Identification and Characterization of Falcilysin, a Metallopeptidase Involved in Hemoglobin Catabolism within the Malaria Parasite Plasmodium falciparum
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.32411
ISSN1083-351X
AutoresKathleen Kolakovich Eggleson, Kevin L. Duffin, Daniel E. Goldberg,
Tópico(s)Trypanosoma species research and implications
ResumoThe malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the α and β chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4′ positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases. The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the α and β chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4′ positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases. 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane liquid chromatography 2-(4-morpholino)ethanesulfonic acid mass spectrometry phenylmethanesulphonyl fluoride processing peptidase 4-(4-dimethylaminophenylazo)benzoic acid 5-[(2-aminoethyl)amino]naphthalene-1 sulfonic acid polyacrylamide gel electrophoresis high performance liquid chromatography Plasmodium falciparum is a protozoan parasite that causes the most lethal form of human malaria. Upon invasion of a human erythrocyte, the parasite grows and matures surrounded by cytosol consisting predominantly of a single protein, hemoglobin. Amino acids derived from the proteolysis of hemoglobin are incorporated into parasite proteins and parasites require supplementation with only a few amino acids that are absent or deficient in hemoglobin for normal growth in culture (1Divo A.A. Geary G., T. Davis N.L. Jensen J.B. J. Protozool. 1985; 32: 59-64Crossref PubMed Scopus (172) Google Scholar, 2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar). Hemoglobin proteolysis occurs within an acidic organelle, the food vacuole. This compartment has a pH estimated at 5.0–5.4 (3Yayon A. Cabantchik Z.I. Ginsburg H. EMBO J. 1984; 3: 2695-2700Crossref PubMed Scopus (268) Google Scholar, 4Krogstad D.J. Schlesinger P.H. Gluzman I.Y. J. Cell Biol. 1985; 101: 2302-2309Crossref PubMed Scopus (214) Google Scholar).Nonproteolytic acid hydrolases could not be detected in food vacuoles isolated from P. falciparum (5Goldberg D.E. Slater A.F.G. Cerami A. Henderson G.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2931-2935Crossref PubMed Scopus (396) Google Scholar). Thus, it appears that the food vacuole of P. falciparum does not function in degradation and recycling of macromolecules in general. The catabolic capability of this organelle is focused on hemoglobin. Disruption of hemoglobin catabolism causes parasite death in an animal model and in culture (2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 6Rosenthal P.J. Lee G.K. Smith R.E. J. Clin. Invest. 1993; 91: 1052-1056Crossref PubMed Scopus (162) Google Scholar, 7Rosenthal P.J. Wollish W.S. Palmer J.T. Rasnick D. J. Clin. Invest. 1991; 88: 1467-1472Crossref PubMed Scopus (178) Google Scholar). The vital and specialized process of hemoglobin degradation within the food vacuole provides promising targets for the development of novel antimalarial drugs, greatly needed in the face of increasing resistance to existing chemotherapeutic agents (8Olliaro P. Cattani J. Wirth D. J. Am. Med. Assoc. 1996; 275: 230-233Crossref PubMed Google Scholar).Multiple proteases within the food vacuole facilitate the degradation of hemoglobin to peptide fragments. Three acidic proteases have been identified, purified from food vacuoles, and characterized (2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 9Goldberg D.E. Slater A.F.G. Beavis R. Cerami A. Chait B. Henderson G.B. J. Exp. Med. 1991; 173: 961-969Crossref PubMed Scopus (232) Google Scholar, 10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar, 11Dame J.B. Reddy G.R. Yowell C.A. Dunn B.M. Kay J. Berry C. Mol. Biol. Parasitol. 1994; 64: 177-190Crossref PubMed Scopus (106) Google Scholar, 12Rosenthal P.J. McKerrow J.H. Aikawa M. Nagasawa H. Leech J.H. J. Clin. Invest. 1988; 82: 1560-1566Crossref PubMed Scopus (306) Google Scholar, 13Rosenthal P.J. Nelson R.G. Mol. Biochem. Parasitol. 1992; 51: 143-152Crossref PubMed Scopus (96) Google Scholar, 14Vander Jagt D.L. Hunsaker L.A. Campos N.M. Scaletti J.V. Biochim. Biophys. Acta. 1992; 1122: 256-264Crossref PubMed Scopus (47) Google Scholar). Two aspartic proteases, plasmepsin I and plasmepsin II, can cleave native hemoglobin. A cysteine protease, falcipain, is able to cleave denatured globin but not native hemoglobin (10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar, 15Francis S.E. Gluzman I.Y. Oksman A. Banerjee D. Goldberg D.E. Mol. Biol. Parasitol. 1996; 83: 189-200Crossref PubMed Scopus (64) Google Scholar). Exopeptidase activity capable of generating individual amino acids from peptide fragments is absent from the food vacuole (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). However, peptides may traverse the food vacuole membrane and could be converted to amino acids by exopeptidase activity in the parasite cytoplasm. An aminopeptidase that functions at neutral pH has been purified from parasites and characterized (17Florent I. Derhy Z. Allary M. Monsigny M. Mayer R. Schrevel J. Mol. Biol. Parasitol. 1998; 97: 149-160Crossref PubMed Scopus (65) Google Scholar, 18Curley G.P. O'Donovan S.M. McNally J. Mullally M. O'Hara H. Troy A. O'Callaghan S.A. Dalton J.P. J. Eukaryot. Microbiol. 1994; 41: 119-123Crossref PubMed Scopus (52) Google Scholar, 19Vander Jagt D.L. Baack B.R. Hunsaker L.A. Mol. Biol. Parasitol. 1984; 10: 45-54Crossref PubMed Scopus (39) Google Scholar).When hemoglobin was incubated with food vacuole lysate at acidic pH, a series of discrete peptide fragments was generated (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). Cleavage sites along the hemoglobin α and β chains were located an average of eight amino acids apart. Many cleavage sites corresponded to the peptide bonds previously identified as sites for the known vacuolar proteases. Twenty-four cleavage sites that could not be attributed to the known proteases of the vacuole were identified. Unlike the preference of plasmepsin I, plasmepsin II, and falcipain for hydrophobic residues, many of the novel cleavage sites contained polar residues at the P1 and/or P1′ positions. These results suggested that one or more vacuolar endopeptidases had remained unidentified (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). In this report, we describe the discovery of a novel proteolytic activity, purification of the enzyme responsible for this activity, characterization of the purified enzyme, and the corresponding molecular sequence data. Furthermore, we provide evidence that this enzyme, falcilysin, has a distinct, downstream role in the semiordered hemoglobin degradation pathway of P. falciparum.DISCUSSIONOur previous work suggested that there may be an additional unknown enzyme(s) in the food vacuole that participates in hemoglobin degradation (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). This paper describes the identification of a novel metallopeptidase activity in P. falciparum. Falcilysin was purified from food vacuoles and characterized. It is an oligoendopeptidase of the M16 family which contains two subfamilies. Subfamily B contains consists of the mitochondrial processing peptidases and chloroplast stromal processing peptidases that cleave NH2-terminal signal peptides from proteins (35Rawlings N.D. Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Bath, United Kingdom1998: 1360-1362Google Scholar). Subfamily A of family M16 consists of large proteins (approximately 100 kDa or more) that function as oligoendopeptidases. Substrates for these enzymes include peptides such as a-factor (40Fujita A. Oka C. Arikawa Y. Katagai T. Tonouchi A. Kuhara S. Misumi Y. Nature. 1994; 372: 567-570Crossref PubMed Scopus (119) Google Scholar, 41Adames N. Blundell K. Ashby M.N. Boone C. Science. 1995; 270: 464-466Crossref PubMed Scopus (122) Google Scholar), insulin (42Shii K. Yokono K. Baba S. Roth R.A. Diabetes. 1986; 35: 675-683Crossref PubMed Scopus (83) Google Scholar), glucagon (43Kirschner R.J. Goldberg A.L. J. Biol. Chem. 1983; 258: 967-976Abstract Full Text PDF PubMed Google Scholar), atrial natriuretic peptide (44Muller D. Baumeister H. Buck F. Richter D. Eur. J. Biochem. 1991; 202: 285-292Crossref PubMed Scopus (68) Google Scholar), transforming growth factor α (45Gehm B.D. Rosner M.R. Endocrinology. 1991; 128: 1603-1610Crossref PubMed Scopus (62) Google Scholar), β-amyloid protein (46Kurochkin I. Goto S. FEBS Lett. 1994; 345: 33-37Crossref PubMed Scopus (342) Google Scholar), somatostatin 28 (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar), β-galactosidase (48Cheng Y.E. Zisper D. J. Biol. Chem. 1979; 254: 4698-4706Abstract Full Text PDF PubMed Google Scholar), and now, peptide fragments of hemoglobin (this work). Inhibition by alkylating agents has been observed in insulysin (42Shii K. Yokono K. Baba S. Roth R.A. Diabetes. 1986; 35: 675-683Crossref PubMed Scopus (83) Google Scholar), nardilysin (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar), mitochondrial processing peptidase ofNeurospora crassa (49Schneider A. Arretz M. Wachter E. Neupert W. J. Biol. Chem. 1990; 265: 9881-9887Abstract Full Text PDF PubMed Google Scholar), and falcilysin (this work). Nardilysin is inhibited by the aminopeptidase inhibitors amastatin and bestatin (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar) but this was not observed in falcilysin.The M16 family of metallopeptidases is growing. Besides the addition of falcilysin presented in this work, BLAST searches revealed sequence characteristics common to M16 family members in several uncharacterized protein sequences from different organisms (Fig.7). In fact, many potential M16 family members share greater sequence identity with falcilysin than the known members. Future investigations of these sequences will reveal which of these proteins function as metallopeptidases and whether one or more new types of enzymes with distinct biological functions are among them.As expected for a metallopeptidase of this class, falcilysin is unable to cleave polypeptides such as the α and β chains of hemoglobin (141 and 146 amino acids in length) but is able to cleave within peptides generated by the other proteases of the food vacuole. In this study falcilysin cleaved peptides up to 20 amino acids and preferred those 11–15 residues in length (Table I). It thus functions downstream of other vacuolar proteases, providing strong evidence for order in the hemoglobin degradation process. Our current understanding of the sequential nature of the proteolytic events is that the initial cleavages of the native hemoglobin molecule are made by the plasmepsins. Denatured or fragmented globin is susceptible to cleavage by falcipain as well as the plasmepsins (15Francis S.E. Gluzman I.Y. Oksman A. Banerjee D. Goldberg D.E. Mol. Biol. Parasitol. 1996; 83: 189-200Crossref PubMed Scopus (64) Google Scholar). Falcilysin cannot cleave hemoglobin or globin, but is able to cleave hemoglobin fragments. It is likely that many peptide intermediates are susceptible to cleavage by multiple enzymes. The distinct but overlapping roles of the proteolytic enzymes of the food vacuole and the location of exopeptidase activity across the food vacuole membrane in the parasite cytoplasm suggest that hemoglobin catabolism within P. falciparum is a semiordered pathway.The importance of zinc to intraerythrocytic P. falciparumhas been demonstrated. Levels of zinc increase in parallel with parasite maturation within the red blood cell (50Ginsburg H. Gorodetsky R. Krugliak M. Biochim. Biophys. Acta. 1986; 886: 337-344Crossref PubMed Scopus (22) Google Scholar). Treatment of cultures with dipicolinic acid does not prevent schizont rupture or reinvasion of host erythrocytes but blocks parasite maturation from the ring to the trophozoite stage, coincident with the onset of hemoglobin degradation (50Ginsburg H. Gorodetsky R. Krugliak M. Biochim. Biophys. Acta. 1986; 886: 337-344Crossref PubMed Scopus (22) Google Scholar, 51Yayon A. Vande Waa J.A. Yayon M. Geary T.G. Jensen J.B. J. Protozool. 1983; 30: 642-647Crossref PubMed Scopus (90) Google Scholar). Inhibition of hemoglobin catabolism is lethal to intraerythrocytic P. falciparum (2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 7Rosenthal P.J. Wollish W.S. Palmer J.T. Rasnick D. J. Clin. Invest. 1991; 88: 1467-1472Crossref PubMed Scopus (178) Google Scholar). The specific role of falcilysin in the essential process of hemoglobin degradation may have encouraging implications for the development of new chemotherapeutic agents. Plasmepsins I and II as well as falcipain prefer to cleave the α and β chains of hemoglobin at sites with hydrophobic residues at the P1 and/or P1′ positions (10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar). In contrast, falcilysin sites are polar in character, with charged residues at the P1 and/or P4′ positions. Internal cleavage of the large peptides generated by the plasmepsins and falcipain at distinct sites is likely to be critical for production of peptides small enough to cross the food vacuole membrane for cytosolic amino acid production. Falcilysin appears to play an integral role in this process. Plasmodium falciparum is a protozoan parasite that causes the most lethal form of human malaria. Upon invasion of a human erythrocyte, the parasite grows and matures surrounded by cytosol consisting predominantly of a single protein, hemoglobin. Amino acids derived from the proteolysis of hemoglobin are incorporated into parasite proteins and parasites require supplementation with only a few amino acids that are absent or deficient in hemoglobin for normal growth in culture (1Divo A.A. Geary G., T. Davis N.L. Jensen J.B. J. Protozool. 1985; 32: 59-64Crossref PubMed Scopus (172) Google Scholar, 2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar). Hemoglobin proteolysis occurs within an acidic organelle, the food vacuole. This compartment has a pH estimated at 5.0–5.4 (3Yayon A. Cabantchik Z.I. Ginsburg H. EMBO J. 1984; 3: 2695-2700Crossref PubMed Scopus (268) Google Scholar, 4Krogstad D.J. Schlesinger P.H. Gluzman I.Y. J. Cell Biol. 1985; 101: 2302-2309Crossref PubMed Scopus (214) Google Scholar). Nonproteolytic acid hydrolases could not be detected in food vacuoles isolated from P. falciparum (5Goldberg D.E. Slater A.F.G. Cerami A. Henderson G.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2931-2935Crossref PubMed Scopus (396) Google Scholar). Thus, it appears that the food vacuole of P. falciparum does not function in degradation and recycling of macromolecules in general. The catabolic capability of this organelle is focused on hemoglobin. Disruption of hemoglobin catabolism causes parasite death in an animal model and in culture (2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 6Rosenthal P.J. Lee G.K. Smith R.E. J. Clin. Invest. 1993; 91: 1052-1056Crossref PubMed Scopus (162) Google Scholar, 7Rosenthal P.J. Wollish W.S. Palmer J.T. Rasnick D. J. Clin. Invest. 1991; 88: 1467-1472Crossref PubMed Scopus (178) Google Scholar). The vital and specialized process of hemoglobin degradation within the food vacuole provides promising targets for the development of novel antimalarial drugs, greatly needed in the face of increasing resistance to existing chemotherapeutic agents (8Olliaro P. Cattani J. Wirth D. J. Am. Med. Assoc. 1996; 275: 230-233Crossref PubMed Google Scholar). Multiple proteases within the food vacuole facilitate the degradation of hemoglobin to peptide fragments. Three acidic proteases have been identified, purified from food vacuoles, and characterized (2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 9Goldberg D.E. Slater A.F.G. Beavis R. Cerami A. Chait B. Henderson G.B. J. Exp. Med. 1991; 173: 961-969Crossref PubMed Scopus (232) Google Scholar, 10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar, 11Dame J.B. Reddy G.R. Yowell C.A. Dunn B.M. Kay J. Berry C. Mol. Biol. Parasitol. 1994; 64: 177-190Crossref PubMed Scopus (106) Google Scholar, 12Rosenthal P.J. McKerrow J.H. Aikawa M. Nagasawa H. Leech J.H. J. Clin. Invest. 1988; 82: 1560-1566Crossref PubMed Scopus (306) Google Scholar, 13Rosenthal P.J. Nelson R.G. Mol. Biochem. Parasitol. 1992; 51: 143-152Crossref PubMed Scopus (96) Google Scholar, 14Vander Jagt D.L. Hunsaker L.A. Campos N.M. Scaletti J.V. Biochim. Biophys. Acta. 1992; 1122: 256-264Crossref PubMed Scopus (47) Google Scholar). Two aspartic proteases, plasmepsin I and plasmepsin II, can cleave native hemoglobin. A cysteine protease, falcipain, is able to cleave denatured globin but not native hemoglobin (10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar, 15Francis S.E. Gluzman I.Y. Oksman A. Banerjee D. Goldberg D.E. Mol. Biol. Parasitol. 1996; 83: 189-200Crossref PubMed Scopus (64) Google Scholar). Exopeptidase activity capable of generating individual amino acids from peptide fragments is absent from the food vacuole (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). However, peptides may traverse the food vacuole membrane and could be converted to amino acids by exopeptidase activity in the parasite cytoplasm. An aminopeptidase that functions at neutral pH has been purified from parasites and characterized (17Florent I. Derhy Z. Allary M. Monsigny M. Mayer R. Schrevel J. Mol. Biol. Parasitol. 1998; 97: 149-160Crossref PubMed Scopus (65) Google Scholar, 18Curley G.P. O'Donovan S.M. McNally J. Mullally M. O'Hara H. Troy A. O'Callaghan S.A. Dalton J.P. J. Eukaryot. Microbiol. 1994; 41: 119-123Crossref PubMed Scopus (52) Google Scholar, 19Vander Jagt D.L. Baack B.R. Hunsaker L.A. Mol. Biol. Parasitol. 1984; 10: 45-54Crossref PubMed Scopus (39) Google Scholar). When hemoglobin was incubated with food vacuole lysate at acidic pH, a series of discrete peptide fragments was generated (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). Cleavage sites along the hemoglobin α and β chains were located an average of eight amino acids apart. Many cleavage sites corresponded to the peptide bonds previously identified as sites for the known vacuolar proteases. Twenty-four cleavage sites that could not be attributed to the known proteases of the vacuole were identified. Unlike the preference of plasmepsin I, plasmepsin II, and falcipain for hydrophobic residues, many of the novel cleavage sites contained polar residues at the P1 and/or P1′ positions. These results suggested that one or more vacuolar endopeptidases had remained unidentified (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). In this report, we describe the discovery of a novel proteolytic activity, purification of the enzyme responsible for this activity, characterization of the purified enzyme, and the corresponding molecular sequence data. Furthermore, we provide evidence that this enzyme, falcilysin, has a distinct, downstream role in the semiordered hemoglobin degradation pathway of P. falciparum. DISCUSSIONOur previous work suggested that there may be an additional unknown enzyme(s) in the food vacuole that participates in hemoglobin degradation (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). This paper describes the identification of a novel metallopeptidase activity in P. falciparum. Falcilysin was purified from food vacuoles and characterized. It is an oligoendopeptidase of the M16 family which contains two subfamilies. Subfamily B contains consists of the mitochondrial processing peptidases and chloroplast stromal processing peptidases that cleave NH2-terminal signal peptides from proteins (35Rawlings N.D. Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Bath, United Kingdom1998: 1360-1362Google Scholar). Subfamily A of family M16 consists of large proteins (approximately 100 kDa or more) that function as oligoendopeptidases. Substrates for these enzymes include peptides such as a-factor (40Fujita A. Oka C. Arikawa Y. Katagai T. Tonouchi A. Kuhara S. Misumi Y. Nature. 1994; 372: 567-570Crossref PubMed Scopus (119) Google Scholar, 41Adames N. Blundell K. Ashby M.N. Boone C. Science. 1995; 270: 464-466Crossref PubMed Scopus (122) Google Scholar), insulin (42Shii K. Yokono K. Baba S. Roth R.A. Diabetes. 1986; 35: 675-683Crossref PubMed Scopus (83) Google Scholar), glucagon (43Kirschner R.J. Goldberg A.L. J. Biol. Chem. 1983; 258: 967-976Abstract Full Text PDF PubMed Google Scholar), atrial natriuretic peptide (44Muller D. Baumeister H. Buck F. Richter D. Eur. J. Biochem. 1991; 202: 285-292Crossref PubMed Scopus (68) Google Scholar), transforming growth factor α (45Gehm B.D. Rosner M.R. Endocrinology. 1991; 128: 1603-1610Crossref PubMed Scopus (62) Google Scholar), β-amyloid protein (46Kurochkin I. Goto S. FEBS Lett. 1994; 345: 33-37Crossref PubMed Scopus (342) Google Scholar), somatostatin 28 (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar), β-galactosidase (48Cheng Y.E. Zisper D. J. Biol. Chem. 1979; 254: 4698-4706Abstract Full Text PDF PubMed Google Scholar), and now, peptide fragments of hemoglobin (this work). Inhibition by alkylating agents has been observed in insulysin (42Shii K. Yokono K. Baba S. Roth R.A. Diabetes. 1986; 35: 675-683Crossref PubMed Scopus (83) Google Scholar), nardilysin (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar), mitochondrial processing peptidase ofNeurospora crassa (49Schneider A. Arretz M. Wachter E. Neupert W. J. Biol. Chem. 1990; 265: 9881-9887Abstract Full Text PDF PubMed Google Scholar), and falcilysin (this work). Nardilysin is inhibited by the aminopeptidase inhibitors amastatin and bestatin (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar) but this was not observed in falcilysin.The M16 family of metallopeptidases is growing. Besides the addition of falcilysin presented in this work, BLAST searches revealed sequence characteristics common to M16 family members in several uncharacterized protein sequences from different organisms (Fig.7). In fact, many potential M16 family members share greater sequence identity with falcilysin than the known members. Future investigations of these sequences will reveal which of these proteins function as metallopeptidases and whether one or more new types of enzymes with distinct biological functions are among them.As expected for a metallopeptidase of this class, falcilysin is unable to cleave polypeptides such as the α and β chains of hemoglobin (141 and 146 amino acids in length) but is able to cleave within peptides generated by the other proteases of the food vacuole. In this study falcilysin cleaved peptides up to 20 amino acids and preferred those 11–15 residues in length (Table I). It thus functions downstream of other vacuolar proteases, providing strong evidence for order in the hemoglobin degradation process. Our current understanding of the sequential nature of the proteolytic events is that the initial cleavages of the native hemoglobin molecule are made by the plasmepsins. Denatured or fragmented globin is susceptible to cleavage by falcipain as well as the plasmepsins (15Francis S.E. Gluzman I.Y. Oksman A. Banerjee D. Goldberg D.E. Mol. Biol. Parasitol. 1996; 83: 189-200Crossref PubMed Scopus (64) Google Scholar). Falcilysin cannot cleave hemoglobin or globin, but is able to cleave hemoglobin fragments. It is likely that many peptide intermediates are susceptible to cleavage by multiple enzymes. The distinct but overlapping roles of the proteolytic enzymes of the food vacuole and the location of exopeptidase activity across the food vacuole membrane in the parasite cytoplasm suggest that hemoglobin catabolism within P. falciparum is a semiordered pathway.The importance of zinc to intraerythrocytic P. falciparumhas been demonstrated. Levels of zinc increase in parallel with parasite maturation within the red blood cell (50Ginsburg H. Gorodetsky R. Krugliak M. Biochim. Biophys. Acta. 1986; 886: 337-344Crossref PubMed Scopus (22) Google Scholar). Treatment of cultures with dipicolinic acid does not prevent schizont rupture or reinvasion of host erythrocytes but blocks parasite maturation from the ring to the trophozoite stage, coincident with the onset of hemoglobin degradation (50Ginsburg H. Gorodetsky R. Krugliak M. Biochim. Biophys. Acta. 1986; 886: 337-344Crossref PubMed Scopus (22) Google Scholar, 51Yayon A. Vande Waa J.A. Yayon M. Geary T.G. Jensen J.B. J. Protozool. 1983; 30: 642-647Crossref PubMed Scopus (90) Google Scholar). Inhibition of hemoglobin catabolism is lethal to intraerythrocytic P. falciparum (2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 7Rosenthal P.J. Wollish W.S. Palmer J.T. Rasnick D. J. Clin. Invest. 1991; 88: 1467-1472Crossref PubMed Scopus (178) Google Scholar). The specific role of falcilysin in the essential process of hemoglobin degradation may have encouraging implications for the development of new chemotherapeutic agents. Plasmepsins I and II as well as falcipain prefer to cleave the α and β chains of hemoglobin at sites with hydrophobic residues at the P1 and/or P1′ positions (10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar). In contrast, falcilysin sites are polar in character, with charged residues at the P1 and/or P4′ positions. Internal cleavage of the large peptides generated by the plasmepsins and falcipain at distinct sites is likely to be critical for production of peptides small enough to cross the food vacuole membrane for cytosolic amino acid production. Falcilysin appears to play an integral role in this process. Our previous work suggested that there may be an additional unknown enzyme(s) in the food vacuole that participates in hemoglobin degradation (16Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biol. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). This paper describes the identification of a novel metallopeptidase activity in P. falciparum. Falcilysin was purified from food vacuoles and characterized. It is an oligoendopeptidase of the M16 family which contains two subfamilies. Subfamily B contains consists of the mitochondrial processing peptidases and chloroplast stromal processing peptidases that cleave NH2-terminal signal peptides from proteins (35Rawlings N.D. Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, Bath, United Kingdom1998: 1360-1362Google Scholar). Subfamily A of family M16 consists of large proteins (approximately 100 kDa or more) that function as oligoendopeptidases. Substrates for these enzymes include peptides such as a-factor (40Fujita A. Oka C. Arikawa Y. Katagai T. Tonouchi A. Kuhara S. Misumi Y. Nature. 1994; 372: 567-570Crossref PubMed Scopus (119) Google Scholar, 41Adames N. Blundell K. Ashby M.N. Boone C. Science. 1995; 270: 464-466Crossref PubMed Scopus (122) Google Scholar), insulin (42Shii K. Yokono K. Baba S. Roth R.A. Diabetes. 1986; 35: 675-683Crossref PubMed Scopus (83) Google Scholar), glucagon (43Kirschner R.J. Goldberg A.L. J. Biol. Chem. 1983; 258: 967-976Abstract Full Text PDF PubMed Google Scholar), atrial natriuretic peptide (44Muller D. Baumeister H. Buck F. Richter D. Eur. J. Biochem. 1991; 202: 285-292Crossref PubMed Scopus (68) Google Scholar), transforming growth factor α (45Gehm B.D. Rosner M.R. Endocrinology. 1991; 128: 1603-1610Crossref PubMed Scopus (62) Google Scholar), β-amyloid protein (46Kurochkin I. Goto S. FEBS Lett. 1994; 345: 33-37Crossref PubMed Scopus (342) Google Scholar), somatostatin 28 (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar), β-galactosidase (48Cheng Y.E. Zisper D. J. Biol. Chem. 1979; 254: 4698-4706Abstract Full Text PDF PubMed Google Scholar), and now, peptide fragments of hemoglobin (this work). Inhibition by alkylating agents has been observed in insulysin (42Shii K. Yokono K. Baba S. Roth R.A. Diabetes. 1986; 35: 675-683Crossref PubMed Scopus (83) Google Scholar), nardilysin (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar), mitochondrial processing peptidase ofNeurospora crassa (49Schneider A. Arretz M. Wachter E. Neupert W. J. Biol. Chem. 1990; 265: 9881-9887Abstract Full Text PDF PubMed Google Scholar), and falcilysin (this work). Nardilysin is inhibited by the aminopeptidase inhibitors amastatin and bestatin (47Chesneau V. Pierotti A.R. Barre N. Creminon C. Tougard C. Cohen P. J. Biol. Chem. 1994; 269: 2056-2061Abstract Full Text PDF PubMed Google Scholar) but this was not observed in falcilysin. The M16 family of metallopeptidases is growing. Besides the addition of falcilysin presented in this work, BLAST searches revealed sequence characteristics common to M16 family members in several uncharacterized protein sequences from different organisms (Fig.7). In fact, many potential M16 family members share greater sequence identity with falcilysin than the known members. Future investigations of these sequences will reveal which of these proteins function as metallopeptidases and whether one or more new types of enzymes with distinct biological functions are among them. As expected for a metallopeptidase of this class, falcilysin is unable to cleave polypeptides such as the α and β chains of hemoglobin (141 and 146 amino acids in length) but is able to cleave within peptides generated by the other proteases of the food vacuole. In this study falcilysin cleaved peptides up to 20 amino acids and preferred those 11–15 residues in length (Table I). It thus functions downstream of other vacuolar proteases, providing strong evidence for order in the hemoglobin degradation process. Our current understanding of the sequential nature of the proteolytic events is that the initial cleavages of the native hemoglobin molecule are made by the plasmepsins. Denatured or fragmented globin is susceptible to cleavage by falcipain as well as the plasmepsins (15Francis S.E. Gluzman I.Y. Oksman A. Banerjee D. Goldberg D.E. Mol. Biol. Parasitol. 1996; 83: 189-200Crossref PubMed Scopus (64) Google Scholar). Falcilysin cannot cleave hemoglobin or globin, but is able to cleave hemoglobin fragments. It is likely that many peptide intermediates are susceptible to cleavage by multiple enzymes. The distinct but overlapping roles of the proteolytic enzymes of the food vacuole and the location of exopeptidase activity across the food vacuole membrane in the parasite cytoplasm suggest that hemoglobin catabolism within P. falciparum is a semiordered pathway. The importance of zinc to intraerythrocytic P. falciparumhas been demonstrated. Levels of zinc increase in parallel with parasite maturation within the red blood cell (50Ginsburg H. Gorodetsky R. Krugliak M. Biochim. Biophys. Acta. 1986; 886: 337-344Crossref PubMed Scopus (22) Google Scholar). Treatment of cultures with dipicolinic acid does not prevent schizont rupture or reinvasion of host erythrocytes but blocks parasite maturation from the ring to the trophozoite stage, coincident with the onset of hemoglobin degradation (50Ginsburg H. Gorodetsky R. Krugliak M. Biochim. Biophys. Acta. 1986; 886: 337-344Crossref PubMed Scopus (22) Google Scholar, 51Yayon A. Vande Waa J.A. Yayon M. Geary T.G. Jensen J.B. J. Protozool. 1983; 30: 642-647Crossref PubMed Scopus (90) Google Scholar). Inhibition of hemoglobin catabolism is lethal to intraerythrocytic P. falciparum (2Francis S.E. Gluzman I.Y. Oksman A. Knickerbocker A. Mueller R. Bryant M.L. Sherman D.R. Russell D.G. Goldberg D.E. EMBO J. 1994; 13: 306-317Crossref PubMed Scopus (250) Google Scholar, 7Rosenthal P.J. Wollish W.S. Palmer J.T. Rasnick D. J. Clin. Invest. 1991; 88: 1467-1472Crossref PubMed Scopus (178) Google Scholar). The specific role of falcilysin in the essential process of hemoglobin degradation may have encouraging implications for the development of new chemotherapeutic agents. Plasmepsins I and II as well as falcipain prefer to cleave the α and β chains of hemoglobin at sites with hydrophobic residues at the P1 and/or P1′ positions (10Gluzman I.Y. Francis S.E. Oksman A. Smith C.E. Duffin K.L. Goldberg D.E. J. Clin. Invest. 1994; 93: 1602-1608Crossref PubMed Scopus (250) Google Scholar). In contrast, falcilysin sites are polar in character, with charged residues at the P1 and/or P4′ positions. Internal cleavage of the large peptides generated by the plasmepsins and falcipain at distinct sites is likely to be critical for production of peptides small enough to cross the food vacuole membrane for cytosolic amino acid production. Falcilysin appears to play an integral role in this process. We thank Anna Oksman for assistance with culturing parasites, Ilya Gluzman for providing recombinant plasmepsin II, Choukri Ben Mamoun for genomic DNA and assistance with computer analyses, and Christoph Turck for determining internal peptide sequences.
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