EXPRESSION AND CHARACTERIZATION OF PLASMEPSIN-I FROM PLASMODIUM-FALCIPARUM

Two aspartic proteinases, plasmepsins I and II, are present in the dig estive vacuole of the human malarial parasite Plasmodium falciparum an d are believed to be essential for parasite degradation of haemoglobin . Here we report the expression and kinetic characterisation of functi onal recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced in to the propart of the zymogen by mutation of Lys110P to Val (P indicat es a propart residue). Appropriate refolding of the mutated zymogen th en permitted pH-dependent autocatalytic processing of the zymogen to t he active mature proteinase. A purification scheme was devised that re moved aggregated and misfolded protein to yield pure, fully processabl e, proplasmepsin I. Kinetic constants for two synthetic peptide substr ates and four inhibitors were determined for both recombinant plasmeps in I and recombinant plasmepsin II. Plasmepsin I had 5-10-fold lower k (cat)/K-m values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhi bit P. falciparum growth, were found to have up to 80-fold lower inhib ition constants for plasmepsin I compared to plasmepsin II. The most a ctive plasmepsin I inhibitors were antagonistic to the antimalarial ac tion of chloroquine on cultured parasites. Northern blot analysis of R NA, isolated from specific stages of the erythrocytic cycle of P. falc iparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest the y are not functionally redundant but play distinct roles in the parasi te.