Plasmodium Food Vacuole Plasmepsins Are Activated by Falcipains

Abstract

Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation. Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation. Human malaria is a disease of devastating proportions. Approximately 40% of the world population is at risk for acquiring it, and an estimated two million people, mostly children, die of it each year (1Snow R.W. Guerra C.A. Noor A.M. Myint H.Y. Hay S.I. Nature. 2005; 434: 214-217Crossref PubMed Scopus (2125) Google Scholar). The most deadly form of malaria is caused by infection with the protozoan parasite Plasmodium falciparum. During the disease-causing stage of its life cycle, the parasite lives within human red blood cells and consumes over two-thirds of the hemoglobin of the host cell (2Goldberg D.E. Curr. Top. Microbiol. Immunol. 2005; 295: 275-291Crossref PubMed Scopus (170) Google Scholar). This massive catabolic process is essential for parasite survival and is facilitated by numerous endo- and exo-peptidases within an acidic organelle termed the food vacuole (3Klemba M. Goldberg D.E. Annu. Rev. Biochem. 2002; 71: 275-305Crossref PubMed Scopus (191) Google Scholar, 4Liu J. Istvan E.S. Gluzman I.Y. Gross J. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8840-8845Crossref PubMed Scopus (264) Google Scholar, 5Dahl E.L. Rosenthal P.J. Mol. Biochem. Parasitol. 2005; 139: 205-212Crossref PubMed Scopus (64) Google Scholar, 6Subramanian S. Sijwali P.S. Rosenthal P.J. J. Biol. Chem. 2007; 282: 24961-24969Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Two families of proteases are involved in the initial steps of hemoglobin catabolism: three cysteine protease falcipains (FP-2, FP-2′, FP-3) and four aspartic protease plasmepsins (PM I, PM II, HAP, and PM IV). The plasmepsins are synthesized as type II integral membrane proteins that are delivered to the food vacuole through the secretory system via the plasma membrane of the parasite. Once at the plasma membrane, they appear to incorporate into the membranes of the forming cytostomes that deliver host cell hemoglobin to the food vacuole (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (109) Google Scholar). Once at the food vacuole, the plasmepsins are activated and released from the membrane by proteolytic cleavage at a conserved sequence site (see Fig. 1A) (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar). Although numerous examples of autocatalytic protease activation exist, PM activation is refractory to inhibitors of aspartic proteases such as pepstatin (a potent inhibitor of plasmepsin activity) as well as to general inhibitors of cysteine, serine, or metalloproteases (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar) both in cell culture and cell-free assays. Processing can, however, be inhibited by tripeptide aldehydes such as N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN). 3The abbreviations used are: ALLN, N-acetyl-l-leucyl-l-leucyl-l-norleucinal; Z, benzyloxycarbonyl; PBS, phosphate-buffered saline; PM, plasmepsin; FP, falcipain. 3The abbreviations used are: ALLN, N-acetyl-l-leucyl-l-leucyl-l-norleucinal; Z, benzyloxycarbonyl; PBS, phosphate-buffered saline; PM, plasmepsin; FP, falcipain. This has led to a model of plasmepsin processing in which a unique ALLN-sensitive trans-processing activity, or maturase, serves to cleave the proplasmepsins to their mature, active form (9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar). We have extended these studies with the identification of the enzymes involved in proplasmepsin processing. These findings significantly revise our current model for this event and have important ramifications for the notion of proplasmepsin processing as a potential drug target. Materials—Unless otherwise noted, all of the reagents were from Sigma-Aldrich. [35S]Cysteine/methionine (1175 Ci/mmol) and [3H]methionine (1000 Ci/mmol) (PerkinElmer Life Sciences), RPMI and Albumax (Invitrogen), brefeldin A (Calbiochem), pepstatin (Roche Applied Science), HHDG peptide (dabcyl-GABA-NYLGSSND-edans; Anaspec), Z-Leu-Arg-AMC peptide (R & D Systems), and tetanolysin (List Biological Laboratories) were used. E-64-bio was synthesized by the method of Greenbaum et al. (10Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Chem. Biol. 2000; 7: 569-581Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Parasite Culture and Purification—P. falciparum clone 3D7 was grown at 37 °C under 3% O2, 3%CO2, 94% N2 in RPMI 1640 medium supplemented with 25 mm HEPES, 30 mg/l hypoxanthine, 0.225% NaHCO3, and 0.5% Albumax I (Invitrogen). Parasites were grown in 2% human red blood cells and were synchronized using successive rounds of 5% sorbitol treatment. The parasites were purified from host red blood cells by incubation with 50 hemolytic units/ml tetanolysin in PBS for 10 min on ice, followed by two cold PBS washes. Immunoprecipitation—Rabbit polyclonal antiserum against plasmepsin II (antibody 737) was made using the peptide HSHSSNDNIELNDFQNIMFYGDAEV derived from the mature N terminus of native plasmepsin II prepared as described (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Labeled trophozoite stage parasites (routinely 5 × 108) were immunoprecipitated using denaturing conditions. Free parasites were lysed by the addition of 1% SDS (100 μl) at 100 °C for 10 min. The solution was adjusted to 150 mm NaCl, 40 mm Tris, pH 7.5, 6 mm EDTA, 0.5% deoxycholate, 1% Triton X-100, and 0.1% SDS; insoluble material was removed by centrifugation. Antibody was added at a 1:250 dilution (determined to be saturating) along with 70 μl of protein A-Sepharose (1:1 bead slurry to immunoprecipitation buffer). The samples were mixed by nutation at 4 °C for 16–20 h, and immunocomplexes were collected, added to an equal volume of 2× nonreducing sample buffer, boiled 5 min, and analyzed by 12% SDS-PAGE with fluorography. In Vivo Processing Assay—Inhibitor experiments were performed with synchronized cultures of trophozoite stage parasites. The cultures were washed one time in RPMI prepared without methionine and cysteine, resuspended in the same medium at a density of 2.0 × 108/ml with [35S]methionine/cysteine (350 μCi/ml), and incubated at 37 °C for 30 min (3% O2, 3%CO2). The cultures were chased for the indicated times by removal of labeling media and addition of complete RPMI. The parasites were harvested by tetanolysin purification and processed immediately for immunoprecipitation. Kinetics of processing was calculated by densitometry and calculation of the fraction of plasmepsin II (PM II) processed at each time point. Band intensities were normalized to account for change in specific activity caused by the loss of 35S-labeled cysteine and methionine residues upon processing. Cell-free Processing Assay—The assay was by the method of Francis et al. (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Trophozoite stage parasites were metabolically labeled as above except that brefeldin A (5 μg/ml) and [35S]methionine/cysteine were added at the same time. The cultures were incubated for 4–5 h and then harvested and lysed in PBS by gentle sonication. The radiolabeled lysates were incubated in the presence of an equal amount of unlabeled parasite lysate plus inhibitors in 100 mm sodium acetate buffer, pH 5.5, for 1 h at 37 °C. The reactions were stopped by addition of one volume of 2% SDS and boiling. PM II was immunoprecipitated as described above. In Vitro Transcription/Translation and Radiosequencing—Capped PM II RNA was prepared using linearized plasmid DNA containing the entire open reading frame of plasmepsin II and T7 polymerase Message Machine kit (Ambion) following the manufacturer's instructions. Transcript was added to nuclease-free rabbit reticulocyte lysate (Promega) in the presence of 0.5 μl of [35S]methionine, 10 μg of brewer's yeast tRNA, and 1.8 μl of canine pancreatic microsomes/25 μl of translation reaction. 2 μl of in vitro translated protein (above) was incubated with parasite lysate treated with either 10 μm E-64 or pepstatin for 3 h at 37 °C. Denaturing immunoprecipitations were performed as above, fractionated by SDS-PAGE, and blotted onto polyvinylidene difluoride membrane. Plasmepsin bands were excised and subjected to Edman degradation. Each cycle from the sequencer was assayed for radioactivity in a scintillation counter. Nonspecific background increases with successive cycles as is typical with Edman degradation. Target Analysis with E-64-biotin—Synchronized trophozoite stage culture (∼5% parasitemia) was incubated with 10 μm inhibitor for 15 min to block accessible active sites followed by purification of parasites by tetanolysin. The parasites were then washed twice with ice-cold PBS. The parasite pellet was resuspended in 100 μl of lysis/labeling mix (50 mm sodium acetate, pH 5.5, 1.0%Triton X-100, 10 mm dithiothreitol, 25 μm E-64-bio) and incubated at 37 °C for 30 min followed by the addition of 25 μl of 5× SDS sample buffer and boiled for 5 min. 30 μl of sample was resolved by SDS-PAGE and transferred to nitrocellulose. Biotinylated proteins were detected using Vectastain ABC-AmP kit and DuoLuX reagent (Vector Laboratories) as per the manufacturer's instructions. Fluorogenic Cleavage Assays—Cleavage of HHDG internally quenched fluorogenic peptide was initiated by the addition of 0.5 μl of purified recombinant FP-2 or FP-3 to a final reaction volume of 200 μl (50 mm sodium acetate, pH 5.5, HHDG peptide, 10 mm dithiothreitol) and monitored over time using a Varian Cary Eclipse fluorimeter fitted with a 96-well plate reader. Excitation and emission wavelengths were 355 and 495 nm, respectively. The reaction rates were plotted relative to substrate concentration, and Km and Vmax values were determined by a Michaelis-Menten curve fit using Kaleidagraph (Synergy Software). Enzyme concentration was determined by absorbance at 280 nm using calculated extinction coefficients of 41,870 and 55,280 m–1 cm–1 for mature FP-2 and FP-3, respectively. Plasmepsin Processing Is Inhibited by Combined Treatment with and experiments on parasites incubated with protease inhibitors that the processing activity is refractory to the general cysteine and aspartic protease E-64 and to as (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar) We of inhibitors and the combination of and pepstatin inhibited II processing activity to a as This the presence of two processing In the of with a potent plasmepsin processing of II This is in however, to which in two processed of PM II, both of which appear to be the of autoprocessing (see both and the can process II, with of II processing of both of these We were that the of processing by the with and pepstatin be to a general and through a of processing a using a cell-free processing assay (9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar) in which II was in by parasites with brefeldin A a labeling with A was then prepared from these parasites and incubated with unlabeled parasite lysate in the presence of Processing of synthesized II was inhibited by the combination of E-64 and pepstatin as well as by This a of these inhibitors on PM processing The in to the in that the processing activity is accessible and to E-64 in the cell-free that proplasmepsin processing in occurs in a accessible to Cleavage of PM II Is Inhibited by that processing of II occurs at the peptide I.Y. Francis S.E. A. Goldberg D.E. J. PubMed Scopus Google Scholar). We were in the cleavage of each of these processing and or to II processing. this by the N terminus of in vitro translated II that been incubated with parasite lysate with either E-64 or pepstatin of these cleavage that the lysate II at the cleavage site by of radiolabeled methionine released 15 the N incubation with lysate in cleavage one to this site at the peptide of radiolabeled methionine released the N that cleavage at (9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google this that the cysteine protease activity the processing activity in Processing Is via with processing is in the presence of E-64 in to that it is to the processing We that this be the of a in the of these two this performed experiments the of II processing and determined that was an in the of II processing in the presence of inhibitor 15 min Fig. This II processing occurs via the activity in in was from the experiments that used in combination with the the was to the processing activity in We to take of this to in the protease of We this by red blood cell cultures with either E-64 or to all protease active sites this the cultures were washed of and the parasites were purified from the host red blood parasite lysate was then prepared and incubated with a of E-64 to the unlabeled active These lysates were then resolved by SDS-PAGE and transferred to and the proteins were detected by Although numerous bands are labeled in the of inhibitor a protein that is by by E-64 was at E-64 was added however, it was to This labeling the of processing to these inhibitors used in combination with Fig. by Greenbaum et al. A. M. Medzihradszky K.F. J. J. Bogyo M. 2002; PubMed Scopus Google Scholar) using an E-64 this as falcipains 2 and a PM II at the Processing the notion that falcipains are for proplasmepsin assayed the activity of recombinant FP-2 and FP-3 on a fluorogenic peptide the cleavage site of FP-2 and FP-3 were to cleave the processing site peptide in a with values of × and × recombinant PM II and PM were were both on this peptide of FP-2 and FP-3 with internally quenched fluorogenic substrate × × × × in a We used to the falcipain cleavage on this peptide incubation of peptide with FP-2 or FP-3, the to and of a cleavage event at the peptide of cleavage sites was These results that both enzymes its level of identity to FP-2 Sijwali P.S. Rosenthal P.J. Parasitol. 2006; PubMed Scopus Google and cleave PM II at the processing Two of Processing in has been of to our to the proplasmepsin processing maturase general protease inhibitors block this activity led to that a unique processing was The reveal that processing is a of a unique activity that proplasmepsin processing can through either of two in processing this pepstatin however, the combination of and pepstatin processing of proplasmepsin processing of both an cysteine protease activity as well as of a aspartic protease The Is from our has determined that a cleavage is conserved for all four food vacuole plasmepsins (9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar) with the of mature plasmepsin II I.Y. Francis S.E. A. Goldberg D.E. J. PubMed Scopus Google Scholar). In the studies have determined using in vitro translated PM II that the activity in parasite lysate processed this the pepstatin activity one to this site These that the activity is to the In the processing is upon with with this is the presence of the processed of PM II that are upon with in or in vitro The is at the for mature plasmepsin II and to the autoprocessing site have one from the site in our in vitro experiments The is is to a autoprocessing site that it is upon pepstatin treatment. We the activity to be the of plasmepsin have that recombinant PM II under acidic J. J. PubMed Scopus Google Scholar, Francis S.E. Gluzman I.Y. Goldberg D.E. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar). et al. S. J. Biochem. 2006; 139: PubMed Scopus Google Scholar) have extended these studies by that recombinant PM II and PM autoprocessing in both and We in the cell-free assay a amount of the in the of inhibitor or in the presence of This that the PM II substrate in this assay is significantly relative to the in that with this is the that S. J. Biochem. 2006; 139: PubMed Scopus Google Scholar). The for the Processing plasmepsins through the secretory system to the plasma membrane of the at which they appear to incorporate into the membranes of the forming cytostomes that deliver host cell hemoglobin to the food vacuole (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (109) Google Scholar). is that an acidic pH is for both the processing and the autoprocessing (9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar). The pH of both the secretory and the Goldberg D. M. Biochem. J. 2007; PubMed Scopus Google Scholar) the of proplasmepsin processing to the food and 3 the food vacuole cysteine protease activity P.J. M. J. PubMed Scopus Google Scholar) and the cysteine within this organelle E.L. Rosenthal P.J. Mol. Biochem. Parasitol. 2005; 139: 205-212Crossref PubMed Scopus (64) Google Scholar, 6Subramanian S. Sijwali P.S. Rosenthal P.J. J. Biol. Chem. 2007; 282: 24961-24969Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). the falcipains appear to be the for the proplasmepsin In our experiments that used in combination with was to processing This that the cysteine protease processing activity in an accessible by with the food vacuole falcipains a to E-64 relative to in our in experiments This that as with to the food This is with the of as a of have however, that E-64 has an on hemoglobin within the parasite and a food vacuole P.J. M. J. PubMed Scopus Google Scholar). This is by the and incubation times used to the in these studies with our in processing assays. these experiments a model by which the falcipains as the proplasmepsin maturase, and upon their as by are of PM II Although both are of proplasmepsin the falcipains appear to of their processing The falcipains to the of cysteine of the substrate for the falcipains between FP-2 the and FP-3 M. Biochem. J. 2006; PubMed Scopus Google both enzymes with at Sijwali P.S. A. Rosenthal P.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P.S. J. A. Rosenthal P.J. Biochem. J. PubMed Scopus Google Scholar). with this it is that both FP-2 and FP-3 are to cleave proplasmepsin II at the cleavage both FP-2 and FP-3 are to cleave our proplasmepsin peptide, as well as recombinant proplasmepsin II is without this same that were with by these if of the falcipain enzymes a in proplasmepsin processing in has been in this our that the enzymes cleave the proplasmepsin peptide with an in × and × m–1 for FP-2 and FP-3, that and FP-3 are in the cycle of the parasite P.S. J. A. Rosenthal P.J. Biochem. J. PubMed Scopus Google Scholar). This that FP-2 as the proplasmepsin maturase, the of and FP-3 be to this be to use the FP-2 and cell J. Istvan E.S. Gluzman I.Y. Gross J. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8840-8845Crossref PubMed Scopus (264) Google Scholar, P.S. J. Rosenthal P.J. Mol. Biochem. Parasitol. 2006; PubMed Scopus Google Scholar) to the proplasmepsin processing of these with to the We have this with FP-2 cells were to is that FP-3 activity was for processing. These experiments are the stage of the parasite This into the of a unique proplasmepsin processing activity as a drug target. We that to the falcipains or the plasmepsins are to be inhibitors of processing that both families block proplasmepsin processing. of this is ALLN, our described maturase inhibitor (7Francis S.E. Banerjee R. Goldberg D.E. J. Biol. Chem. 1997; 272: 14961-14968Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Banerjee R. Francis S.E. Goldberg D.E. Mol. Biochem. Parasitol. 2003; 129: 157-165Crossref PubMed Scopus (52) Google Scholar). can block proplasmepsin processing it is both a inhibitor of the falcipains a inhibitor of the plasmepsins and et al. S. J. Biochem. 2006; 139: PubMed Scopus Google We have that II can be activated by two processing and the conserved processing processing inhibitor and processing that this is the for all food vacuole redundancy to be a in Plasmodium examples can be in the of the parasite 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) and the 2007; PubMed Scopus Google Scholar). to the redundancy can be in their along with the to hemoglobin degradation. the of FP-3, of each plasmepsin and falcipain have been P.S. J. Rosenthal P.J. Mol. Biochem. Parasitol. 2006; PubMed Scopus Google Scholar, J. Gluzman I.Y. Goldberg D.E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. J. Parasitol. 2007; PubMed Scopus Google Scholar). it has been that the falcipains appear to at in for the loss of all four food vacuole plasmepsins C.A. Mol. 2007; PubMed Scopus Google Scholar). of these results is that these enzymes function by to hemoglobin and that has as a for the parasite to a level of plasmepsin and between plasmepsin and falcipain families. We level of functional the of the of the families to the proplasmepsin it the parasite has a between and redundancy by the to the plasmepsins through this processing activity is in the of infection is at this time. We for parasite Istvan and for and for with