cleavage at the Asn80-Ala81 peptide bond. Mature plasmepsin V exhibited a pH optimum of 5.5-7.0 with Km and kcat of 4.6 μM and 0.24s(-1), respectively, at pH 6.0 using the substrate DABCYL-LNKRLLHETQ-E(EDANS). Furthermore, the prosegment of proplasmepsin V was shown to be nonessential for refolding and inhibition. Unexpectedly, unprocessed proplasmepsin V was enzymatically active with slightly reduced substrate affinity (∼ 2-fold), and similar pH optimum as well as turnover compared to the mature form. Both zymogenic and mature plasmepsin V were partially inhibited by pepstatin A as well as several KNI aspartic protease inhibitors while certain metals strongly inhibited activity. Overall, the present study provides the first report on the nonessentiality of the prosegment for plasmepsin V folding and activity, and therefore, subsequent characterization of its structure-function relationships of both zymogen and mature forms in the development of novel inhibitors with potential antimalarial activities is warranted.