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• W2044869144 abstract "In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). Thepfsub-1 gene is expressed in asexual blood stages of P. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development. In the vertebrate host, the malaria parasite invades and replicates asexually within circulating erythrocytes. Parasite proteolytic enzymes play an essential but poorly understood role in erythrocyte invasion. We have identified a Plasmodium falciparum gene, denoted pfsub-1, encoding a member of the subtilisin-like serine protease family (subtilases). Thepfsub-1 gene is expressed in asexual blood stages of P. falciparum, and the primary gene product (PfSUB-1) undergoes post-translational processing during secretory transport in a manner consistent with its being converted to a mature, enzymatically active form, as documented for other subtilases. In the invasive merozoite, the putative mature protease (p47) is concentrated in dense granules, which are secretory organelles located toward the apical end of the merozoite. At some point following merozoite release and completion of erythrocyte invasion, p47 is secreted from the parasite in a truncated, soluble form. The subcellular location and timing of secretion of p47 suggest that it is likely to play a role in erythrocyte invasion. PfSUB-1 is a new potential target for antimalarial drug development. phenylmethylsulfonyl fluoride glycosyl phosphatidylinositol P. falciparum merozoite surface protein-1 polymerase chain reaction brefeldin A fluorescein isothiocyanate glutathione S-transferase indirect immunofluorescence diisopropyl fluorophosphate endoplasmic reticulum phosphate-buffered saline base pair(s) open reading frame polyacrylamide gel electrophoresis. Plasmodium falciparum, the causative agent of the most severe form of human malaria, is an obligate intracellular apicomplexan parasite. The life cycle of the organism includes a number of specialized invasive (zoite) stages. In the vertebrate host, replication of the parasite in circulating erythrocytes is initiated when the cells are invaded by merozoites. The parasite replicates asexually within the infected erythrocyte to produce a number of progeny merozoites. Upon rupture of the host cell, these are released to invade fresh erythrocytes and perpetuate the blood stage cycle. Erythrocyte invasion by the malaria merozoite has been the subject of intensive study, since intervention strategies that prevent invasion would effectively block both replication of the parasite and the associated clinical disease.Electron microscopic studies have shown that erythrocyte invasion by the malaria merozoite takes place in a number of discrete stages. Initial reversible attachment of the parasite to the red cell surface is rapidly followed by reorientation, the formation of an irreversible junction between the apical prominence of the merozoite and the host cell surface, and finally entry of the parasite into the cell by a mechanism resembling a form of induced endocytosis (1Dvorak J.A. Miller L.H. Whitehouse W.C. Shiroishi T. Science. 1975; 187: 748-750Crossref PubMed Scopus (321) Google Scholar, 2Bannister L.H. Butcher G.A. Dennis E.D. Mitchell G.H. Parasitology. 1975; 71: 483-491Crossref PubMed Scopus (117) Google Scholar, 3Aikawa M. Miller L.H. Johnson J.G. Rabbege J. J. Cell Biol. 1978; 77: 72-82Crossref PubMed Scopus (439) Google Scholar, 4Miller L.H. Aikawa M. Johnson J.G. Shiroishi T. J. Exp. Med. 1979; 149: 172-184Crossref PubMed Scopus (257) Google Scholar). The process is facilitated by the controlled release of the contents of three types of secretory organelles, called rhoptries, micronemes, and dense granules, situated at or toward the apical domain of the merozoite (2Bannister L.H. Butcher G.A. Dennis E.D. Mitchell G.H. Parasitology. 1975; 71: 483-491Crossref PubMed Scopus (117) Google Scholar,5Bannister L.H. Mitchell G.H. Butcher G.A. Dennis E.D. Parasitology. 1986; 92: 291-303Crossref PubMed Scopus (106) Google Scholar, 6Torii M. Adams J.H. Miller L.H. Aikawa M. Infect. Immun. 1989; 57: 3230-3233Crossref PubMed Google Scholar). There is extensive evidence indicating an essential role for parasite-derived proteases in invasion. Invasion by P. falciparum merozoites is blocked in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF)1 (7Dejkriengkraikhul P. Wilairat P. Z. Parasitenkd. 1983; 69: 313-317Crossref PubMed Scopus (39) Google Scholar), and invasion by merozoites of a number of Plasmodium species is prevented by chymostatin (8Dutta G.P. Banyal H.S. Indian J. Exp. Biol. 1981; 19: 9-11PubMed Google Scholar, 9Banyal H.S. Misra G.C. Gupta C.M. Dutta G.P. J. Parasitol. 1981; 67: 623-626Crossref PubMed Scopus (67) Google Scholar, 10Dluzewski A.R. Rangachari K. Wilson R.J.M. Exp. Parasitol. 1986; 62: 416-422Crossref PubMed Scopus (80) Google Scholar, 11Braun-Breton C. Blisnick T. Jouin H. Barale J.C. Rabilloud T. Langsley G. Pereira da Silva L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9647-9651Crossref PubMed Scopus (60) Google Scholar, 12McPherson R.A. Donald D.R. Sawyer W.H. Tilley L. Mol. Biochem. Parasitol. 1993; 62: 233-242Crossref PubMed Scopus (27) Google Scholar, 13Schrevel J. Deguercy A. Mayer R. Monsigny M. Blood Cells. 1990; 16: 563-584PubMed Google Scholar). The inhibitory effect of chymostatin on invasion can be reversed by pretreatment of target erythrocytes with chymotrypsin (10Dluzewski A.R. Rangachari K. Wilson R.J.M. Exp. Parasitol. 1986; 62: 416-422Crossref PubMed Scopus (80) Google Scholar), suggesting that the chymostatin-sensitive step in invasion involves an essential, parasite-induced proteolytic modification of the red cell surface (10Dluzewski A.R. Rangachari K. Wilson R.J.M. Exp. Parasitol. 1986; 62: 416-422Crossref PubMed Scopus (80) Google Scholar, 11Braun-Breton C. Blisnick T. Jouin H. Barale J.C. Rabilloud T. Langsley G. Pereira da Silva L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9647-9651Crossref PubMed Scopus (60) Google Scholar, 12McPherson R.A. Donald D.R. Sawyer W.H. Tilley L. Mol. Biochem. Parasitol. 1993; 62: 233-242Crossref PubMed Scopus (27) Google Scholar). A glycosylphosphatidylinositol (GPI)-anchored malarial serine protease activity has been described that may be involved in this modification (14Braun-Breton C. Rosenberry T.L. Pereira da Silva L. Nature. 1988; 332: 457-459Crossref PubMed Scopus (129) Google Scholar). Treatment of isolated, invasive merozoites of the simian malariaP. knowlesi with N-tosyl phenylalanylchloromethyl ketone or N-tosyl lysylchloromethyl ketone prevents primary attachment of the parasites to host cells, whereas chymostatin blocks a later stage in the invasion pathway, indicating that more than one distinct protease activity may be involved (15Hadley T. Aikawa M. Miller L.H. Exp. Parasitol. 1983; 55: 306-311Crossref PubMed Scopus (109) Google Scholar). Consistent with this, a P. falciparum serine protease activity that mediates an essential processing and shedding of a major merozoite surface protein (merozoite surface protein-1; MSP-1) at invasion is highly sensitive to inhibition by PMSF but not by chymostatin (16Blackman M.J. Holder A.A. Mol. Biochem. Parasitol. 1992; 50: 307-316Crossref PubMed Scopus (185) Google Scholar, 17Blackman M.J. Chappel J.A. Shai S. Holder A.A. Mol. Biochem. Parasitol. 1993; 62: 103-114Crossref PubMed Scopus (49) Google Scholar). Parasite proteases involved in invasion are attractive potential targets for new rational approaches to antimalarial chemotherapy.Here we report the identification of a novel, single copy P. falciparum gene (denoted pfsub-1) encoding a member of the subtilisin-like serine protease superfamily (subtilases). The primary gene product is expressed in the latter stages of intracellular merozoite maturation, and is post-translationally modified during secretory transport in a manner consistent with it being processed to form a mature, enzymatically active product. The putative mature protease is concentrated in a subset of dense granules within the apical domain of free merozoites and then is released in a soluble form during erythrocyte invasion, suggesting that it may play a role in invasion. This is the first molecular characterization of a putative apicomplexan serine protease.DISCUSSIONThis report describes the first gene to be identified of any apicomplexan parasite that encodes a serine protease-like protein. We have not directly demonstrated any proteolytic activity associated with any pfsub-1 gene product, but a number of indications lead us to propose that p47 or its truncated, secreted product is likely to be an enzymatically active protease and that it may play a role in erythrocyte invasion.First, at the primary sequence level PfSUB-1 shows significant homology to known subtilases, and possesses all of the features known to be required of an active subtilase. Only four amino acid residues are completely conserved among known subtilases (34Siezen R.J. Leunissen J.A.M. Protein Sci. 1997; 6: 501-523Crossref PubMed Scopus (770) Google Scholar); these are the catalytic triad residues (PfSUB-1 residues Asp374, His430, and Ser608) and a glycine residue (Gly606 in the PfSUB-1 sequence) two positions N-terminal to the active site serine. PfSUB-1 also possesses an asparagine (Asn522) at the position of the oxyanion hole residue; the only substitution acceptable at this position is that of an aspartate, found in the PC2 subfamily of kexin-like convertases (34Siezen R.J. Leunissen J.A.M. Protein Sci. 1997; 6: 501-523Crossref PubMed Scopus (770) Google Scholar). While many bacterial subtilisins are devoid of cysteine residues, an increasingly large number of subtilases are known to possess up to two intramolecular disulfide bonds within the catalytic domain (34Siezen R.J. Leunissen J.A.M. Protein Sci. 1997; 6: 501-523Crossref PubMed Scopus (770) Google Scholar). There are seven cysteine residues within the putative catalytic domain of PfSUB-1, and the reduction-sensitive mobility on SDS-PAGE of p47 is consistent with the presence of intramolecular disulfide bonds. Detailed homology-based molecular modeling of the putative PfSUB-1 catalytic domain 3M. Hirshberg, K. Henrick, C. Withers-Martinez, and M. Blackman, manuscript in preparation. supports this postulate. Thus, at the structural level, PfSUB-1 resembles a subtilase.Second, the proteolytic processing to which PfSUB-1 is subjected during secretory transport probably represents a process of enzyme activation common among subtilases. In a number of well studied examples, co-translational signal peptide cleavage allows folding of the subtilase catalytic domain, mediated by the intramolecular chaperone activity of the pro-domain (36Shinde U. Inouye M. Trends Biochem. Sci. 1993; 18: 442-446Abstract Full Text PDF PubMed Scopus (162) Google Scholar, 48Baker D. Shiau A.K. Agard D.A. Curr. Opin. Cell Biol. 1993; 5: 966-970Crossref PubMed Scopus (149) Google Scholar). This is often (but not always) rapidly followed by autocatalytic cleavage of the pro-domain from the catalytic domain; this cleavage can take place in cis(intramolecular) or in trans (intermolecular) and in some cases simple pro-domain cleavage is sufficient to allow zymogen activation (49Ikemura H. Inouye M. J. Biol. Chem. 1988; 263: 12959-12963Abstract Full Text PDF PubMed Google Scholar, 50Volkov A. Jordan F. J. Mol. Biol. 1996; 262: 595-599Crossref PubMed Scopus (25) Google Scholar). However, there is a growing body of evidence that subtilase activation is often a substantially more complex process, which in eukaryotes may be intimately linked to correct routing of the proenzyme through the secretory pathway and final compartmentalization of the active enzyme (51Shennan K.I.J. Taylor N.A. Jermany J.L. Matthews G. Docherty K. J. Biol. Chem. 1995; 270: 1402-1407Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 52Zhou A. Paquet L. Mains R.E. J. Biol. Chem. 1995; 270: 21509-21516Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 53Powner D. Davey J. Biochem. Soc. Trans. 1997; 25: 230SCrossref PubMed Scopus (3) Google Scholar, 54Anderson E.D. VanSlyke J.K. Thulin C.D. Jean F. Thomas G. EMBO J. 1997; 16: 1508-1518Crossref PubMed Scopus (197) Google Scholar). Here we have shown that the primarypfsub-1 gene product is subjected to at least two major post-translational processing steps. The first of these, conversion of the 82-kDa form, possibly via a 60/61-kDa intermediate, to the p54 form, takes place very rapidly following synthesis and therefore could represent an autocatalytic processing step triggered by signal peptide cleavage and co-translational folding within the lumen of the ER. The p54 form is then quantitatively converted to p47 in a slower, BFA-sensitive process. Further work is required to establish whether either or both of these processing events represent enzyme activation, but certainly they are wholly consistent with a putative activation pathway. If so, the process is clearly more complex than a simple one-step pro-domain removal. BFA sensitivity is a common feature of transport to secretory organelles in apicomplexan parasites (55Sadak A. Taghy Z. Fortier B. Dubremetz J.F. Mol. Biochem. Parasitol. 1988; 29: 203-211Crossref PubMed Scopus (161) Google Scholar, 56Achbarou A. Mercereau-Puijalon O. Autheman J.M. Fortier B. Camus D. Dubremetz J.F. Mol. Biochem. Parasitol. 1991; 47: 223-234Crossref PubMed Scopus (100) Google Scholar, 57Ogun S. Holder A.A. Exp. Parasitol. 1994; 79: 270-278Crossref PubMed Scopus (42) Google Scholar, 58Howard R.F. Schmidt C.M. Mol. Biochem. Parasitol. 1995; 74: 43-54Crossref PubMed Scopus (43) Google Scholar, 59Sam-Yellowe T.Y. Parasitol. Today. 1996; 12: 308-316Abstract Full Text PDF PubMed Scopus (146) Google Scholar), but little is known about the structural requirements for correct sorting to these organelles (59Sam-Yellowe T.Y. Parasitol. Today. 1996; 12: 308-316Abstract Full Text PDF PubMed Scopus (146) Google Scholar); it is possible that part of the PfSUB-1 sequence could provide a targeting signal, as has been determined for other subtilases and yeast carboxypeptidase Y (52Zhou A. Paquet L. Mains R.E. J. Biol. Chem. 1995; 270: 21509-21516Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,60Valls L.A. Winther J.R. Stevens T.H. J. Cell Biol. 1990; 111: 361-368Crossref PubMed Scopus (115) Google Scholar).Third, the terminal intracellular PfSUB-1 processing product, p47, localizes to a subpopulation of dense granule-like organelles within the extreme apical domain of the merozoite and appears to be shed in a truncated, soluble form following merozoite release but before completion of erythrocyte invasion; no PfSUB-1-derived proteins were detectable in the newly invaded erythrocyte. These observations are in apparent conflict with the majority of the available data suggesting that dense granule secretion in apicomplexan parasites is a postinvasion event (2Bannister L.H. Butcher G.A. Dennis E.D. Mitchell G.H. Parasitology. 1975; 71: 483-491Crossref PubMed Scopus (117) Google Scholar, 6Torii M. Adams J.H. Miller L.H. Aikawa M. Infect. Immun. 1989; 57: 3230-3233Crossref PubMed Google Scholar, 41Culvenor J.G. Day K.P. Anders R.F. Infect. Immun. 1991; 59: 1183-1187Crossref PubMed Google Scholar, 42Entzeroth R. Dubremetz J.F. Hodick D. Ferreira E. Eur. J. Cell Biol. 1986; 41: 182-188PubMed Google Scholar, 43Dubremetz J.F. Achbarou A. Bermudes D. Joiner K. Parasitol. Res. 1993; 79: 402-408Crossref PubMed Scopus (173) Google Scholar, 44Carruthers V.B. Sibley L.D. Eur. J. Cell Biol. 1997; 73: 114-123PubMed Google Scholar). However, dense granules in Plasmodium are currently defined essentially on morphological criteria; there is a paucity of specific markers for these organelles, and indeed only two other malarial proteins, RESA/Pf155 and a 14-kDa protein denoted RIMA, have been previously localized to merozoite dense granules (41Culvenor J.G. Day K.P. Anders R.F. Infect. Immun. 1991; 59: 1183-1187Crossref PubMed Google Scholar, 61Aikawa M. Torii M. Sjölander A. Berzins K. Perlmann P. Miller L.H. Exp. Parasitol. 1990; 71: 326-329Crossref PubMed Scopus (88) Google Scholar, 63Trager W. Rozario C. Shio H. Williams J. Perkins M.E. Infect. Immun. 1992; 60: 4656-4661Crossref PubMed Google Scholar). It is therefore possible that distinct subpopulations of dense granules may exist in Plasmodium; there is evidence for this in the apicomplexan parasite Cryptosporidium parvum (64Bonnin A. Gut J. Dubremetz J.F. Nelson R.G. Camerlynck P. J. Eukaryot. Microbiol. 1995; 42: 395-401Crossref PubMed Scopus (29) Google Scholar), and if so, these subpopulations may be functionally distinct and may undergo exocytosis at different stages of the invasion process. Our evidence that PfSUB-1 is released at invasion suggests that it could play a role in the process of erythrocyte entry. Anti-PfSUB-1m antibodies had no inhibitory effect on erythrocyte invasion by released merozoitesin vitro, but it is unclear whether these antibodies have access to PfSUB-1 in the intact merozoite or whether they can interfere with its function. The demonstration of enzymatic activity in native, merozoite-derived p47 or its truncated soluble product would be a step toward addressing this issue. Consistent with observations of other workers (65Rosenthal P.J. Kim K. McKerrow J.H. Leech J.H. J. Exp. Med. 1987; 166: 816-821Crossref PubMed Scopus (75) Google Scholar), we have not been able to detect activity corresponding to p47 on gelatin substrate SDS-PAGE. 4M. Blackman, unpublished observations. However, the enzyme may be irreversibly denatured by exposure to SDS, and the insolubility of p47 in nonionic detergents precludes ready isolation and analysis of the merozoite-derived protein in a native state. These limitations could be overcome by recombinant expression of enzymatically active PfSUB-1, and this is a major priority of current work.If PfSUB-1 is involved in erythrocyte invasion, what precise role might the enzyme perform? The best characterized proteolytic event associated with erythrocyte invasion is the processing and shedding of a merozoite surface protein complex derived from the precursor protein MSP-1. A single proteolytic cleavage at a Leu-Asn motif within the membrane-bound component of this complex, known as secondary processing, releases the complex quantitatively from the surface of the merozoite as it enters the host erythrocyte (16Blackman M.J. Holder A.A. Mol. Biochem. Parasitol. 1992; 50: 307-316Crossref PubMed Scopus (185) Google Scholar, 17Blackman M.J. Chappel J.A. Shai S. Holder A.A. Mol. Biochem. Parasitol. 1993; 62: 103-114Crossref PubMed Scopus (49) Google Scholar, 18Blackman M.J. Heidrich H.G. Donachie S. McBride J.S. Holder A.A. J. Exp. Med. 1990; 172: 379-382Crossref PubMed Scopus (478) Google Scholar, 29Blackman M.J. Dennis E.D. Hirst E.M. Kocken C.H. Scott-Finnigan T.J. Thomas A.W. Exp. Parasitol. 1996; 83: 229-239Crossref PubMed Scopus (41) Google Scholar). MSP-1 secondary processing is thought to be essential for successful erythrocyte invasion, and it is mediated by a parasite-derived, calcium-dependent serine protease (16Blackman M.J. Holder A.A. Mol. Biochem. Parasitol. 1992; 50: 307-316Crossref PubMed Scopus (185) Google Scholar, 17Blackman M.J. Chappel J.A. Shai S. Holder A.A. Mol. Biochem. Parasitol. 1993; 62: 103-114Crossref PubMed Scopus (49) Google Scholar). Interestingly, this activity is exquisitely sensitive to inhibition by PMSF, but it is only poorly inhibited by even very high concentrations (up to 10 mm) of DFP (19Blackman M.J. Methods Cell Biol. 1994; 45: 213-220Crossref PubMed Scopus (79) Google Scholar). Given its apparent nonreactivity with DFP, its location in the merozoite, and the timing of its secretion, PfSUB-1 is a good candidate for this activity. This intriguing possibility clearly merits further investigation and is supported by molecular modeling of the proposed catalytic domain of PfSUB-1, which has indicated that a heptapeptide corresponding to the sequence flanking the MSP-1 secondary processing site (i.e.Gln-Gly-Met-Leu-Asn-Ile-Ser) fits well into the active site groove of the model, with the Leu-Asn scissile bond in the appropriate position for cleavage.3 The only other serine protease activity previously localized to P. falciparum merozoites is a DFP-reactive enzyme thought to be activated following cleavage of a GPI anchor (14Braun-Breton C. Rosenberry T.L. Pereira da Silva L. Nature. 1988; 332: 457-459Crossref PubMed Scopus (129) Google Scholar); it is unlikely that PfSUB-1 corresponds to this activity, since it shows no DFP reactivity and the sequence exhibits no C-terminal hydrophobic domain typical of GPI signal sequences (66Udenfriend S. Kodukula K. Annu. Rev. Biochem. 1995; 64: 563-591Crossref PubMed Scopus (434) Google Scholar).Of the two major families of active site serine endoproteases, the chymotrypsin-like and the subtilisin-like families (32Rawlings N.D. Barrett A.J. Biochem. J. 1993; 290: 205-218Crossref PubMed Scopus (698) Google Scholar), chymotrypsin-like proteases are common in higher eukaryotes but appear to be rare in lower eukaryotes and prokaryotic organisms. Sakanariet al. (67Sakanari J.A. Staunton C.E. Eakin A.E. Craik C.S. McKerrow J.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4863-4867Crossref PubMed Scopus (112) Google Scholar) have reported evidence for the existence of a chymotrypsin-like gene in Trypanosoma cruzi, but there is no other previous genetic data on protozoal serine proteases. Extensive attempts in this laboratory to isolate chymotrypsin-like P. falciparum genes using a number of different PCR-based approaches have been unsuccessful.4 It is conceivable that, likeSaccharomyces cerevisiae (40The Yeast Genome Directory (1997) Nature 387, suppl., 1–105Google Scholar), the malaria parasite (and perhaps other apicomplexan parasites) does not possess genes of this class. Proteases play a crucial role in the life cycle of the blood stage malaria parasite and are undoubtedly potential targets for the design of protease inhibitor-based drugs. Expression of PfSUB-1 in an enzymatically active form should allow the development and evaluation of such inhibitors. Plasmodium falciparum, the causative agent of the most severe form of human malaria, is an obligate intracellular apicomplexan parasite. The life cycle of the organism includes a number of specialized invasive (zoite) stages. In the vertebrate host, replication of the parasite in circulating erythrocytes is initiated when the cells are invaded by merozoites. The parasite replicates asexually within the infected erythrocyte to produce a number of progeny merozoites. Upon rupture of the host cell, these are released to invade fresh erythrocytes and perpetuate the blood stage cycle. Erythrocyte invasion by the malaria merozoite has been the subject of intensive study, since intervention strategies that prevent invasion would effectively block both replication of the parasite and the associated clinical disease. Electron microscopic studies have shown that erythrocyte invasion by the malaria merozoite takes place in a number of discrete stages. Initial reversible attachment of the parasite to the red cell surface is rapidly followed by reorientation, the formation of an irreversible junction between the apical prominence of the merozoite and the host cell surface, and finally entry of the parasite into the cell by a mechanism resembling a form of induced endocytosis (1Dvorak J.A. Miller L.H. Whitehouse W.C. Shiroishi T. Science. 1975; 187: 748-750Crossref PubMed Scopus (321) Google Scholar, 2Bannister L.H. Butcher G.A. Dennis E.D. Mitchell G.H. Parasitology. 1975; 71: 483-491Crossref PubMed Scopus (117) Google Scholar, 3Aikawa M. Miller L.H. Johnson J.G. Rabbege J. J. Cell Biol. 1978; 77: 72-82Crossref PubMed Scopus (439) Google Scholar, 4Miller L.H. Aikawa M. Johnson J.G. Shiroishi T. J. Exp. Med. 1979; 149: 172-184Crossref PubMed Scopus (257) Google Scholar). The process is facilitated by the controlled release of the contents of three types of secretory organelles, called rhoptries, micronemes, and dense granules, situated at or toward the apical domain of the merozoite (2Bannister L.H. Butcher G.A. Dennis E.D. Mitchell G.H. Parasitology. 1975; 71: 483-491Crossref PubMed Scopus (117) Google Scholar,5Bannister L.H. Mitchell G.H. Butcher G.A. Dennis E.D. Parasitology. 1986; 92: 291-303Crossref PubMed Scopus (106) Google Scholar, 6Torii M. Adams J.H. Miller L.H. Aikawa M. Infect. Immun. 1989; 57: 3230-3233Crossref PubMed Google Scholar). There is extensive evidence indicating an essential role for parasite-derived proteases in invasion. Invasion by P. falciparum merozoites is blocked in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF)1 (7Dejkriengkraikhul P. Wilairat P. Z. Parasitenkd. 1983; 69: 313-317Crossref PubMed Scopus (39) Google Scholar), and invasion by merozoites of a number of Plasmodium species is prevented by chymostatin (8Dutta G.P. Banyal H.S. Indian J. Exp. Biol. 1981; 19: 9-11PubMed Google Scholar, 9Banyal H.S. Misra G.C. Gupta C.M. Dutta G.P. J. Parasitol. 1981; 67: 623-626Crossref PubMed Scopus (67) Google Scholar, 10Dluzewski A.R. Rangachari K. Wilson R.J.M. Exp. Parasitol. 1986; 62: 416-422Crossref PubMed Scopus (80) Google Scholar, 11Braun-Breton C. Blisnick T. Jouin H. Barale J.C. Rabilloud T. Langsley G. Pereira da Silva L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9647-9651Crossref PubMed Scopus (60) Google Scholar, 12McPherson R.A. Donald D.R. Sawyer W.H. Tilley L. Mol. Biochem. Parasitol. 1993; 62: 233-242Crossref PubMed Scopus (27) Google Scholar, 13Schrevel J. Deguercy A. Mayer R. Monsigny M. Blood Cells. 1990; 16: 563-584PubMed Google Scholar). The inhibitory effect of chymostatin on invasion can be reversed by pretreatment of target erythrocytes with chymotrypsin (10Dluzewski A.R. Rangachari K. Wilson R.J.M. Exp. Parasitol. 1986; 62: 416-422Crossref PubMed Scopus (80) Google Scholar), suggesting that the chymostatin-sensitive step in invasion involves an essential, parasite-induced proteolytic modification of the red cell surface (10Dluzewski A.R. Rangachari K. Wilson R.J.M. Exp. Parasitol. 1986; 62: 416-422Crossref PubMed Scopus (80) Google Scholar, 11Braun-Breton C. Blisnick T. Jouin H. Barale J.C. Rabilloud T. Langsley G. Pereira da Silva L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9647-9651Crossref PubMed Scopus (60) Google Scholar, 12McPherson R.A. Donald D.R. Sawyer W.H. Tilley L. Mol. Biochem. Parasitol. 1993; 62: 233-242Crossref PubMed Scopus (27) Google Scholar). A glycosylphosphatidylinositol (GPI)-anchored malarial serine protease activity has been described that may be involved in this modification (14Braun-Breton C. Rosenberry T.L. Pereira da Silva L. Nature. 1988; 332: 457-459Crossref PubMed Scopus (129) Google Scholar). Treatment of isolated, invasive merozoites of the simian malariaP. knowlesi with N-tosyl phenylalanylchloromethyl ketone or N-tosyl lysylchloromethyl ketone prevents primary attachment of the parasites to host cells, whereas chymostatin blocks a later stage in the invasion pathway, indicating that more than one distinct protease activity may be involved (15Hadley T. Aikawa M. Miller L.H. Exp. Parasitol. 1983; 55: 306-311Crossref PubMed Scopus (109) Google Scholar). Consistent with this, a P. falciparum serine protease activity that mediates an essential processing and shedding of a major merozoite surface protein (merozoite surface protein-1; MSP-1) at invasion is highly sensitive to inhibition by PMSF but not by chymostatin (16Blackman M.J. Holder A.A. Mol. Biochem. Parasitol. 1992; 50: 307-316Crossref PubMed Scopus (185) Google Scholar, 17Blackman M.J. Chappel J.A. Shai S. Holder A.A. Mol. Biochem. Parasitol. 1993; 62: 103-114Crossref PubMed Scopus (49) Google Scholar). Parasite proteases involved in invasion are attractive potential targets for new rational approaches to antimalarial chemotherapy. Here we report the identification of a novel, single copy P. falciparum gene (denoted pfsub-1) encoding a member of the subtilisin-like serine protease superfamily (subtilases). The primary gene product is expressed in the latter stages of intracellular merozoite maturation, and is post-translationally modified during secretory transport in a manner consistent with it being processed to form a mature, enzymatically active product. The putative mature protease is concentrated in a subset of dense granules within the apical domain of free merozoites and then is released in a soluble form during erythrocyte invasion, suggesting that it may play a role in invasion. This is the first molecular characterization of a putative apicomplexan serine protease. DISCUSSIONThis report describes the first gene to be identified of any apicomplexan parasite that encodes a serine protease-like protein. We have not directly demonstrated any pro" @default.
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