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Identification and Characterization of Falcilysin, a Metallopeptidase Involved in Hemoglobin Catabolism Within the Malaria Parasite Plasmodium Falciparum*

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Identification and Characterization of Falcilysin, a Metallopeptidase Involved in Hemoglobin Catabolism Within the Malaria Parasite Plasmodium Falciparum* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 45, Issue of November 5, pp. 32411–32417, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Identification and Characterization of Falcilysin, a Metallopeptidase Involved in Hemoglobin Catabolism within the Malaria Parasite Plasmodium falciparum* (Received for publication, May 25, 1999, and in revised form, August 24, 1999) Kathleen Kolakovich Eggleson, Kevin L. Duffin‡, and Daniel E. Goldberg§ From the Howard Hughes Medical Institute, Washington University, Departments of Molecular Microbiology and Medicine, St. Louis, Missouri 63110 and ‡Monsanto Corporate Research, Monsanto Company, St. Louis, Missouri 63198 The malaria parasite Plasmodium falciparum de- organelle, the food vacuole. This compartment has a pH esti- grades hemoglobin in its acidic food vacuole for use as a mated at 5.0–5.4 (3, 4). major nutrient source. A novel metallopeptidase activ- Nonproteolytic acid hydrolases could not be detected in food ity, falcilysin, was purified from food vacuoles and char- vacuoles isolated from P. falciparum (5). Thus, it appears that acterized. Falcilysin appears to function downstream of the food vacuole of P. falciparum does not function in degrada- the aspartic proteases plasmepsins I and II and the cys- tion and recycling of macromolecules in general. The catabolic Downloaded from teine protease falcipain in the hemoglobin proteolytic capability of this organelle is focused on hemoglobin. Disrup- pathway. It is unable to cleave hemoglobin or denatured tion of hemoglobin catabolism causes parasite death in an globin but readily destroys peptide fragments of hemo- globin. Falcilysin cleavage sites along the a and b chains animal model and in culture (2, 6, 7). The vital and specialized of hemoglobin are polar in character, with charged res- process of hemoglobin degradation within the food vacuole idues located in the P1 and/or P4* positions. In contrast, provides promising targets for the development of novel anti- plasmepsins I and II and falcipain prefer hydrophobic malarial drugs, greatly needed in the face of increasing resist- www.jbc.org residues around the scissile bond. The gene encoding ance to existing chemotherapeutic agents (8). falcilysin has been cloned. Its coding sequence exhibits Multiple proteases within the food vacuole facilitate the deg- features characteristic of clan ME family M16 metal- radation of hemoglobin to peptide fragments. Three acidic pro- lopeptidases, including an “inverted” HXXEH active site teases have been identified, purified from food vacuoles, and at Washington University on August 9, 2006 motif. Falcilysin shares primary structural features characterized (2, 9–14). Two aspartic proteases, plasmepsin I with M16 family members such as insulysin, mitochon- and plasmepsin II, can cleave native hemoglobin. A cysteine drial processing peptidase, nardilysin, and pitrilysin as protease, falcipain, is able to cleave denatured globin but not well as with data base hypothetical proteins that are potential M16 family members. The characterization of native hemoglobin (10, 15). Exopeptidase activity capable of falcilysin increases our understanding of hemoglobin generating individual amino acids from peptide fragments is catabolism in P. falciparum and the unusual M16 family absent from the food vacuole (16). However, peptides may tra- of metallopeptidases. verse 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 Plasmodium falciparum is a protozoan parasite that causes purified from parasites and characterized (17–19). the most lethal form of human malaria. Upon invasion of a When hemoglobin was incubated with food vacuole lysate at human erythrocyte, the parasite grows and matures sur- acidic pH, a series of discrete peptide fragments was generated rounded by cytosol consisting predominantly of a single pro- (16). Cleavage sites along the hemoglobin a and b chains were tein, hemoglobin. Amino acids derived from the proteolysis of located an average of eight amino acids apart. Many cleavage hemoglobin are incorporated into parasite proteins and para- sites corresponded to the peptide bonds previously identified as sites require supplementation with only a few amino acids that sites for the known vacuolar proteases. Twenty-four cleavage are absent or deficient in hemoglobin for normal growth in sites that could not be attributed to the known proteases of the culture (1, 2). Hemoglobin proteolysis occurs within an acidic vacuole were identified. Unlike the preference of plasmepsin I, plasmepsin II, and falcipain for hydrophobic residues, many of * This work was supported in part by National Institutes of Health the novel cleavage sites contained polar residues at the P1 Grant AI-31615. Preliminary sequence data for P. falciparum chromo- and/or P19 positions. These results suggested that one or more some 14 was obtained from The Institute for Genomic Research web- vacuolar endopeptidases had remained unidentified (16). In site. Sequencing of chromosome 14 was part of the International Ma- this report, we describe the discovery of a novel proteolytic laria Genome Sequencing Project and was supported by awards from the Burroughs Wellcome Fund and the U. S. Department of Defense. activity, purification of the enzyme responsible for this activity, The costs of publication of this article were defrayed in part by the characterization of the purified enzyme, and the corresponding payment of page charges. This article must therefore be hereby marked molecular sequence data. Furthermore, we provide evidence “advertisement” in accordance with 18 U.S.C. Section 1734 solely to that this enzyme, falcilysin, has a distinct, downstream role in indicate this fact. The nucleotide sequence for the falcilysin gene and the amino acid the semiordered hemoglobin degradation pathway of P. sequence for the falcilysin protein have been deposited in the GenBank falciparum. data base under GenBank accession number AF123458. § Recipient of a Burroughs Wellcome Fund Scholar Award in Molec- EXPERIMENTAL PROCEDURES ular Parasitology. To whom correspondence should be addressed: Wash- ington University School of Medicine, Box 8230, 660 S. Euclid Ave., St. Reagents—Human hemoglobin, human globin, pepstatin, E64, Louis, MO 63110. Tel.: 314-362-1514; Fax: 314-362-1232; E-mail: EDTA, dipicolinic acid, amastatin, bestatin, bacitracin, 1,10-phenan- [email protected]. throline, cobalt sulfate, magnesium sulfate, manganese sulfate, zinc This paper is available on line at http://www.jbc.org 32411 32412 Falcilysin, a Vacuolar Metallopeptidase from P. falciparum sulfate, BisTris,1 Klentaq LA, and Triton X-100 were obtained from Brilliant Blue staining. Endoproteinase Lys-C was used to perform an Sigma. Endoproteinase Lys-C and PMSF were from Roche Molecular in-gel digest of the falcilysin SDS-PAGE band and the resulting pep- Biochemicals (Indianapolis, IN). N-Ethylmaleimide was from Fluka tides were extracted (24). The peptide mixture was loaded onto a mi- (Buchs, Switzerland). Coomassie Brilliant Blue, gel filtration standard crobore C18 column. The column was washed for 15 min with 0.1% vials, high range SDS-PAGE standards, and Tween 20 were from Bio- trifluoroacetic acid and the peptides were eluted by applying a linear Rad (Hercules, CA). Silver nitrate was from Eastman Kodak (Roches- gradient of acetonitrile from 0 to 50% in 0.1% trifluoroacetic acid over ter, NY). The Ultrasphere C18 HPLC column was from P. J. Cobert 1 h at a flow rate of 100 ml/min. 0.5-min fractions were collected and the Associates, Inc. (St. Louis, MO). The Microbore C18 column was from peptides were detected at 215 nm on a Hewlett-Packard 1090 HPLC The Separations Group (Hesperia, CA). Mono S PC 1.6/5, Superose 12 system (Hewlett-Packard, Palo Alto, CA). Peptides were sequenced on a HR 10/30, DEAE-Sepharose, and CM-Sepharose were from Amersham PROCISE 492 protein sequencer (PE Biosystems, Foster City, CA). Pharmacia Biotech. MES and acetonitrile (CH3CN) were from Fisher Metal Requirement—The apoenzyme form of falcilysin was generated (Pittsburgh, PA). Millex HV polyvinylidine difluoride 0.45-mm filters by incubation in dilute form at 4 °C until no activity against the a71–78 were from Millipore (Bedford, MA). Synthetic oligonucleotides were substrate could be detected. Sulfate salts of Co21,Mg21,Mn21,orZn21 from Integrated DNA Technologies (Coralville, IA). The quenched flu- were added to the apoenzyme at various concentrations and tested for orescence substrates were from AnaSpec (San Jose, CA). their ability to restore activity in the fluorogenic assay. An amount of Culture—P. falciparum clones HB3 (gift of W. Trager, Rockefeller apoenzyme equivalent to 0.47 units of original holoenzyme activity University) and 3D7 (gift of P. Rathod, Catholic University) were grown (where 1 unit of activity equals 1 pmol of quenched fluorescence sub- by the method of Trager and Jensen (20) in serum-free medium sup- strate cleaved/min) was used in each reaction. plemented with 0.5% AlbuMAX I from Life Technologies, Inc. (Grand Falcilysin was tested for inhibition by 10 mM amastatin, 10 mM Island, NY). Synchrony was maintained by sorbitol treatment (21). bestatin, 1 mM pepstatin, 10 mM E64, 1 mM PMSF, 5 mM N-ethylmale- Food Vacuole Preparation—Food vacuoles were isolated from P. fal- imide, 1 mM EDTA, 500 mM dipicolinic acid, and 1 mM 1,10-phenanthro-
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