The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum.

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.

Naturally-occurring and recombinant forms of the aspartic proteinases plasmepsins I and II from the human malaria parasite Plasmodium falciparum.

Comparable kinetic parameters were derived for the hydrolysis of peptide substrates and the interaction of synthetic inhibitors with recombinant and naturally-occurring forms of plasmepsin II. In contrast, recombinant plasmepsin I was extended by 12 residues at its N-terminus relative to its naturally-occurring counterpart and a 3-10-fold diminution in the k(cat) values was measured for substrate hydrolysis by the recombinant protein. However, comparable Ki values were derived for the interaction of two distinct inhibitors with both forms of plasmepsin I, thereby validating the use of recombinant material for drug screening. The value of plasmepsin I inhibitors was determined by assessing their selectivity using human aspartic proteinases.

A distinct member of the aspartic proteinase gene family from the human malaria parasite Plasmodium falciparum.

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.

Expression and characterisation of plasmepsin I from Plasmodium falciparum.

Two aspartic proteinases, plasmepsins I and II, 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 I. 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 Lys110P 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 I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5-10-fold lower k(cat)/Km values than plasmepsin II 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 II. 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 P. falciparum, 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 they are not functionally redundant but play distinct roles in the parasite.

Actin in the oomycetous fungus Phytophthora infestans is the product of several genes.

Actin (ACT) in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and other filamentous fungi where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. The complete nucleotide sequence of both genes has been determined. Unlike the actin-encoding genes (act) of other filamentous fungi, no introns are obvious in the coding region, a feature shared with the act genes of certain protists. Northern blotting and primer extension studies of the mRNA show that actA and actB are actively transcribed in mycelium, sporangia and germinating cysts but only at a low level in the case of actB. Both genes display bias in their codon usage. This is more extreme in actA. The deduced ACTB protein is strikingly similar to that of the Phytophthora megasperma actin and is more diverged from other actins than ACTA.