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Malarial proteases: assignment of function to activity
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tification of Plas,nodium knowlesi erythrocyte binding proteins. Mol. Biochem. Parasit., 31,217-222. Mons, B. (1990), Preferential invasion of malarial merozoites into young red blood cells. Blood Cells, 16, 299-312. Orlandi, P.A., Sim, B.K.L., Chulay, J.D. & Haynes, J.D. (1990), Characterization of the 175 kilodalton erythrocyte binding antigen of Plasmodium falciparum. Mol. Biochem. Parasit., 40, 285-294. Pasvol, G.~ Jungery, M., Weatheral, D.J., Parsons, S.F., Anstee, D.J. & Tanner, M.J.A. (1982), Glycophorin as a possible receptor for Plasmodium falciparum. Lancet, II, 947-950. Perkins, M.E. (1981), Inhibitory effects of erythrocyte membrane proteins on the in vitro invasion of the human malaria parasite Plasmodium falciparum into its host cell. J. Cell. Biol., 90, 563-570. Perkins, M.E. & Rocco, L.J. (1988), Sialic acid-dependent binding of Plasmodiumfa!ciparum antigen, Pf 200, to human erythrocytes. J. Immunol., 141, 3190-3196. Pirson, P.J. & Perkins, M.E. (1985), Characterization with monoclonal antibodies of a surface antigen of Piasmodium falciparum merozoites. J. Immunol., 134, 1946-1951.
Schrevel, J., Deguercy, A., Mayer, R. & Monsigney, M. (1990), Proteases in malaria-infected red blood cells. Blood Cells, 16, 563-584. Sim, B.K.L., Orlandi, P.A., Haynes, J.D., Klotz, F.W., Carter, J.M., Camus, D., Zegans, M.E. & Chulay, J.D. (1990), Primary structure of the 174 K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion. J. Cell Biol., 111, 1877-1884. Tanner, M.J.A., Anstee, D.J., Mallinson, G., Ridgewell, K., Martin, P.G., Arent, N.D. & Parsons, S. (1988), Effect of endoglycosidase F-peptidyl N-glycosidase F on preparations of the surface components of the human erythrocyte. Carbohydr. Res., 178, 203-212. Torii, M., Adams, J.H., Miller, L.H. & Aikawa, M. (1989), Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi. Infect. lmmun., 57, 3230-3233. Wertheimer, S.P. & Barnwell, J.W. (1989), Plasmodium vivax interaction with the human Duffy blood group glycoprotein: identification of a parasite receptor-like protein. Exp. Parasit., 69, 340-350.
Malarial proteases: assignment of function to activity J . - C . Barale o), G. L a n g s l e y (I)(*), W . F . M a n g e l (2) a n d c . B r a u n - B r e t o n (D
a) Unit6 de Parasitologie expdrimentale, Ddpartement d'Immunologie, Institut Pasteur, 75724 Paris Cedex 15, and t2) Biology Department, Brookhaven National Laboratories, Upton, N Y 11973 (USA)
Introduction As classical drug treatment of malaria becomes less efficient due to the appearance of resistant parasites, the development of new antimalarial agents is of importance. Enzymes involved irl essential parasitic physiological pathways represent an interesting target, since their specific inhibition should block parasite growth. Whether in prokaryotic or eukaryotic or.~anisms, proteases have been shown to be involved in many important physiological events, such as protein matu(*) To whom correspondence should be addressed.
ration, gene regulation, metabolic pathway regulation, blood coagulation and immune reactions. Parasite proteases have been described as being involved in host protein degradation, protein processing, invasion of host cells and immune evasion. In this context, plasmodia proteolytic activities could be considered as targets for new drugs based on highly specific inhibitors. The design of highly specific inhibitors depends on the determination of both the cleavage site and efficient peptidic substrates of a peptidase. For this reason, we will only focus on endopeptidases, from
A MALARIA DIVERTIMENTO hereon named proteases. Aside from its cleavage sites, a protease is usually characterized by the pH at which its is me.ximally active, its molecular weight, pI, localization and especially by the composition of its active site. The composition of the active site defines a specific catalytic mechanism and has led to the classification of endopeptidases into four groups. Each group is defined by a series of specific inhibitors (table I). When the reactive amino acid is a serine, the enzyme is named a serine-protease; likewise, there are cysteine proteases and asparte proteases. If the active site involves a divalent cation, usually Zn ~-+, the enzyme is a metalloprotease. The active site structure and the general catalytic mechanism are closely related for serine and cysteine proteases. Both types have active sites organized around three amino acids; aspartate, histidine and serine or cysteine. Serine, cysteine and histidine are localized in a stretch of well conserved residues (consensus sequences), whereas the aspartate region is more degenerate. The catalytic mechanism involves two steps, a quick acylation and a slow deacylati, m. Histidine and aspartic acid are directly involved in the catalytic process removing the proton from tlae serine, thereby rendering the oxygen of the serine, or the sulphur of the cysteine nucleoph~lic. The serine or cysteine is involved in a covalent (enzyme-
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substrate) transition complex. The above properties have been used to design specific irreversible inhibitots by modifying natural substrates (like peptidyl fluoromethanes). The modified substrate binds covalently to the active site and, because it is not hydrolysed, irreversibly blocks the enzyme activity (Boyer, 1971 and Stryer, 1988). It has been recently proposed that cysteine and serine proteases are evolutionarily related, since viral cysteine proteases have active sites homologous to classical serine protease active sites. The histidine and aspartate consensus sequences are conserved, as well as the usual serine consensus sequence, with the single exchange of the serine catalytic residue by a cysteine (Pearl and Taylor, 1987; Brenner, 1988). Aspartate proteases and metalloproteases do not form a covalent transition. Their catalytic process is based on classical acid-base mechanism. Aspartate proteases are well conserved all along their sequence. They have a bilobal structure, each domain contains an aspartate site (Tang and Wong, 1987). These two domains are folded in the native enzyme, in order to form the ~o.tive site. It has been proposed that this bilobal structure is the consequence of the duplication and fusion of a single domain ancestor gene (Holm et aL, 1984). These proteases are often named acidic proteases, since their optimal pH is low (2-5).
Table I. The four classes of proteases and their specific inhibitors.
Inhibitors t*) lr
Reversible Aspartate proteases
Pepstatin
Metalloproteases
EDTA, Phosphoramidon, l, 10-phenanthroline
Cysteine proteases
Leupeptin, antipain
Serine Leupeptin, chymostatin (for proteases chymotrypsin-like enzymes)
Irreversible
E-64, NEM, TLCK, Cbz-Phe-AIa-CH2N, Iodoacetate PMSF, DFP, a2-macroglobulin, TPCK (for chymotrypsin-like enzymes) and TLCK (for trypsin-like enzymes)
of proteases Pepsin, renin, Pf 40 kDa, Pf 10 kDa, Pf Datin-like protease Thermolysin, carboxypeptidase A, GP63 t**~(leishmania) Papain, Pf 68 kDa, Pf 37 kDa, Pf 28 kDa, Pb 68 kDa, lab 37 kDa Trypsin, chymotrypsin, factor Xa, Pf 76 kDa
For each class, at least one general example is given. In bold are mentioned the proteases that are cited ~n t.le text. Pf-~ P. f:~cipq:~o;~, Pl., - P. berghi. E-64: L-trans-epoxysuccinyl-leucilamido(4-amino)butane; NEM : N-ethyl-maleimide; DFP: di-isoprop.df oorophosphate; PMSF: phenylmethanesulphonylfluoride; TPCK : L-l-chloro3-(4-tosylamido)7-amino-2-heptanone; TLCK: L-l-chloro3A4-tosylamido)4-phenyl2-butanone; EDTA: ethylenediaminotetraacetic acid). (*) Stryer 0988) and Boyer (1971). (**) Bouvier et al. 0989).
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The active site of metalloproteases is constituted by at least five amino acids dispersed in the protein sequence and forming a water-filled cavity. Three of these amino acids, two histidines and a glutamine, bind Zn ÷ +. This tetrahedron is considered to be involved in the catalytic process and the other residues are thought to stabilize the (enzyme-substrate) complex. Proteases are often synthesized as an inactive precursor, called a zymogen. The active site is formed as a consequence of the specific cleavage of the zymogen. This processing can be achieved by another protease and for example, biochemical cascades involving serine proteases have been described for b'""'~ coagulation and fibrinolysis (Bergmeyer et al., 1984). The processing can also be due to an autocatalytic process, eg. pepsinogen is autoactivated into pepsin following acidification of the medium (Tang and Wong, 1987). The first description of a malaria pEoteolytic activity was published in 1946, and since then, at least 25 other proteases of all four classes have been reported in the different plasmodia species (Moulder and Evans, 1946; and see Schrevel et al., 1990, for a review). In spite of this, however, the definition of a natural substrate appears to be more delicate as only one has been clearly identified (Goldberg et al., 1991). Commonly, malaria proteases are divided into two general groups: a) If the optimal pH is acidic, the protease is presumed to be involved in haemoglobin degradation, since it should be active in the acidic environment (pH 5) of the digestive vacuole. Several proteases have been described as being able to degrade haemoglobin (Vander Jagt et aL, 1986; Rosenthal et al., 1989). b) If the protease is specifically active in schizonts and/or merozoites, then a role in merozoite maturation or red blood cell invasion is often invoked. Such a malarial protease would be involved in the regulation of parasite development. Inhibitor studies have clearly implicated proteases in red blood cell invasion (Banyal et al., 1981; Hadley et al., 1983; Dluzewski et al., 1986; Rosenthal et al., 1987; BraunBreton et al., 1988; Deguercy et al., 1990; Schrevel et al., 1990). In this paper, we will consider a few examples of malaria proteases implicated in both of these two general functions: l) haemoglobin degradation; ano 2) regulation of parasite development.
Proteases and haemoglobin degradation Using a P. gallinaceum extract~ Moulder and Evans reported in 1946 that raalarial proteases might be implicated in haemoglobin degradation, and one
of the first ideo,'_ified physiological functions of the parasite was its aoility to degrade host haemoglobin (Groman, 1951). The parasite degrades 25 °70 to 75 07o of the red blood cell (rbc) haemoglobin. This degradation might contribute to a volume compensation necessary for parasite growth inside the rbc (Ginsburg, 1990), and also in the generation of the necessary amino acids for parasite development (Sherman, 1979). This is nowadays widely accepted and, among the many s~.udies that have been performed, we will focus on three that illustrate the general approach which has been followed by many laboratories. Goldberg et ai. (1991) have described a trophozoite-specific 40-kDa protease with an optimal pH of 5. The purified enzyme is able to cleave in vitro haemoglobin (Hb) between Phe33 and Leu34 of the alpha chain; 50 07o inhibition was observed with 5-nM p "pstatin, an aspartate protease inhibitor isolated from Streptomyces. The same cleavage of Hb was achieved by incubation with an extract of purified digestive vacuoles, the pH of which is around 5. This last reaction was also inhibited by pepstatin. These results indicated that the 40-kDa protease is located in the food vacuole and is responsible for one of the steps in Hb degradation. Residues 33-34 are located in an important and conserved structural reg',on of haemoglobin, the hinge. As a first s t ~ of Hb degradation, hinge cleavage could induce unraveling of haemoglogin and make it accessible to further proteolytic degradation. Since the hinge region is highly conserved in vertebrate Hb, proteolytic cleavage of the hinge might also be conserved in the different Plasmodia species. A ' I U. .W. . .IIIUI~I,.,UI¢I.I . ' .... ' . . . .W. l.~-_L, . . . . . -. . . . . . :,: . . . . .p .i p ; l ~ l l t , IJl~l..131.atllll~-31~ll31LIV~:, tease (10-kDa) purified from P. f a l c i p a r u m trophozoites has been described by Vander Jagt et al. (1986). This aspartate prote~',e, ca!leA protease S, displays maximal Hb degradaf~3n at pH 4.5. It has been considereed as being located in the food vacu~ pie. The activity is inhibited in vitro by ferriprotoporphyrin IX, a degradation product of the haem, as well as by the ferriprotoporp aTrin IX-chlorocluine compI..-x. This last observatioh might reflect ~:::e of the mechanisms by which ch!oroqt:ine is toxic for malaria parasites. Accumulation of chloroquine in the food vacuole is due to its abi2 ty to complex ferriprotoporphyrin IX; it prevents the incorporation of ferriprotoporphyrin IX into the parasite pigment and leads to the inhibition of haemoglobin degradation, with consequent inhibition of parasite growth. A trophozoite-specific 28-kDa cysteine protease k,.as also been implicated in Hb metabolism (Rosenthai et al., 1989). The degradation of different arginine-containing fluorogenic substrates by trophozoite extracts has been tested. Cbz-Phe-ArgAMC and Cbz-Val-Leu-Arg-AMC were selected as the best substrates with an optimal pH of 5. The only proteolytic activity detected by gelatin-PAGE and af-
A MALARIA DIVERTIMENTO
fected by 1 ~M Cbz-Phe-AIa-CH2F was the 28-kDa protease. Due to its substrate specificity, the 28-kDa cysteine protease is likely to be a lysosomal cathepsin B, or L type protease (Rosenthal et al., 1989). Independent studies using similar cysteine protease inhibitors have shown that Cbz-Leu-Tyr-CH2F is dramatically more inhibitory than Cbz-Phe-AIaCH2F for intraerythrocytic development of P. falciparum (Rockett et al., 1990). Enlargment of the parasite food vacuole was observed, consistent with an inhibition of a food vacuole protease(s). However, the target of the inhibitor has not yet been identified. These inhibition studies indicate that whether identical to, or different from, the 28-kDa protease, it is not likely to be a cathepsin-L-like protease (Rockett et al., 1990). Only the 40-kDa aspartate protease has been clearly shown to be involved in Hb degradation. It is not yet known whether the other activities are responsible for Hb degradation in vivo and how many different activities are involved in this degradation. It is also clear that the food vacuole probably contains many proteases, not only for Hb degradation, but also for the proteolysis of other substrates. Obviously, these activities could also be crucial for the parasite growth.
Proteases and antigen processing Proteases are known to be involved in posttranslational modifications of proteins. Malaria parasite antigens have been described that appear to undergo a controlled number of processing steps: p 126 (Delplace et al., 1987), GBPI30 (Perkins, 1988), MSA-I (Freeman and Holder, 1983; Holder, 1988, for a review). Due to its position on the merozoite surface and its use as a potential vaccine, MSA-I (also referred to as p190/195, PSA or PMMSA) has been the subject of intensive study (Holder, 1988). This antigen is synthesized as a high molecular weight precursor in schizonts and processed about the time of merozoite release. The precise time of processing is not known. It has been shown, however, that even when processed, the products remain associated as a complex at the surface of merozoites (Freeman and Holder, 1983; David e t al., 1984; McBride et ai., 1985 ; Lyon et al., 1986, 1087). Although the intact molecule is polymorphic, a conserved pattern of degradation is observed (Holder, 1988). This observation suggests that the enzymes involved in this processing are relatively conserved in different Plasmodia strains and species. The processing of MSA-I was inhibited by protease inhibitors (Pirson and Perkins, 1985), but not affected by relatively specific inhibitors like
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chymostatin, leupeptin, pepstatin and antipain (Lyon and Haynes, 1986). Therefore, the protease(s) involved in this processing appears to be either a metalloprotease(s) (since no inhibitor of this type was tested), or an unusual enzyme(s) of the other three classes of proteases. The cleavage sites within the high molecular weight precursor have been recently determined : the amino-terminal sequence of each processing product was compared with the amino acid sequence of the precursor, deduced from the gene sequence (Strych et al., 1987 and Blackman et aL, 1991). These results indicate that only the last processing step (one that cuts behind a hydrophobic residue) can be attributed to a serine protease, since the other cleavage sites do not resemble those reported for any known protease. For this reason, the enzyme(s) might be sufficiently different from host proteases to be the target(s) of specific inhibitors. If the processing takes place at the surface of the released merozoite in the blood, the enzyme certainly has a specificity different from that of host proteases, since it is active in an environment where 10 % of the serum proteins are protease inhibitors. The last processing step seems to involve a chymotrypsin-like enzyme, since it occurs at a sequence homologous to a chymotrypsin cleavage site (Blackman et al., 1991). The only parasite chymotrypsin-like protease that has been described as being active at the time of merozoite release is the P. falciparum p76 (Braun-Breton et al., 1988). However, this last MSA-I processing step is known t,a t~lcp pl~r.,~ rh~rlng rbc invasion, and if it occurs within the newly formed parasitophorous vacuole, then it might also be performed by a host enzyme. Autocatalytic processing has been reported for proteases and we have to consider that MSA-1 might self-process. The DNA sequence of MSA-I has been determined for an impressive number of isolates (reviewed by Anders and Smythe, 1989). No apparent consensus sites of proteases have been found in the predicted amino acid sequence. This observation does not exclude the possibility that MSA-I is either a metalloprotease for which consensus sites have not been reported or an unusual protease of the serine, cysteine, or aspartate types. Indeed, a similar case of autocatalytic processing has been proposed for Hsp70, a member of the family of heat shock proteins. Degradation fragments of 40-44 kDa and 18-22 kDa have been observed in a preparation of pure Hsp70 (Mitchell et al., 1985). The amino acids histidine, aspartate, and serine were present in the sequence in an appropriate arrangement to be a serine protease, but no proper serine protease consensus sites were observed. Autocatalytic processing might be invoked for the parasitophorous vacuole antigen p126, or SERP.
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This antigen is processed into 50-kDa, 47-kDa and 18-kDa fragments at the very end of the intraerythrocytic cycle (Delplace et al., 1985, 1987). The processing is inhibited by leupeptin; TLCK and TPCK are also inhibitors of this reaction, but they seem to be toxic for the parasite (Debrabant and Delplace, 1989). A computer analysis of pI26/SERP gene sequence revealed the presence of consensus sites of cathepsin L cysteine protease, where the active cysteine has been exchanged for a serine (Knapp et al., 1989; Higgins et al., 1989; Eakin et al., 1989; Mottram et al., 1989). Eakin et al. (1989) have proposed that this protein is a serine protease with a cysteine protease conformation. There is a precedent for this, as in viral cysteine trypsin-like proteases, the cysteine has replaced serine in the serine consensus sequence. Although an elegant observation, it remains theorectical, as to date no proteolytic activity has been assigned to pI26/SERP.
Proteases, merozoite maturation and red blood cell invasion
Merozoite maturation concerns both the processing of parasite antigens necessary to produce infectious merozoites and the efficient release of these merozoites into the blood stream. Proteases are involved in antigen processing, as reported in the previ. ous paragraph. However, the involvement of proteases in the red blood cell lysis is not conclusive. The lysis does not seem to be sensitive to protease inhibitors, and thus this step might involve haemolytic activity comparable to the haemolysins of Trypanosoma cruzi, or Listeria monocytogenes, which allow these organisms to escape from the phagocytic vacuole to the cytoplasm of the host cells (Andrews et al., 1990 and Bielecki et al., 1990). In contrast to lysis, the release of individual merozoites appeared to be sensitive to leupeptin, a general protease inhibitor. Leupeptin treated P. knowlesi schizonts failed to release merozoites, whereas the red blood cell plasma membrane seemed to be disrupted (Hadley et al., 1983). The merozoites remained aggregated as clusters, probably surrounded by a membrane of host origin. Lyon and Haynes (1986) have obtained analogous results when P. falciparum schizonts were incubated with 10 ~tg/ml of chymostatin, leupeptin, antipain, pepstatin; and Banyal et al. (1981) reported similar observations with 50 ~tg/ml leupeptin. The proteolytic activity or activities implicated for merozoite release has/have not yet been identified. Both the binding of merozoites and their entry into the red cell are sensitive to protease inhibitors. Hadley et al. (1983) have shown that TPCK and TLCK inhibit the attachment of purified P. knowlesi merozoites to the red blood cell surface. Chymosta-
tin, however, does not seem to affect binding, but entry. Invasion appeared to be blocked after the reorientation of the merozoite and tight junction formation with the erythrocyte surface. In conclusion, more than one proteolytic activity seems to be implicated in the invasion process. At least one activity (inhibited by TPCK and TLCK) is necessary for the efficient recognition of red blood cell receptors by merozoite surface molecules. A second (chymostatin-sensitive) activity appears necessary for the entry of the parasite into the red cell. The first might be implicated in processings of parasite and/or rbc surface molecules and the second might be involved in the degradation of red blood cell surface molecules, or cytoskeleton proteins, or release of rhoptry contents. In a study using P. falciparum, Dluzewski et al. (1986) have contributed to the characterization of the second enzyme, as pretreatment of rbc by chymotrypsin reversed the inhibition of invasion by chymostatin. Furthermore, when rbc were loaded with chymostatin prior to invasion, inhibition was again observed. It was comparable to that obtained when chymostatin was added directly to the culture medium. Due to its sensitivity to chymos~atin, the protease involved in this step of parasite entry is likely to be a chymotrypsin-like serine protease. Only p76 has been reported to be a merozoitespecific serine pro~ease (Braun-Breton et al., 1988). Both p76 and the P. chabaudi analogt~e, p65, are glycosyl-phosphatidylinositol(GPI)-anchored. In schizonts, the proteins are GPI-anchored and catalytically inert. The presence of a conformationally formed active site can be detected by binding of tritiated di-isopropylfluorophosphate (DFP), but no proteolytic activity associated with the membrane forms of these proteins has been detected using gelatin gel assays (Braun-Breton and Pereira da Silva, 1988). In merozoites, however, the proteins are partially recovered as soluble forms, exhibiting serine protease-like activity and harbouring a cross-reacting epitope specific for the presence of a phosphatidylinositol-phospholipase C (PI-PLC) cleaved glycolipid anchor (Braun-Breton et al., 1988; BraunBreton and Pereira da Silva, 1988). This last result suggests that the solubilization and concomitant activation of the enzyme is due to an endogenous PI-PLC. A developmentally regulated glycosyl-PIPLC (GPI-PLC) activity has been reported in P. falciparum and P. chabaudi merozoites, which might be responsible for the release of active p76 and p65 serine proteases (Braun-Breton et al., 1990, 1991). The precise location of these proteins is not known. A monoclonal antibody Hb3 lcl 3, which immunoprecipitates the p76 serine protease activity, has been reported to be rhoptry specific (Perrin and Dayal, 1982). Since this monoclonal immunoprecipitates both the p76 antigen and a 4 l-kDa antigen, 1o-
A MALARIA DIVERTIMENTO cation of the p76 in the rhoptries has not been conclusively established. However, the active p76 and p65 enzymes can be released by exogenous PI-PLC treatment of intact merozoites, indicating that the protein anchors are accessible to externally added reagents (Braun-Breton et al., !988). The natural substrates of these enzymes have not yet been identified. Schrevel et al. (1990) have characterized two cysteine proteases, a 37-kDa and a 68-kDa enzyme, present in P. berghei and P. falciparum merozoites. The authors proposed that the 37-kDa enzyme is involved in the cleavage of erythrocyte cytoskeletal components, since it is able to cleave host spectrin in vitro. Interestingly, specific peptidic inhibitors of the 68-kDa enzyme block invasion when added to an in vitro culture of P. falciparum parasites. Due to the fact that purified P. falciparum merozoites are poorly invasive, these results were obtained by addi-
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tion of the inhibitors to mature schizonts. Therefore, the precise step at which the inhibitors are efficient is not known. The effect ot these inhibitors on the parasite development in vivo has not yet been reported. Proteases and gene regulation
One of the best examples of proteolytic activity being important in gene regulation is that of C 1 digestion of the phage lambda repressor (Roberts and Roberts, 1975). However, a more topical example is the recently described cleavage of the NF-kB activating factor by the HIV-encoded aspartate protease (Rivi6re et al., 1991). As part of our general analysis of proteases, we have identified a 27-kDa aspartate protease in P. falciparum and P. chabaudi schizonts (see fig. 1). Given that the malaria genome is AT-rich
I Is a malaria 27-kDa asoartate to ~ ,r,.o. ~ . . protease . . eouivalent . . . AT-specific DNA-binding protein?
Malaria
Yeast Datin * *
i. Genome 80% AT-rich
AT-specific DNA-binding protein
2. 27-kDa aspartate protease
27-kDa protein with aspartate protease consensus sequence
3. Inhibited by pepstatin
DNA-binding inhibited by pepstatin
4. Sediments then solubilized
Datin may be associated
following DNase 1 treatment
with nuclear scaffold
5. pl of 4 ** Properties of Datin taken from Winter and Varshavsky, 1989
Fig. 1. Identification of a P. falciparum 27-kDa aspartate protease. A freeze/thaw lysate of segmented P. falciparum schizonts was analysed by SDS-PAGE containing 0.1 070 gelatin. The SDS was removed by incubation in a solution of 2.5 °70 Triton-Xl00 for at least 1 h at room temperature. The proteolytic activity was revealed following staining with amidoblack. The aspartate protease nature of the indicated activity was confirmed by inhibition with pepstatin.
I
i I
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(Pollack eta!., 1982), we became interested in the report of a yeast 27.kDa protein (Datin) that specifically binds to AT-rich DNA and contains aspartate protease consensus sequences (Winter and Varshavsky, 1959). The fact that the 27-kOa malaria aspartate protease and yeast Datin -hare a number of features (see fig. 1), led us try and identify the P. falciparum equivalent of Datin. A 270-bp part of the yeast Datin gone which encodes both the aspartate protease consensus sequence, as well as the DNA-binding activity, was used on Southern blots of P. falciparum DNA. As presented in figure 2, ',his specific region of the Datin gone has a homologous counterpart in the P. falciparum genome. Only time and DNA sequencing will tell to wha: degree the two sequences are homologous. However, preliminary gel retardation experiments using the partially purified 27-kDa P. chabaudi aspartate protease and an AT-rich fragment from the 5'-end of the 11-1 gene (Scherf et al.,
I
p
1988) implies that the protease may have pepstatinsensitive DNA-binding activity (data not shown). Drawing on the published examples of how proteases are implicated in gene expression, it seems reasonable to propose a similar model for the Plasmodium Datin-like aspartate protease; namely, the protease binds to DNA and, once bound, cleaves the in situ repressor/activator resulting in the necessary alteration of transcription.
Conclusions The crucial role of parasite proteases in the parasite life cycle has been established. Despite the in,: easing number of characterized parasite-specific proteolytic activities, only one activity has been clearly assigned to a specific function, i.e. haemoglobin degradation. However, correlations have been made between the ability to degrade a substrate (hae-
t'alc~arvm has a sequence homologous to the yeast Datin cjene
s°ut"e,l
I
N-terminol end of Dotin that contains both DNA binding activity and the aspartate protease consensus sequence D-T-G
of Hind l I cut DNA
9k. ----> o
270 bp Probe
ATG GCG AAA ACT TTG GCA CAA GGA AGG A A A M A K T L A Q G R K CCT GGA AGC GGC AGA AAG CCC GGA AAA GGG P G S G R K P G K G AAG ACG TTG AGA GAG GGA AGA AAG CCT GGC K T L R E G R K P G AGT GGT AGG AGG AGG AGG CAA GAT ACT GGG S G R R R R Q D I" IB GGT A A A GAG ACC GAC GGG TCT CAG CAA GAT G K E T D G S Q Q D CAG GAG TCG CGT CTT ATT AGT TCC AGG GAC Q E S R L I S S R D ATG GAA GCT GTG GAC GCA CTG AGA GAG TTG M E A V D A L R E L ACG CAG AGC CCG TCG TCT CAC TCA GCT CAT T H S P S S H S A H AAT TCA TCA GCA GEA CCA CCG CCG CAT N S S A A P P P H
Fig. 2. Identification of a homologous sequence to Datin in the P. falciparum genome. Four ~tg of P. falciparum genomic DNA were digested with Hindlll and then size-separated by 1 070agarose gel electrophoresis; the gel was transferred to Hybond N (Amersham). The 270-bp probe was generated following digestion of the whole Datin gene with ?,'col and SphI and the isolated fragment radiolabelled by random priming. The filter was hybridized with the Datin probed in 6 x SSC at 65°C and the washes were performed at increasing stringency, with the final wash at 2 x SSC at 55°C.
A ML4LARL4 D I V E R 7 tMtzN7
moglobin, spectrin) in vitro and the potential natural (in vivo) substrate. The design of highly specific inhibitors should help in the determination of the biological role for a given proteases. Specific inhibitors have only been used in the analysis of the 28- and 68-kDa cysteine proteases. These inhibitors block parasite growth in vitro. However, relatively low concentrations of Cbz-Leu-Tyr-CH2F (59 nM) and Cbz-Phe-Ala-CH2F (2 ~M), which are specific for the 28-kDa, produced only 50 °70 inhibition of parasite growth and a 100-~M concentration of Cbz-Phe-AIa-CH2F was necessary to totally inhibit parasite development. This may, in part, be due to poor transport of the inhibitor to its target. To improve transport, small molecules are routinely used and such molecules may be very rapidly cleared from the blood. The concentrations used in vivo may therefore have to be much higher than in vitro, with possible toxic effects to the host. In this context, amino acid derivatives of chloromethyl ketones react with nucleophilic residues of many cellular proteins and some of their toxic effects in vivo might be attributed to their general electrophilic nature, rather than to their selective inhibitory action towards certain host proteinases. This type of problem somewhat compromises the interest of intracellular proteases as targets of anti-parasite reagents. Surface proteases seem to be involved i~, merozoite maturation and red blood cell invasion. These parasite enzymes have to be rather unique, since they are active in an hostile environment; IO °7o of serum proteins are protease inhibitors whose function is to limit the half lives of host proteases. Given that
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u
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References Anders, R.F. & Smythe, J.A. (1989), Polymorphic antigens in P. falciparum. Blood, 74, 1865-1875. Andrews, N.W., Abrams, C.K., Slatin, S.L. & Griffiths, G. (1990), A T. cruzi-secreted protein immunologicaily related to the complement component C9: evidence for membrane pore-forming at low pH. Cell, 61, 1277-1287. Banyal, H.S., Misra, G.C., Gupta, C.M. & Dutta, G.P. (1981), Involvement of malaria proteases in the interaction between the parasite and host erythrocyte in Plasmodium knowlesi infections. J. Parasit., 67, (5) 623-626. Bergmeyer, H.U. (1983), Enzymes 3" peptidases, proteinases and their inhibitors in methods of enzymatic analysis (third edition), Volume V (Bergrneyer, H.U.). Verlag Chemie, Weinheim. Bielecki, J., Youngman, P., Connelly, P. & Portnoy: D.A. (1990), Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells. Nature (Lond.), 345, 175-176. Blackman, M.J., Ling, I.T., Whittle, H. & Holder, A. (1991), Processing of the Plasmodium falciparum merozoite surface protein 1" identification of a 33 kilodalton product of a secondary proteolytic processing event and partial characterisation of the protease involved. Paper present at the British Parasitological Society meeting, in Edinburgh, Scotland. Boyer, P.D. (1971), Hydrolysis" peptide bonds, in "The Enzymes", 3, Academic Press, New York, London. Braun-Breton, C., Rosenberry, T.L. & Pereira da Silva, L. (1988), Induction of the proteolytic activity of a membrane protein in P. falciparum by phosphatidyl inositol-specific phospholipase C. Nature (Lond.), 332, 457-459.
t~ h~ ~ot;w~
makes it theoretically possible to design specific inhibitors which will not affect host physiological processes. The surface location of parasite proteases makes the use of higher molecular weight inhibitors possible. Large inhibitors should not enter host cells, thus decreasing any potential toxic side effects. The problems of immunogenicity and degradation of such molecules might be circumvented by modifying natural host inhibitors, rendering them specific for parasite proteases. These molecules would represent a new family of antimalarial agents.
Acknowledgements We would like to thank Prof. L. Pereira da Silva and all the members of the Unit of Experimental Parasitology for their support. This investigation received financial support from the UNDP/World Bank/WHO Special programme for Research and Training in Tropical Diseases and grants from the Minist~re de la Recherche et I'Enseignement Sup~rieur (Aid,. no. 87W0043-Eureka).
tion of a Piasmodium falciparum protease correlated with merozoite maturation and erythrocyte invasion. Biol. Cell, 64, 223-231. Braun-Breton, C., Langsley, G., Barale, J.C. & Pereira da Silva, L. (1999), A malaria phosphatidylinositolspecific phospholipase C" a possible role in merozoite maturation and erythrocyte invasion, in "Cellular and molecular biology of normal and abnormal erythroid membranes" (pp. 315-332). Alan R. Liss, New York. Braun-Breton, C., Blisnick, T., Barbot, P., Billow, R., Pereira da Silva, L. & Langsley, G. (1991), Characterisation of a glycosyl-phosphatidylinositol-specific phospholipase C from malaria parasites. Exp. Parasit. (in press). Brenner, S. (1988), The molecular evolution of genes and proteins" a tale of two serines. Nature (Lond.), 334, 528-530. David, P.H., Hadley, T.J., Aikawa, M. & Miller, L.H. (1984), Processing of a major parasite surface glycoprotein during the ultimate stages of differentiation in Plasmodium knowlesi. Mol. Biochem. Parasit., 11,267-282. Debrabant, A. & Delplace, P. (1989), I eupeptin alters the proteolytic processing of p126, the major parasitophorous vacuole antigen of Plasmodium falciparum. Mol. Biochem. Parasit., 33, 151-158. Deguercy, A., Hommel, M. & Schr~vel, J. (1990), Purifi-
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cation and characterization of 37-kilodalton proteases from Plasmodium falciparum and Plasmodium berghei which cleave erythrocyte cytoskeletal components. Mol. Biochem. Parasit., 38, 233-244. Delplace, P., Dubremetz, J.-F., Fortier, B. & Vernes, A. (1985), A 50 kilodalton exoantigen specific to the merozoite release-reinvasion stage of Piasmodiumfalciparum. Mol. Biochem. Parasit., 17, 239-251. Delplace, P., Fortier, B., Tronchin, G., Dubremetz, J.-F. & Vernes, A. (1987), Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of P. falciparum. Mol. Biochem. Porasit., 23, 193-201. Dluzewski, A.R., Rangachari, K., Wilson, R.J.M. & Gratzer, W.B. (1986), Plasmodiumfalciparum: protease inhibitors and inhibition of erythrocyte invasion. Exp. Parasit., 62, 416-422. Eakin, A.E., Higaki, J.N., McKerrow, J.H. & Craik, C.S. (1989), Cysteine or serine proteinase? Nature (Land.), 342, 132. Freeman, R. & Holder, A. (1983), Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000-mol wt expressed on the surface of Plasmodiumfalciparum merozoites. 3'. exp. Med., 158, 1647-1653. Ginsburg, H. (1990), Some reflections concerning host erythrocyt.e-malarial parasite interrelationships. Blood Cells, 16, 225-235. Goldberg, D.E., Slater, A.F.G., Beavis, R., Chait, B., Cerami, A. & Henderson, G.B. (1991), Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease. J. exp. Med., 173,961-969. Groman, N.B. (1951), Dynamic aspects of the nitrogen metabolism of Piasmodium gallinaceum in viva and in vitro. J. infect. Dis., 88, 126-150. Hadley, T., Aikawa, M. & Miller, L.H. (1983), Studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors. Exp. Parasit., 55, 306-311. Higgins, D.G., McConnel, D.J. & Sharp, P.M. (1989), M~arial proteases? Nature (Land.), 340, 604. Holder, A.A. (1988), The precursor to major merozoite surface antigens: structure and role in immunity. Progr. Allergy, 41, 72-97. Holm, I., Ollo, R., Panthier, J. & Rougeon, F. (1984), Evolution of aspartly proteases by gene duplication: the mouse renin gene is organized in two homologous clusters of four exons. EMBO J., 3, 557-562. Knapp, B., Hundt, E., Nau, U. & Kiipper, H.A. (1989), Molecular cloning, genomic structure and localisation of a blood stage antigen of P. falciparum characterised by a serine stretch. Mol. Biochem. Parasit., 32, 73-84. Lyon, J.A., Geller, R.H., Haynes, J.D., Chulay, J.D. & Weber, J.L. (1986), Epitop~ map and processing scheme for the 195,000-da~ton surface glycoprotein of Plasmodiumfalciparum merozoites deduced from cloned overlapping segments of the gene. PrOConat. Acad. Sci. (Wash.), 83, 2989-2993. Lyon, J.A. & Haynes, J.D. (1986), Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the resence of protease inhibitors. J. Immunol., 136, 2245-2251. Lyon, J.A., Haynes, J.D., Diggs, C.L., Chulay, J.D.,
Haidaris, C.G. & Pratt-Rossiter, J. (1987), Monoclonal antibody characterisation of the 195-kilodalton major surface glycoprotein of P. falciparum malaria schizonts and merozoites: identification of additional processed products and a serotype restricted repetitive epitope. J. Immunol., 138, 859-90. McBride, J.S., Newbold, C.I. & Anand, R. (1985), Polymorphism of a high molecular weight schizont antigen of the human malaria parasite Piasmodium faiciparum. J. exp. Med., 161, 160-180. Mitchel, H.K., Petersen, N.S. & Buzin, C.H. (1985), Selfdegradation of heat shock proteins. Prcc. nat. Acad. $ci. (Wash.), 82, 4969-4973. Mottram, J.C., Coombs, G.H. & North, M.J. (1989), Cysteine or serine proteinase ? Nature (Land.), 342, 132. Moulder, J.W. & Evans, E.A.J. (1946), The biochemistry of the malaria parasite. 1 VI. Studies on nitrogen metabolism of the malaria parasite. J. biol. Chem., 164, 135-157. Pearl, L.H. & Taylor, W.R. (1987), A structural model for the retroviral proteases. Nalure (Land.), 329, 351-354. Perkins, M.E. & Rocco, L.J. (1988), Sialic acid-dependent binding of Plasmodiumfalciparum merozoite surface antigen: Pf200, to human erythrocyte. J. Immunoi., 141, 3190-3196. Perrin, L.H. & Dayal, R. (1982), Immunity to sexual erythrocytic stages of P. falciparum: role of defined antigens in the humoral response, lmmunoi. Rev., 61, 245-270. Pirson, P.J. & Perkins, M.E. (1985), Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites. J. Immunol., 134, 1946-1951. Pollack, Y., Katzen, A., Spira, D.T. & Golenser, J. (1982), The genome of Plasmodiumf~lciparum - - 1. DNA base composition. Nucl. Acids Res., !,a, ~ _ ~ . Rivi6re, Y., Blank, V., Kourilsky, P. & Israel, A. (1991), Processing of the precursor of the NF-KB by the HIV-I protease during acute infection. Nature (Land.), 350, 625-626. Roberts, J.W. & Roberts, C.W. (1975), Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc. nat. Acad. ScL (Wash.), 72, 147-151. Rockett, K.A., Playfair, J.H.L., Ashall, F., Targett, G.A.T., Angliker, H. & Shaw, E. (1990), Inhibition of intraerythrocytic development of Plasmodium faiciparum by proteinase inhibitors. FEBS, 259, 257-259. Rosenthal, P.J., Kim, K., McKerrow, J.H. & Leech, J. (1987), Identification of three stage-specific proteinases of Plasmodium falciparum. J. exp. Med., 166, 816-821. Rosenthal, P.J., McKerrow, J.H., Rasnick, D. & Leech, J.H. (1989), Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development. Mol. Biochem. Parasit., 35, 177-184. Scherf, A., Hilbich, C., Seig, K., Mattei, D., MercereauPuijalon, O. & Mfiller-Hill, B. (1988), The 11-1 gene of P. falciparum codes for distinct fast evolving domains. EMBO J., 1129-1137. Schr~vel, J., Deguercy, A., Mayer, R. & Monsigny, M. (1990), Proteases in malaria-infected red blood cells. Blood Cells, 16, 563-584.
A M A L A R I A D I V E R TIMENTO Sherman, I.W. (1979), Biochemistry of Plasmodium (malaria parasites). Microbiol. Rev., 43, 453-495. Strych, W., Miettinen-Baumann, A., Lottspeich, F. & Heidrich, H. (1987), Isolation and characterisation of the 80,000 dalton Plasmodium falciparum merozoite surface antigen. Parasit. Res., 73, 435441. Stryer, L. (1988), Mechanisms of enzyme action and control of enzymatic activity. Biochemistry, third editien. W.H. Freeman & Co., San Francisco.
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Tang, J. & Wong, R.N.S. (1987), Evolution in the structure and function of aspartic proteases. J. Cell. BioL, 33, 135-145. Vander Jagt, D.L., Hunsaker, L.A. & Campos, N.M. (1986), Characterisation of a hemoglobin-degrading, low molecular weight protease from P. falciparum. MoL Biochem. Parasit., 18, 389-400. Winter, E. & Varshavsky, A. (1989), A DNA binding protein that recognizes oligo(dA).oligo(dT) tracts. EMBO J., 8, 1867-1877.
Plasmodium falciparum cytoadherence J. Leech
Parasitology Laboratory, Department o f Medicine, University o f California-San Francisco and San Francisco General Hospital, San Francisco CA 94110 (USA)
Plasmodium falciparum cytoadherence refers to the specific binding that occurs between trophozoiteand schizont-infected erythrocytes and host endothelial cells. In vivo, cytoadherence results in the removal of mature asexual parasites from the general circulation and their localization within the postcapillary venuies of multiple organs, predominantly the brain, heart, and skeletal muscle (Miller, 1969). This localization is referred to as sequestration. Sequestration appears to contribute to the consequences of P. falciparum malaria in multiple ways, and the molecules involved in cytoadherence may be important targets of naturally acquired or vaccine-induced immunity to this disease. In vitro, the phenomenon of cytoadherence has been investigated with a variety of model systems, including cultured endothelial cells (Udeinya et al., 1981), C32 melanoma cells (Schmidt et al., 1982), monocytes (Ockenhouse and Chulay, 1988), transfected COS cells (Oquendo et al., 1989) and with purified candidate receptor molecules applied to plastic surfaces. Use of these model systems has produced information about the physiology of cytoadherence and the molecules involved. In this article, I will first discuss the role of cytoadherence in the clinical and biological consequences of P. falciparum malaria. Second, I will discuss the inhibition of cytoadherence by antibody and third, the
antigenic differences among cytoadherence antigens from different parasite isolates. The question of antigenic variation will be discussed. Fourth, I will discuss recent work on the molecular basis of cytoadherence. Throughout this discussion, I will use the following terminology. "Cytoadherence antig e m " are the molecule(s) on the surface of im%cted erythrocytes that are the target of antibody that inhibits cytoadherence. "Cytoadherence ligands" are the molecule(s) that mediate binding to the host cell. "Cytoadherence receptors" are the host molecules that bind cytoadherence ligands. This article will emphasize recent work and will be opinionated and speculative. Readers desiring more comprehensive and historical reviews are referred to several recent, excellent articles. The phenomenon of rosetting between infected erythrocytes and between infected and uninfected erythrocytes will not be discussed in this article. The role of cytoadherence in the biological and clinical consequences of P. falciparum malaria The evidence that cytoadherence and sequestration have important roles in P. falciparum biology is teleological and experimental. Teleologically, the
Address for correspondence: J. Leech, Building 30, Room 408, Say Francisco General Hospital, 1001Potrero Ave., San Francisco, CA 94110.
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tification of Plas,nodium knowlesi erythrocyte binding proteins. Mol. Biochem. Parasit., 31,217-222. Mons, B. (1990), Preferential invasion of malarial merozoites into young red blood cells. Blood Cells, 16, 299-312. Orlandi, P.A., Sim, B.K.L., Chulay, J.D. & Haynes, J.D. (1990), Characterization of the 175 kilodalton erythrocyte binding antigen of Plasmodium falciparum. Mol. Biochem. Parasit., 40, 285-294. Pasvol, G.~ Jungery, M., Weatheral, D.J., Parsons, S.F., Anstee, D.J. & Tanner, M.J.A. (1982), Glycophorin as a possible receptor for Plasmodium falciparum. Lancet, II, 947-950. Perkins, M.E. (1981), Inhibitory effects of erythrocyte membrane proteins on the in vitro invasion of the human malaria parasite Plasmodium falciparum into its host cell. J. Cell. Biol., 90, 563-570. Perkins, M.E. & Rocco, L.J. (1988), Sialic acid-dependent binding of Plasmodiumfa!ciparum antigen, Pf 200, to human erythrocytes. J. Immunol., 141, 3190-3196. Pirson, P.J. & Perkins, M.E. (1985), Characterization with monoclonal antibodies of a surface antigen of Piasmodium falciparum merozoites. J. Immunol., 134, 1946-1951.
Schrevel, J., Deguercy, A., Mayer, R. & Monsigney, M. (1990), Proteases in malaria-infected red blood cells. Blood Cells, 16, 563-584. Sim, B.K.L., Orlandi, P.A., Haynes, J.D., Klotz, F.W., Carter, J.M., Camus, D., Zegans, M.E. & Chulay, J.D. (1990), Primary structure of the 174 K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion. J. Cell Biol., 111, 1877-1884. Tanner, M.J.A., Anstee, D.J., Mallinson, G., Ridgewell, K., Martin, P.G., Arent, N.D. & Parsons, S. (1988), Effect of endoglycosidase F-peptidyl N-glycosidase F on preparations of the surface components of the human erythrocyte. Carbohydr. Res., 178, 203-212. Torii, M., Adams, J.H., Miller, L.H. & Aikawa, M. (1989), Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi. Infect. lmmun., 57, 3230-3233. Wertheimer, S.P. & Barnwell, J.W. (1989), Plasmodium vivax interaction with the human Duffy blood group glycoprotein: identification of a parasite receptor-like protein. Exp. Parasit., 69, 340-350.
Malarial proteases: assignment of function to activity J . - C . Barale o), G. L a n g s l e y (I)(*), W . F . M a n g e l (2) a n d c . B r a u n - B r e t o n (D
a) Unit6 de Parasitologie expdrimentale, Ddpartement d'Immunologie, Institut Pasteur, 75724 Paris Cedex 15, and t2) Biology Department, Brookhaven National Laboratories, Upton, N Y 11973 (USA)
Introduction As classical drug treatment of malaria becomes less efficient due to the appearance of resistant parasites, the development of new antimalarial agents is of importance. Enzymes involved irl essential parasitic physiological pathways represent an interesting target, since their specific inhibition should block parasite growth. Whether in prokaryotic or eukaryotic or.~anisms, proteases have been shown to be involved in many important physiological events, such as protein matu(*) To whom correspondence should be addressed.
ration, gene regulation, metabolic pathway regulation, blood coagulation and immune reactions. Parasite proteases have been described as being involved in host protein degradation, protein processing, invasion of host cells and immune evasion. In this context, plasmodia proteolytic activities could be considered as targets for new drugs based on highly specific inhibitors. The design of highly specific inhibitors depends on the determination of both the cleavage site and efficient peptidic substrates of a peptidase. For this reason, we will only focus on endopeptidases, from
A MALARIA DIVERTIMENTO hereon named proteases. Aside from its cleavage sites, a protease is usually characterized by the pH at which its is me.ximally active, its molecular weight, pI, localization and especially by the composition of its active site. The composition of the active site defines a specific catalytic mechanism and has led to the classification of endopeptidases into four groups. Each group is defined by a series of specific inhibitors (table I). When the reactive amino acid is a serine, the enzyme is named a serine-protease; likewise, there are cysteine proteases and asparte proteases. If the active site involves a divalent cation, usually Zn ~-+, the enzyme is a metalloprotease. The active site structure and the general catalytic mechanism are closely related for serine and cysteine proteases. Both types have active sites organized around three amino acids; aspartate, histidine and serine or cysteine. Serine, cysteine and histidine are localized in a stretch of well conserved residues (consensus sequences), whereas the aspartate region is more degenerate. The catalytic mechanism involves two steps, a quick acylation and a slow deacylati, m. Histidine and aspartic acid are directly involved in the catalytic process removing the proton from tlae serine, thereby rendering the oxygen of the serine, or the sulphur of the cysteine nucleoph~lic. The serine or cysteine is involved in a covalent (enzyme-
673
substrate) transition complex. The above properties have been used to design specific irreversible inhibitots by modifying natural substrates (like peptidyl fluoromethanes). The modified substrate binds covalently to the active site and, because it is not hydrolysed, irreversibly blocks the enzyme activity (Boyer, 1971 and Stryer, 1988). It has been recently proposed that cysteine and serine proteases are evolutionarily related, since viral cysteine proteases have active sites homologous to classical serine protease active sites. The histidine and aspartate consensus sequences are conserved, as well as the usual serine consensus sequence, with the single exchange of the serine catalytic residue by a cysteine (Pearl and Taylor, 1987; Brenner, 1988). Aspartate proteases and metalloproteases do not form a covalent transition. Their catalytic process is based on classical acid-base mechanism. Aspartate proteases are well conserved all along their sequence. They have a bilobal structure, each domain contains an aspartate site (Tang and Wong, 1987). These two domains are folded in the native enzyme, in order to form the ~o.tive site. It has been proposed that this bilobal structure is the consequence of the duplication and fusion of a single domain ancestor gene (Holm et aL, 1984). These proteases are often named acidic proteases, since their optimal pH is low (2-5).
Table I. The four classes of proteases and their specific inhibitors.
Inhibitors t*) lr
Reversible Aspartate proteases
Pepstatin
Metalloproteases
EDTA, Phosphoramidon, l, 10-phenanthroline
Cysteine proteases
Leupeptin, antipain
Serine Leupeptin, chymostatin (for proteases chymotrypsin-like enzymes)
Irreversible
E-64, NEM, TLCK, Cbz-Phe-AIa-CH2N, Iodoacetate PMSF, DFP, a2-macroglobulin, TPCK (for chymotrypsin-like enzymes) and TLCK (for trypsin-like enzymes)
of proteases Pepsin, renin, Pf 40 kDa, Pf 10 kDa, Pf Datin-like protease Thermolysin, carboxypeptidase A, GP63 t**~(leishmania) Papain, Pf 68 kDa, Pf 37 kDa, Pf 28 kDa, Pb 68 kDa, lab 37 kDa Trypsin, chymotrypsin, factor Xa, Pf 76 kDa
For each class, at least one general example is given. In bold are mentioned the proteases that are cited ~n t.le text. Pf-~ P. f:~cipq:~o;~, Pl., - P. berghi. E-64: L-trans-epoxysuccinyl-leucilamido(4-amino)butane; NEM : N-ethyl-maleimide; DFP: di-isoprop.df oorophosphate; PMSF: phenylmethanesulphonylfluoride; TPCK : L-l-chloro3-(4-tosylamido)7-amino-2-heptanone; TLCK: L-l-chloro3A4-tosylamido)4-phenyl2-butanone; EDTA: ethylenediaminotetraacetic acid). (*) Stryer 0988) and Boyer (1971). (**) Bouvier et al. 0989).
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The active site of metalloproteases is constituted by at least five amino acids dispersed in the protein sequence and forming a water-filled cavity. Three of these amino acids, two histidines and a glutamine, bind Zn ÷ +. This tetrahedron is considered to be involved in the catalytic process and the other residues are thought to stabilize the (enzyme-substrate) complex. Proteases are often synthesized as an inactive precursor, called a zymogen. The active site is formed as a consequence of the specific cleavage of the zymogen. This processing can be achieved by another protease and for example, biochemical cascades involving serine proteases have been described for b'""'~ coagulation and fibrinolysis (Bergmeyer et al., 1984). The processing can also be due to an autocatalytic process, eg. pepsinogen is autoactivated into pepsin following acidification of the medium (Tang and Wong, 1987). The first description of a malaria pEoteolytic activity was published in 1946, and since then, at least 25 other proteases of all four classes have been reported in the different plasmodia species (Moulder and Evans, 1946; and see Schrevel et al., 1990, for a review). In spite of this, however, the definition of a natural substrate appears to be more delicate as only one has been clearly identified (Goldberg et al., 1991). Commonly, malaria proteases are divided into two general groups: a) If the optimal pH is acidic, the protease is presumed to be involved in haemoglobin degradation, since it should be active in the acidic environment (pH 5) of the digestive vacuole. Several proteases have been described as being able to degrade haemoglobin (Vander Jagt et aL, 1986; Rosenthal et al., 1989). b) If the protease is specifically active in schizonts and/or merozoites, then a role in merozoite maturation or red blood cell invasion is often invoked. Such a malarial protease would be involved in the regulation of parasite development. Inhibitor studies have clearly implicated proteases in red blood cell invasion (Banyal et al., 1981; Hadley et al., 1983; Dluzewski et al., 1986; Rosenthal et al., 1987; BraunBreton et al., 1988; Deguercy et al., 1990; Schrevel et al., 1990). In this paper, we will consider a few examples of malaria proteases implicated in both of these two general functions: l) haemoglobin degradation; ano 2) regulation of parasite development.
Proteases and haemoglobin degradation Using a P. gallinaceum extract~ Moulder and Evans reported in 1946 that raalarial proteases might be implicated in haemoglobin degradation, and one
of the first ideo,'_ified physiological functions of the parasite was its aoility to degrade host haemoglobin (Groman, 1951). The parasite degrades 25 °70 to 75 07o of the red blood cell (rbc) haemoglobin. This degradation might contribute to a volume compensation necessary for parasite growth inside the rbc (Ginsburg, 1990), and also in the generation of the necessary amino acids for parasite development (Sherman, 1979). This is nowadays widely accepted and, among the many s~.udies that have been performed, we will focus on three that illustrate the general approach which has been followed by many laboratories. Goldberg et ai. (1991) have described a trophozoite-specific 40-kDa protease with an optimal pH of 5. The purified enzyme is able to cleave in vitro haemoglobin (Hb) between Phe33 and Leu34 of the alpha chain; 50 07o inhibition was observed with 5-nM p "pstatin, an aspartate protease inhibitor isolated from Streptomyces. The same cleavage of Hb was achieved by incubation with an extract of purified digestive vacuoles, the pH of which is around 5. This last reaction was also inhibited by pepstatin. These results indicated that the 40-kDa protease is located in the food vacuole and is responsible for one of the steps in Hb degradation. Residues 33-34 are located in an important and conserved structural reg',on of haemoglobin, the hinge. As a first s t ~ of Hb degradation, hinge cleavage could induce unraveling of haemoglogin and make it accessible to further proteolytic degradation. Since the hinge region is highly conserved in vertebrate Hb, proteolytic cleavage of the hinge might also be conserved in the different Plasmodia species. A ' I U. .W. . .IIIUI~I,.,UI¢I.I . ' .... ' . . . .W. l.~-_L, . . . . . -. . . . . . :,: . . . . .p .i p ; l ~ l l t , IJl~l..131.atllll~-31~ll31LIV~:, tease (10-kDa) purified from P. f a l c i p a r u m trophozoites has been described by Vander Jagt et al. (1986). This aspartate prote~',e, ca!leA protease S, displays maximal Hb degradaf~3n at pH 4.5. It has been considereed as being located in the food vacu~ pie. The activity is inhibited in vitro by ferriprotoporphyrin IX, a degradation product of the haem, as well as by the ferriprotoporp aTrin IX-chlorocluine compI..-x. This last observatioh might reflect ~:::e of the mechanisms by which ch!oroqt:ine is toxic for malaria parasites. Accumulation of chloroquine in the food vacuole is due to its abi2 ty to complex ferriprotoporphyrin IX; it prevents the incorporation of ferriprotoporphyrin IX into the parasite pigment and leads to the inhibition of haemoglobin degradation, with consequent inhibition of parasite growth. A trophozoite-specific 28-kDa cysteine protease k,.as also been implicated in Hb metabolism (Rosenthai et al., 1989). The degradation of different arginine-containing fluorogenic substrates by trophozoite extracts has been tested. Cbz-Phe-ArgAMC and Cbz-Val-Leu-Arg-AMC were selected as the best substrates with an optimal pH of 5. The only proteolytic activity detected by gelatin-PAGE and af-
A MALARIA DIVERTIMENTO
fected by 1 ~M Cbz-Phe-AIa-CH2F was the 28-kDa protease. Due to its substrate specificity, the 28-kDa cysteine protease is likely to be a lysosomal cathepsin B, or L type protease (Rosenthal et al., 1989). Independent studies using similar cysteine protease inhibitors have shown that Cbz-Leu-Tyr-CH2F is dramatically more inhibitory than Cbz-Phe-AIaCH2F for intraerythrocytic development of P. falciparum (Rockett et al., 1990). Enlargment of the parasite food vacuole was observed, consistent with an inhibition of a food vacuole protease(s). However, the target of the inhibitor has not yet been identified. These inhibition studies indicate that whether identical to, or different from, the 28-kDa protease, it is not likely to be a cathepsin-L-like protease (Rockett et al., 1990). Only the 40-kDa aspartate protease has been clearly shown to be involved in Hb degradation. It is not yet known whether the other activities are responsible for Hb degradation in vivo and how many different activities are involved in this degradation. It is also clear that the food vacuole probably contains many proteases, not only for Hb degradation, but also for the proteolysis of other substrates. Obviously, these activities could also be crucial for the parasite growth.
Proteases and antigen processing Proteases are known to be involved in posttranslational modifications of proteins. Malaria parasite antigens have been described that appear to undergo a controlled number of processing steps: p 126 (Delplace et al., 1987), GBPI30 (Perkins, 1988), MSA-I (Freeman and Holder, 1983; Holder, 1988, for a review). Due to its position on the merozoite surface and its use as a potential vaccine, MSA-I (also referred to as p190/195, PSA or PMMSA) has been the subject of intensive study (Holder, 1988). This antigen is synthesized as a high molecular weight precursor in schizonts and processed about the time of merozoite release. The precise time of processing is not known. It has been shown, however, that even when processed, the products remain associated as a complex at the surface of merozoites (Freeman and Holder, 1983; David e t al., 1984; McBride et ai., 1985 ; Lyon et al., 1986, 1087). Although the intact molecule is polymorphic, a conserved pattern of degradation is observed (Holder, 1988). This observation suggests that the enzymes involved in this processing are relatively conserved in different Plasmodia strains and species. The processing of MSA-I was inhibited by protease inhibitors (Pirson and Perkins, 1985), but not affected by relatively specific inhibitors like
675
chymostatin, leupeptin, pepstatin and antipain (Lyon and Haynes, 1986). Therefore, the protease(s) involved in this processing appears to be either a metalloprotease(s) (since no inhibitor of this type was tested), or an unusual enzyme(s) of the other three classes of proteases. The cleavage sites within the high molecular weight precursor have been recently determined : the amino-terminal sequence of each processing product was compared with the amino acid sequence of the precursor, deduced from the gene sequence (Strych et al., 1987 and Blackman et aL, 1991). These results indicate that only the last processing step (one that cuts behind a hydrophobic residue) can be attributed to a serine protease, since the other cleavage sites do not resemble those reported for any known protease. For this reason, the enzyme(s) might be sufficiently different from host proteases to be the target(s) of specific inhibitors. If the processing takes place at the surface of the released merozoite in the blood, the enzyme certainly has a specificity different from that of host proteases, since it is active in an environment where 10 % of the serum proteins are protease inhibitors. The last processing step seems to involve a chymotrypsin-like enzyme, since it occurs at a sequence homologous to a chymotrypsin cleavage site (Blackman et al., 1991). The only parasite chymotrypsin-like protease that has been described as being active at the time of merozoite release is the P. falciparum p76 (Braun-Breton et al., 1988). However, this last MSA-I processing step is known t,a t~lcp pl~r.,~ rh~rlng rbc invasion, and if it occurs within the newly formed parasitophorous vacuole, then it might also be performed by a host enzyme. Autocatalytic processing has been reported for proteases and we have to consider that MSA-1 might self-process. The DNA sequence of MSA-I has been determined for an impressive number of isolates (reviewed by Anders and Smythe, 1989). No apparent consensus sites of proteases have been found in the predicted amino acid sequence. This observation does not exclude the possibility that MSA-I is either a metalloprotease for which consensus sites have not been reported or an unusual protease of the serine, cysteine, or aspartate types. Indeed, a similar case of autocatalytic processing has been proposed for Hsp70, a member of the family of heat shock proteins. Degradation fragments of 40-44 kDa and 18-22 kDa have been observed in a preparation of pure Hsp70 (Mitchell et al., 1985). The amino acids histidine, aspartate, and serine were present in the sequence in an appropriate arrangement to be a serine protease, but no proper serine protease consensus sites were observed. Autocatalytic processing might be invoked for the parasitophorous vacuole antigen p126, or SERP.
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This antigen is processed into 50-kDa, 47-kDa and 18-kDa fragments at the very end of the intraerythrocytic cycle (Delplace et al., 1985, 1987). The processing is inhibited by leupeptin; TLCK and TPCK are also inhibitors of this reaction, but they seem to be toxic for the parasite (Debrabant and Delplace, 1989). A computer analysis of pI26/SERP gene sequence revealed the presence of consensus sites of cathepsin L cysteine protease, where the active cysteine has been exchanged for a serine (Knapp et al., 1989; Higgins et al., 1989; Eakin et al., 1989; Mottram et al., 1989). Eakin et al. (1989) have proposed that this protein is a serine protease with a cysteine protease conformation. There is a precedent for this, as in viral cysteine trypsin-like proteases, the cysteine has replaced serine in the serine consensus sequence. Although an elegant observation, it remains theorectical, as to date no proteolytic activity has been assigned to pI26/SERP.
Proteases, merozoite maturation and red blood cell invasion
Merozoite maturation concerns both the processing of parasite antigens necessary to produce infectious merozoites and the efficient release of these merozoites into the blood stream. Proteases are involved in antigen processing, as reported in the previ. ous paragraph. However, the involvement of proteases in the red blood cell lysis is not conclusive. The lysis does not seem to be sensitive to protease inhibitors, and thus this step might involve haemolytic activity comparable to the haemolysins of Trypanosoma cruzi, or Listeria monocytogenes, which allow these organisms to escape from the phagocytic vacuole to the cytoplasm of the host cells (Andrews et al., 1990 and Bielecki et al., 1990). In contrast to lysis, the release of individual merozoites appeared to be sensitive to leupeptin, a general protease inhibitor. Leupeptin treated P. knowlesi schizonts failed to release merozoites, whereas the red blood cell plasma membrane seemed to be disrupted (Hadley et al., 1983). The merozoites remained aggregated as clusters, probably surrounded by a membrane of host origin. Lyon and Haynes (1986) have obtained analogous results when P. falciparum schizonts were incubated with 10 ~tg/ml of chymostatin, leupeptin, antipain, pepstatin; and Banyal et al. (1981) reported similar observations with 50 ~tg/ml leupeptin. The proteolytic activity or activities implicated for merozoite release has/have not yet been identified. Both the binding of merozoites and their entry into the red cell are sensitive to protease inhibitors. Hadley et al. (1983) have shown that TPCK and TLCK inhibit the attachment of purified P. knowlesi merozoites to the red blood cell surface. Chymosta-
tin, however, does not seem to affect binding, but entry. Invasion appeared to be blocked after the reorientation of the merozoite and tight junction formation with the erythrocyte surface. In conclusion, more than one proteolytic activity seems to be implicated in the invasion process. At least one activity (inhibited by TPCK and TLCK) is necessary for the efficient recognition of red blood cell receptors by merozoite surface molecules. A second (chymostatin-sensitive) activity appears necessary for the entry of the parasite into the red cell. The first might be implicated in processings of parasite and/or rbc surface molecules and the second might be involved in the degradation of red blood cell surface molecules, or cytoskeleton proteins, or release of rhoptry contents. In a study using P. falciparum, Dluzewski et al. (1986) have contributed to the characterization of the second enzyme, as pretreatment of rbc by chymotrypsin reversed the inhibition of invasion by chymostatin. Furthermore, when rbc were loaded with chymostatin prior to invasion, inhibition was again observed. It was comparable to that obtained when chymostatin was added directly to the culture medium. Due to its sensitivity to chymos~atin, the protease involved in this step of parasite entry is likely to be a chymotrypsin-like serine protease. Only p76 has been reported to be a merozoitespecific serine pro~ease (Braun-Breton et al., 1988). Both p76 and the P. chabaudi analogt~e, p65, are glycosyl-phosphatidylinositol(GPI)-anchored. In schizonts, the proteins are GPI-anchored and catalytically inert. The presence of a conformationally formed active site can be detected by binding of tritiated di-isopropylfluorophosphate (DFP), but no proteolytic activity associated with the membrane forms of these proteins has been detected using gelatin gel assays (Braun-Breton and Pereira da Silva, 1988). In merozoites, however, the proteins are partially recovered as soluble forms, exhibiting serine protease-like activity and harbouring a cross-reacting epitope specific for the presence of a phosphatidylinositol-phospholipase C (PI-PLC) cleaved glycolipid anchor (Braun-Breton et al., 1988; BraunBreton and Pereira da Silva, 1988). This last result suggests that the solubilization and concomitant activation of the enzyme is due to an endogenous PI-PLC. A developmentally regulated glycosyl-PIPLC (GPI-PLC) activity has been reported in P. falciparum and P. chabaudi merozoites, which might be responsible for the release of active p76 and p65 serine proteases (Braun-Breton et al., 1990, 1991). The precise location of these proteins is not known. A monoclonal antibody Hb3 lcl 3, which immunoprecipitates the p76 serine protease activity, has been reported to be rhoptry specific (Perrin and Dayal, 1982). Since this monoclonal immunoprecipitates both the p76 antigen and a 4 l-kDa antigen, 1o-
A MALARIA DIVERTIMENTO cation of the p76 in the rhoptries has not been conclusively established. However, the active p76 and p65 enzymes can be released by exogenous PI-PLC treatment of intact merozoites, indicating that the protein anchors are accessible to externally added reagents (Braun-Breton et al., !988). The natural substrates of these enzymes have not yet been identified. Schrevel et al. (1990) have characterized two cysteine proteases, a 37-kDa and a 68-kDa enzyme, present in P. berghei and P. falciparum merozoites. The authors proposed that the 37-kDa enzyme is involved in the cleavage of erythrocyte cytoskeletal components, since it is able to cleave host spectrin in vitro. Interestingly, specific peptidic inhibitors of the 68-kDa enzyme block invasion when added to an in vitro culture of P. falciparum parasites. Due to the fact that purified P. falciparum merozoites are poorly invasive, these results were obtained by addi-
677
tion of the inhibitors to mature schizonts. Therefore, the precise step at which the inhibitors are efficient is not known. The effect ot these inhibitors on the parasite development in vivo has not yet been reported. Proteases and gene regulation
One of the best examples of proteolytic activity being important in gene regulation is that of C 1 digestion of the phage lambda repressor (Roberts and Roberts, 1975). However, a more topical example is the recently described cleavage of the NF-kB activating factor by the HIV-encoded aspartate protease (Rivi6re et al., 1991). As part of our general analysis of proteases, we have identified a 27-kDa aspartate protease in P. falciparum and P. chabaudi schizonts (see fig. 1). Given that the malaria genome is AT-rich
I Is a malaria 27-kDa asoartate to ~ ,r,.o. ~ . . protease . . eouivalent . . . AT-specific DNA-binding protein?
Malaria
Yeast Datin * *
i. Genome 80% AT-rich
AT-specific DNA-binding protein
2. 27-kDa aspartate protease
27-kDa protein with aspartate protease consensus sequence
3. Inhibited by pepstatin
DNA-binding inhibited by pepstatin
4. Sediments then solubilized
Datin may be associated
following DNase 1 treatment
with nuclear scaffold
5. pl of 4 ** Properties of Datin taken from Winter and Varshavsky, 1989
Fig. 1. Identification of a P. falciparum 27-kDa aspartate protease. A freeze/thaw lysate of segmented P. falciparum schizonts was analysed by SDS-PAGE containing 0.1 070 gelatin. The SDS was removed by incubation in a solution of 2.5 °70 Triton-Xl00 for at least 1 h at room temperature. The proteolytic activity was revealed following staining with amidoblack. The aspartate protease nature of the indicated activity was confirmed by inhibition with pepstatin.
I
i I
40th FOR UM I N I M M U N O L O G Y
678
(Pollack eta!., 1982), we became interested in the report of a yeast 27.kDa protein (Datin) that specifically binds to AT-rich DNA and contains aspartate protease consensus sequences (Winter and Varshavsky, 1959). The fact that the 27-kOa malaria aspartate protease and yeast Datin -hare a number of features (see fig. 1), led us try and identify the P. falciparum equivalent of Datin. A 270-bp part of the yeast Datin gone which encodes both the aspartate protease consensus sequence, as well as the DNA-binding activity, was used on Southern blots of P. falciparum DNA. As presented in figure 2, ',his specific region of the Datin gone has a homologous counterpart in the P. falciparum genome. Only time and DNA sequencing will tell to wha: degree the two sequences are homologous. However, preliminary gel retardation experiments using the partially purified 27-kDa P. chabaudi aspartate protease and an AT-rich fragment from the 5'-end of the 11-1 gene (Scherf et al.,
I
p
1988) implies that the protease may have pepstatinsensitive DNA-binding activity (data not shown). Drawing on the published examples of how proteases are implicated in gene expression, it seems reasonable to propose a similar model for the Plasmodium Datin-like aspartate protease; namely, the protease binds to DNA and, once bound, cleaves the in situ repressor/activator resulting in the necessary alteration of transcription.
Conclusions The crucial role of parasite proteases in the parasite life cycle has been established. Despite the in,: easing number of characterized parasite-specific proteolytic activities, only one activity has been clearly assigned to a specific function, i.e. haemoglobin degradation. However, correlations have been made between the ability to degrade a substrate (hae-
t'alc~arvm has a sequence homologous to the yeast Datin cjene
s°ut"e,l
I
N-terminol end of Dotin that contains both DNA binding activity and the aspartate protease consensus sequence D-T-G
of Hind l I cut DNA
9k. ----> o
270 bp Probe
ATG GCG AAA ACT TTG GCA CAA GGA AGG A A A M A K T L A Q G R K CCT GGA AGC GGC AGA AAG CCC GGA AAA GGG P G S G R K P G K G AAG ACG TTG AGA GAG GGA AGA AAG CCT GGC K T L R E G R K P G AGT GGT AGG AGG AGG AGG CAA GAT ACT GGG S G R R R R Q D I" IB GGT A A A GAG ACC GAC GGG TCT CAG CAA GAT G K E T D G S Q Q D CAG GAG TCG CGT CTT ATT AGT TCC AGG GAC Q E S R L I S S R D ATG GAA GCT GTG GAC GCA CTG AGA GAG TTG M E A V D A L R E L ACG CAG AGC CCG TCG TCT CAC TCA GCT CAT T H S P S S H S A H AAT TCA TCA GCA GEA CCA CCG CCG CAT N S S A A P P P H
Fig. 2. Identification of a homologous sequence to Datin in the P. falciparum genome. Four ~tg of P. falciparum genomic DNA were digested with Hindlll and then size-separated by 1 070agarose gel electrophoresis; the gel was transferred to Hybond N (Amersham). The 270-bp probe was generated following digestion of the whole Datin gene with ?,'col and SphI and the isolated fragment radiolabelled by random priming. The filter was hybridized with the Datin probed in 6 x SSC at 65°C and the washes were performed at increasing stringency, with the final wash at 2 x SSC at 55°C.
A ML4LARL4 D I V E R 7 tMtzN7
moglobin, spectrin) in vitro and the potential natural (in vivo) substrate. The design of highly specific inhibitors should help in the determination of the biological role for a given proteases. Specific inhibitors have only been used in the analysis of the 28- and 68-kDa cysteine proteases. These inhibitors block parasite growth in vitro. However, relatively low concentrations of Cbz-Leu-Tyr-CH2F (59 nM) and Cbz-Phe-Ala-CH2F (2 ~M), which are specific for the 28-kDa, produced only 50 °70 inhibition of parasite growth and a 100-~M concentration of Cbz-Phe-AIa-CH2F was necessary to totally inhibit parasite development. This may, in part, be due to poor transport of the inhibitor to its target. To improve transport, small molecules are routinely used and such molecules may be very rapidly cleared from the blood. The concentrations used in vivo may therefore have to be much higher than in vitro, with possible toxic effects to the host. In this context, amino acid derivatives of chloromethyl ketones react with nucleophilic residues of many cellular proteins and some of their toxic effects in vivo might be attributed to their general electrophilic nature, rather than to their selective inhibitory action towards certain host proteinases. This type of problem somewhat compromises the interest of intracellular proteases as targets of anti-parasite reagents. Surface proteases seem to be involved i~, merozoite maturation and red blood cell invasion. These parasite enzymes have to be rather unique, since they are active in an hostile environment; IO °7o of serum proteins are protease inhibitors whose function is to limit the half lives of host proteases. Given that
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679
References Anders, R.F. & Smythe, J.A. (1989), Polymorphic antigens in P. falciparum. Blood, 74, 1865-1875. Andrews, N.W., Abrams, C.K., Slatin, S.L. & Griffiths, G. (1990), A T. cruzi-secreted protein immunologicaily related to the complement component C9: evidence for membrane pore-forming at low pH. Cell, 61, 1277-1287. Banyal, H.S., Misra, G.C., Gupta, C.M. & Dutta, G.P. (1981), Involvement of malaria proteases in the interaction between the parasite and host erythrocyte in Plasmodium knowlesi infections. J. Parasit., 67, (5) 623-626. Bergmeyer, H.U. (1983), Enzymes 3" peptidases, proteinases and their inhibitors in methods of enzymatic analysis (third edition), Volume V (Bergrneyer, H.U.). Verlag Chemie, Weinheim. Bielecki, J., Youngman, P., Connelly, P. & Portnoy: D.A. (1990), Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells. Nature (Lond.), 345, 175-176. Blackman, M.J., Ling, I.T., Whittle, H. & Holder, A. (1991), Processing of the Plasmodium falciparum merozoite surface protein 1" identification of a 33 kilodalton product of a secondary proteolytic processing event and partial characterisation of the protease involved. Paper present at the British Parasitological Society meeting, in Edinburgh, Scotland. Boyer, P.D. (1971), Hydrolysis" peptide bonds, in "The Enzymes", 3, Academic Press, New York, London. Braun-Breton, C., Rosenberry, T.L. & Pereira da Silva, L. (1988), Induction of the proteolytic activity of a membrane protein in P. falciparum by phosphatidyl inositol-specific phospholipase C. Nature (Lond.), 332, 457-459.
t~ h~ ~ot;w~
makes it theoretically possible to design specific inhibitors which will not affect host physiological processes. The surface location of parasite proteases makes the use of higher molecular weight inhibitors possible. Large inhibitors should not enter host cells, thus decreasing any potential toxic side effects. The problems of immunogenicity and degradation of such molecules might be circumvented by modifying natural host inhibitors, rendering them specific for parasite proteases. These molecules would represent a new family of antimalarial agents.
Acknowledgements We would like to thank Prof. L. Pereira da Silva and all the members of the Unit of Experimental Parasitology for their support. This investigation received financial support from the UNDP/World Bank/WHO Special programme for Research and Training in Tropical Diseases and grants from the Minist~re de la Recherche et I'Enseignement Sup~rieur (Aid,. no. 87W0043-Eureka).
tion of a Piasmodium falciparum protease correlated with merozoite maturation and erythrocyte invasion. Biol. Cell, 64, 223-231. Braun-Breton, C., Langsley, G., Barale, J.C. & Pereira da Silva, L. (1999), A malaria phosphatidylinositolspecific phospholipase C" a possible role in merozoite maturation and erythrocyte invasion, in "Cellular and molecular biology of normal and abnormal erythroid membranes" (pp. 315-332). Alan R. Liss, New York. Braun-Breton, C., Blisnick, T., Barbot, P., Billow, R., Pereira da Silva, L. & Langsley, G. (1991), Characterisation of a glycosyl-phosphatidylinositol-specific phospholipase C from malaria parasites. Exp. Parasit. (in press). Brenner, S. (1988), The molecular evolution of genes and proteins" a tale of two serines. Nature (Lond.), 334, 528-530. David, P.H., Hadley, T.J., Aikawa, M. & Miller, L.H. (1984), Processing of a major parasite surface glycoprotein during the ultimate stages of differentiation in Plasmodium knowlesi. Mol. Biochem. Parasit., 11,267-282. Debrabant, A. & Delplace, P. (1989), I eupeptin alters the proteolytic processing of p126, the major parasitophorous vacuole antigen of Plasmodium falciparum. Mol. Biochem. Parasit., 33, 151-158. Deguercy, A., Hommel, M. & Schr~vel, J. (1990), Purifi-
680
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cation and characterization of 37-kilodalton proteases from Plasmodium falciparum and Plasmodium berghei which cleave erythrocyte cytoskeletal components. Mol. Biochem. Parasit., 38, 233-244. Delplace, P., Dubremetz, J.-F., Fortier, B. & Vernes, A. (1985), A 50 kilodalton exoantigen specific to the merozoite release-reinvasion stage of Piasmodiumfalciparum. Mol. Biochem. Parasit., 17, 239-251. Delplace, P., Fortier, B., Tronchin, G., Dubremetz, J.-F. & Vernes, A. (1987), Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of P. falciparum. Mol. Biochem. Porasit., 23, 193-201. Dluzewski, A.R., Rangachari, K., Wilson, R.J.M. & Gratzer, W.B. (1986), Plasmodiumfalciparum: protease inhibitors and inhibition of erythrocyte invasion. Exp. Parasit., 62, 416-422. Eakin, A.E., Higaki, J.N., McKerrow, J.H. & Craik, C.S. (1989), Cysteine or serine proteinase? Nature (Land.), 342, 132. Freeman, R. & Holder, A. (1983), Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000-mol wt expressed on the surface of Plasmodiumfalciparum merozoites. 3'. exp. Med., 158, 1647-1653. Ginsburg, H. (1990), Some reflections concerning host erythrocyt.e-malarial parasite interrelationships. Blood Cells, 16, 225-235. Goldberg, D.E., Slater, A.F.G., Beavis, R., Chait, B., Cerami, A. & Henderson, G.B. (1991), Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease. J. exp. Med., 173,961-969. Groman, N.B. (1951), Dynamic aspects of the nitrogen metabolism of Piasmodium gallinaceum in viva and in vitro. J. infect. Dis., 88, 126-150. Hadley, T., Aikawa, M. & Miller, L.H. (1983), Studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors. Exp. Parasit., 55, 306-311. Higgins, D.G., McConnel, D.J. & Sharp, P.M. (1989), M~arial proteases? Nature (Land.), 340, 604. Holder, A.A. (1988), The precursor to major merozoite surface antigens: structure and role in immunity. Progr. Allergy, 41, 72-97. Holm, I., Ollo, R., Panthier, J. & Rougeon, F. (1984), Evolution of aspartly proteases by gene duplication: the mouse renin gene is organized in two homologous clusters of four exons. EMBO J., 3, 557-562. Knapp, B., Hundt, E., Nau, U. & Kiipper, H.A. (1989), Molecular cloning, genomic structure and localisation of a blood stage antigen of P. falciparum characterised by a serine stretch. Mol. Biochem. Parasit., 32, 73-84. Lyon, J.A., Geller, R.H., Haynes, J.D., Chulay, J.D. & Weber, J.L. (1986), Epitop~ map and processing scheme for the 195,000-da~ton surface glycoprotein of Plasmodiumfalciparum merozoites deduced from cloned overlapping segments of the gene. PrOConat. Acad. Sci. (Wash.), 83, 2989-2993. Lyon, J.A. & Haynes, J.D. (1986), Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the resence of protease inhibitors. J. Immunol., 136, 2245-2251. Lyon, J.A., Haynes, J.D., Diggs, C.L., Chulay, J.D.,
Haidaris, C.G. & Pratt-Rossiter, J. (1987), Monoclonal antibody characterisation of the 195-kilodalton major surface glycoprotein of P. falciparum malaria schizonts and merozoites: identification of additional processed products and a serotype restricted repetitive epitope. J. Immunol., 138, 859-90. McBride, J.S., Newbold, C.I. & Anand, R. (1985), Polymorphism of a high molecular weight schizont antigen of the human malaria parasite Piasmodium faiciparum. J. exp. Med., 161, 160-180. Mitchel, H.K., Petersen, N.S. & Buzin, C.H. (1985), Selfdegradation of heat shock proteins. Prcc. nat. Acad. $ci. (Wash.), 82, 4969-4973. Mottram, J.C., Coombs, G.H. & North, M.J. (1989), Cysteine or serine proteinase ? Nature (Land.), 342, 132. Moulder, J.W. & Evans, E.A.J. (1946), The biochemistry of the malaria parasite. 1 VI. Studies on nitrogen metabolism of the malaria parasite. J. biol. Chem., 164, 135-157. Pearl, L.H. & Taylor, W.R. (1987), A structural model for the retroviral proteases. Nalure (Land.), 329, 351-354. Perkins, M.E. & Rocco, L.J. (1988), Sialic acid-dependent binding of Plasmodiumfalciparum merozoite surface antigen: Pf200, to human erythrocyte. J. Immunoi., 141, 3190-3196. Perrin, L.H. & Dayal, R. (1982), Immunity to sexual erythrocytic stages of P. falciparum: role of defined antigens in the humoral response, lmmunoi. Rev., 61, 245-270. Pirson, P.J. & Perkins, M.E. (1985), Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites. J. Immunol., 134, 1946-1951. Pollack, Y., Katzen, A., Spira, D.T. & Golenser, J. (1982), The genome of Plasmodiumf~lciparum - - 1. DNA base composition. Nucl. Acids Res., !,a, ~ _ ~ . Rivi6re, Y., Blank, V., Kourilsky, P. & Israel, A. (1991), Processing of the precursor of the NF-KB by the HIV-I protease during acute infection. Nature (Land.), 350, 625-626. Roberts, J.W. & Roberts, C.W. (1975), Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc. nat. Acad. ScL (Wash.), 72, 147-151. Rockett, K.A., Playfair, J.H.L., Ashall, F., Targett, G.A.T., Angliker, H. & Shaw, E. (1990), Inhibition of intraerythrocytic development of Plasmodium faiciparum by proteinase inhibitors. FEBS, 259, 257-259. Rosenthal, P.J., Kim, K., McKerrow, J.H. & Leech, J. (1987), Identification of three stage-specific proteinases of Plasmodium falciparum. J. exp. Med., 166, 816-821. Rosenthal, P.J., McKerrow, J.H., Rasnick, D. & Leech, J.H. (1989), Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development. Mol. Biochem. Parasit., 35, 177-184. Scherf, A., Hilbich, C., Seig, K., Mattei, D., MercereauPuijalon, O. & Mfiller-Hill, B. (1988), The 11-1 gene of P. falciparum codes for distinct fast evolving domains. EMBO J., 1129-1137. Schr~vel, J., Deguercy, A., Mayer, R. & Monsigny, M. (1990), Proteases in malaria-infected red blood cells. Blood Cells, 16, 563-584.
A M A L A R I A D I V E R TIMENTO Sherman, I.W. (1979), Biochemistry of Plasmodium (malaria parasites). Microbiol. Rev., 43, 453-495. Strych, W., Miettinen-Baumann, A., Lottspeich, F. & Heidrich, H. (1987), Isolation and characterisation of the 80,000 dalton Plasmodium falciparum merozoite surface antigen. Parasit. Res., 73, 435441. Stryer, L. (1988), Mechanisms of enzyme action and control of enzymatic activity. Biochemistry, third editien. W.H. Freeman & Co., San Francisco.
681
Tang, J. & Wong, R.N.S. (1987), Evolution in the structure and function of aspartic proteases. J. Cell. BioL, 33, 135-145. Vander Jagt, D.L., Hunsaker, L.A. & Campos, N.M. (1986), Characterisation of a hemoglobin-degrading, low molecular weight protease from P. falciparum. MoL Biochem. Parasit., 18, 389-400. Winter, E. & Varshavsky, A. (1989), A DNA binding protein that recognizes oligo(dA).oligo(dT) tracts. EMBO J., 8, 1867-1877.
Plasmodium falciparum cytoadherence J. Leech
Parasitology Laboratory, Department o f Medicine, University o f California-San Francisco and San Francisco General Hospital, San Francisco CA 94110 (USA)
Plasmodium falciparum cytoadherence refers to the specific binding that occurs between trophozoiteand schizont-infected erythrocytes and host endothelial cells. In vivo, cytoadherence results in the removal of mature asexual parasites from the general circulation and their localization within the postcapillary venuies of multiple organs, predominantly the brain, heart, and skeletal muscle (Miller, 1969). This localization is referred to as sequestration. Sequestration appears to contribute to the consequences of P. falciparum malaria in multiple ways, and the molecules involved in cytoadherence may be important targets of naturally acquired or vaccine-induced immunity to this disease. In vitro, the phenomenon of cytoadherence has been investigated with a variety of model systems, including cultured endothelial cells (Udeinya et al., 1981), C32 melanoma cells (Schmidt et al., 1982), monocytes (Ockenhouse and Chulay, 1988), transfected COS cells (Oquendo et al., 1989) and with purified candidate receptor molecules applied to plastic surfaces. Use of these model systems has produced information about the physiology of cytoadherence and the molecules involved. In this article, I will first discuss the role of cytoadherence in the clinical and biological consequences of P. falciparum malaria. Second, I will discuss the inhibition of cytoadherence by antibody and third, the
antigenic differences among cytoadherence antigens from different parasite isolates. The question of antigenic variation will be discussed. Fourth, I will discuss recent work on the molecular basis of cytoadherence. Throughout this discussion, I will use the following terminology. "Cytoadherence antig e m " are the molecule(s) on the surface of im%cted erythrocytes that are the target of antibody that inhibits cytoadherence. "Cytoadherence ligands" are the molecule(s) that mediate binding to the host cell. "Cytoadherence receptors" are the host molecules that bind cytoadherence ligands. This article will emphasize recent work and will be opinionated and speculative. Readers desiring more comprehensive and historical reviews are referred to several recent, excellent articles. The phenomenon of rosetting between infected erythrocytes and between infected and uninfected erythrocytes will not be discussed in this article. The role of cytoadherence in the biological and clinical consequences of P. falciparum malaria The evidence that cytoadherence and sequestration have important roles in P. falciparum biology is teleological and experimental. Teleologically, the
Address for correspondence: J. Leech, Building 30, Room 408, Say Francisco General Hospital, 1001Potrero Ave., San Francisco, CA 94110.