Two aspartic proteinases, plasmepsins 1 and 11, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin 1. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of LysllOP to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH-dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin 1. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin 11. Plasmepsin I had 5-10-fold lower kc.,,/K,n values than plasmepsin I1 for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P falciparum growth, were found to have up to 80-fold lower inhibition constants for plasmepsin I compared to plasmepsin 11. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of I? falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin I1 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 they are not functionally redundant but play distinct roles in the parasite.

A gene (hap) transcribed during the intra-erythrocytic life cycle stages of the human malaria parasite Plasmodium falciparum was cloned and sequenced. It was found to encode a protein belonging to the aspartic proteinase family but which carried replacements of catalytically crucial residues in the hallmark sequences contributing to the active site of this type of proteinase. Consideration is given as to whether this protein is the first known parasite equivalent of the pregnancy-associated glycoproteins that have been documented in ungulate mammals. Alternatively, it may be operative as a new type of proteinase with a distinct catalytic mechanism. In this event, since no counterpart is known to exist in humans, it affords an attractive potential target against which to develop new anti-malarial drugs.z 1999 Federation of European Biochemical Societies.

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.z 1999 Federation of European Biochemical Societies.

A cross-sectional sero-epidemiological study was performed in Magoda, Tanzania, an area where malaria is holoendemic. Blood samples were collected from children (1-4 years) and tested for IgG antibody reactivity against 2 recombinant protein fragments of Plasmodium falciparum Rhoptry-Associated Protein-1 (rRAP-1). The data were related to the prevalence of malarial disease and single P. falciparum or mixed Plasmodium infections. Fever (> or = 37.5 degrees C) in combination with parasite densities > 5000/microliter were used to distinguish between children with asymptomatic malaria infections and those with acute clinical disease. Furthermore, C-reactive protein (CRP) was applied as a surrogate marker of malaria morbidity. The prevalence of Plasmodium infections was 96.0%. Eleven children were defined as clinical malaria cases, all with single P. falciparum infections. The density of P. falciparum was significantly lower in children with mixed Plasmodium infections compared to those with single P. falciparum infections. Children with asymptomatic P. falciparum infections had higher IgG reactivities to rRAP-1, compared to IgG reactivities of children with malarial disease. Children with mixed Plasmodium infections generally showed elevated IgG reactivity to rRAP-1, when compared to children with single P. falciparum infections. The possible relationship between mixed species infections, clinical outcome of the disease and antibody responses to RAP-1 is discussed.

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