High level expression and characterisation of Plasmepsin II, an aspartic proteinase from <i>Plasmodium falciparum</i>

Hill, John; Tyas, Lorraine; Phylip, Lowri H.; Kay, John; Dunn, Ben M.; Berry, Colin

doi:10.1016/0014-5793(94)00940-6

Cited by 116 publications

(86 citation statements)

References 19 publications

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“…The pET3a plasmid containing the PfPM4 gene was transformed into BL21 (DE3) pLysS Escherichia coli cells (Invitrogen) for expression (12). All plasmepsin enzymes were expressed in E. coli and purified in inclusion-body form using the methods previously described (23). The inclusion bodies were isolated, solubilized, refolded, and purified using published protocols (21).…”

Section: Methodsmentioning

confidence: 99%

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

et al. 2005

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Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009 Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1′ library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1′-Gln-(Xaa). In both cases, P1 and P1′ were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1′ residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatographymass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1′-Phe pools revealed the favored amino acids at the P3, P2, P2′, and P3′ positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

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Section: Methodsmentioning

confidence: 99%

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

et al. 2005

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“…Recombinant proplasmepsin I does not undergo ph-dependent autocatalytic processing. Proplasmepsin I (from pETpM1) and proplasmepsin I1 [11] were refotded at pH 8.5, incubated at pH 4.7 and analysed by SDSIPAGE. Lanes 1 and 2, proplasmepsin I before and after acidification); lanes 3 and 4, proplasmepsin I1 before and after acidification.…”

Section: Expression Of Authentic Proplasmepsin Imentioning

confidence: 99%

Expression and Characterisation of Plasmepsin I from Plasmodium falciparum

Moon

Tyas

Certa

et al. 1997

European Journal of Biochemistry

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112

124

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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.

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“…These three hemoglobinases have been cloned and sequenced (4 -6). A 43-kDa recombinant precursor of plasmepsin II lacking the first 76 residues of full-length proplasmepsin II has been expressed in Escherichia coli, and its acidification results in the production of a 38-kDa protein by autocatalytic cleavage (7). The 38-kDa enzyme showed kinetic properties similar to those of native plasmepsin II (8).…”

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Plasmepsin II, an Acidic Hemoglobinase from thePlasmodium falciparum Food Vacuole, Is Active at Neutral pH on the Host Erythrocyte Membrane Skeleton

Bonniec¹,

Deregnaucourt²,

Redeker³

et al. 1999

Journal of Biological Chemistry

101

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Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin ␣-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate the involvement of plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.Malaria is a tropical disease caused by the protozoan parasite Plasmodium. In 1996, the World Health Organization estimated the annual mortality at 1.7-2.5 million and persons living in risk areas at ϳ2 billion. Resistance of Plasmodium to antimalarial drugs and vector resistance to insecticides are increasing problems in fighting the parasite. New approaches to chemotherapy based on blocking essential metabolic pathways of the parasite are necessary. Toward this end, the parasite proteases that are involved in crucial steps of Plasmodium development appear to be good targets. Several have been described that are involved in hemoglobin hydrolysis (the so-called hemoglobinases), antigen maturation, merozoite release, or invasion of new red blood cells by merozoites (1).Among the Plasmodium proteases, the hemoglobinases have been extensively studied (2). In the food vacuole (pH 5-5.4), the 37-kDa aspartic proteases plasmepsin I and plasmepsin II initiate cleavage of the native hemoglobin tetramers, whereas the 28-kDa cysteine protease falcipain appears to digest the denatured or fragmented substrate (3). These three hemoglobinases have been cloned and sequenced (4 -6). A 43-kDa recombinant precursor of plasmepsin II lacking the first 76 residues of full-length proplasmepsin II has been expressed in Escherichia coli, and its acidification results in the production of a 38-kDa protein by a...

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High level expression and characterisation of Plasmepsin II, an aspartic proteinase from Plasmodium falciparum

Cited by 116 publications

References 19 publications

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

Expression and Characterisation of Plasmepsin I from Plasmodium falciparum

Plasmepsin II, an Acidic Hemoglobinase from thePlasmodium falciparum Food Vacuole, Is Active at Neutral pH on the Host Erythrocyte Membrane Skeleton

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