Expression of Recombinant Plasmodium falciparumSubtilisin-like Protease-1 in Insect Cells

Abstract

Serine proteases play crucial roles in erythrocyte invasion by merozoites of the malaria parasite. Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) is synthesized during maturation of the intraerythrocytic parasite and accumulates in a set of merozoite secretory organelles, suggesting that it may play a role in host cell invasion or post-invasion events. We describe the production, purification, and characterization of recombinant PfSUB-1 and comparison with the authentic protease detectable in parasite extracts. The recombinant protease requires high levels of calcium for optimum activity and has an alkaline pH optimum. Using a series of decapeptide and protein substrates, PfSUB-1 was found to have a relaxed substrate specificity with regard to the P1 position but is unable to efficiently cleave substrates with a P1 leucine residue. Similarly, replacement of a P4 valine with alanine severely reduced cleavage efficiency, whereas its replacement with lysine abolished cleavage. In all respects investigated, the recombinant protease was indistinguishable from parasite-derived enzyme. Three-dimensional homology modeling of the PfSUB-1 catalytic domain based on an alignment with closely related bacterial subtilisins and an orthologue from the rodent malaria Plasmodium yoelii suggests that the protease has at least three potential calcium ion-binding sites, three intramolecular disulfide bridges, and a single free cysteine within the enzyme S1 pocket. A predicted highly polar S1 pocket and a hydrophobic S4 subsite are in broad agreement with the experimentally determined substrate specificity.Malaria is caused by protozoon parasites of the genus Plasmodium. In humans, the clinical disease is caused by replication of the blood stages of the parasite. The invasive blood stage form, the merozoite, recognizes and binds a circulating erythrocyte, invades it with the concomitant formation of a parasitophorous vacuole, and then undergoes mitotic replication within this vacuole. Eventual rupture of the host cell releases a new wave of merozoites to repeat the cycle. Erythrocyte invasion and a number of the macromolecular modifications associated with it are sensitive to serine protease inhibitors, and there is a large body of evidence indicating an important role for parasite serine proteases in this critical stage of the parasite life cycle (1). Several putative malarial serine protease genes have been identified, some of which are expressed in the blood stages (1), but in no case is the biological function of the respective gene product known. PfSUB-1 1 is a Plasmodium falciparum protease belonging to the subtilisin-like superfamily of serine proteases or subtilases (2). The primary structure of the PfSUB-1 putative catalytic domain (MEROPS identification number S08.012; Ref.3) defines it phylogenetically as probably belonging to a relatively small group of bacterial-like eukaryotic enzymes in the subtilisin or pyrolysin subtilase families (2, 3). PfSUB-1 is synthesized during maturation of the intracellular meroz...

Functional Characterization of the Propeptide of Plasmodium falciparum Subtilisin-like Protease-1

“…PfSUB-1 Enzyme Activity and Inhibition Assays-Kinetic experiments assessing the effect of recombinant propeptide constructs on the activity of baculovirus-derived rPfSUB-1 were as described previously, based on the cleavage of the tetramethylrhodamine-labeled fluorogenic peptide substrate pepF1-6R (12,21). Briefly, 90 l of pepF1-6R (2 M final concentration) in 50 mM Tris-HCl (pH 8.2), 12 mM CaCl 2 , and 0.05% (w/v) Nonidet P-40 was added to a 3 ϫ 3-mm path length cell.…”

“…A mouse polyclonal antiserum was raised against the purified protein using standard procedures. Expression of baculovirus-derived rPfSUB-1 in insect cells and protease purification were as described previously (12).…”

“…falciparum schizont extracts containing endogenous PfSUB-1 were prepared by successive solubilizations with Triton X-100 and CHAPS as described previously (11,12). To determine inhibition of native PfSUB-1 by rp31, the schizont extract was incubated with an unlabeled synthetic peptide substrate called PEP1 (2 mM final concentration) in the absence or presence of purified rp31 (84 nM final concentration) for 4 h at 37°C, and then cleavage of PEP1 was assessed by RP-HPLC as described previously (12).…”

“…PfSUB-1 is expressed in the latter stages of schizogony, is located in a subset of dense granules at the apical end of the merozoite, and is secreted during erythrocyte invasion (8), consistent with an involvement in invasion. Sequence comparisons and homology modeling have shown that PfSUB-1 is structurally most closely related to the bacterial subtilisins; unlike most bacterial subtilisins, however, PfSUB-1 has a highly restricted substrate specificity, suggesting a highly specialized, non-degradative physiological role in the parasite (11,12). It may be a suitable target for drug development.…”

“…PfSUB-1 maturation during secretory transport in the parasite involves two sequential autocatalytic processing events that convert the precursor first to a p54 form by cleavage of the propeptide and subsequently to a p47 form by further cleavage within the N-terminal segment of p54 (11). Interestingly, when produced in a baculovirus expression system, recombinant PfSUB-1 (rPfSUB-1) is mostly secreted in the p54 form, a substantial fraction of which is noncovalently linked to its cognate propeptide, p31 (11,12).…”