Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production
Introduction
Malaria parasites are able to obtain amino acids for intraerythrocytic growth and maturation in three ways: biosynthesis from other carbon sources, uptake from the external medium, and catabolism of host hemoglobin 1, 2. Radiolabeled amino acids that have been exogenously supplied or derived from labeled hemoglobin can be incorporated into parasite proteins [3]. Radiolabeled glucose, pyruvate, or acetate and 14CO2 can be metabolized by the parasite into glutamate, aspartate, alanine, and leucine (reviewed by Scheibel et al. [2]). Intraerythrocytic Plasmodium in semi-defined medium requires only five exogenously supplied amino acids for normal growth: isoleucine, methionine, cysteine, glutamate, and glutamine 4, 5. These five amino acids are rare or lacking in hemoglobin. The relative contributions of biosynthesis, uptake, and hemoglobin catabolism to the parasite amino acid pool in vivo are unknown but are likely to vary by the availability of carbon sources and free amino acids in serum as well as the abundance of a given residue in hemoglobin [1].
Hemoglobin proteolysis occurs in a specialized organelle, the acidic digestive vacuole. Aspartic and cysteine protease activities in the digestive vacuole account for the vast majority of the organelle's ability to degrade hemoglobin [6]. Two aspartic proteases, plasmepsin I and plasmepsin II, and a cysteine protease, falcipain, have been purified from Plasmodium falciparum and characterized 5, 6, 7, 8, 9, 10. Their synergistic action as well as individual cleavage specificities in the degradation of human hemoglobin have been described 6, 11. While the initial cleavage events that generate peptide fragments from hemoglobin in the digestive vacuole have been well studied, later steps that convert peptide fragments into amino acids for metabolic use by the parasite have remained uncharacterized.
It has been presumed that amino acids are generated by exopeptidase activity in the digestive vacuole and transported to the cytoplasm for incorporation into parasite proteins, but no direct evidence supports this 2, 12. Although an aminopeptidase activity was previously isolated from P. falciparum 13, 14, its near-neutral pH optimum is not compatible with efficient function at the acidic pH of the digestive vacuole (slightly above pH 5) 15, 16. In this report, we provide evidence against the degradation of hemoglobin peptide fragments into individual amino acids by vacuolar proteases. No vacuolar exopeptidase could be detected in assays with hemoglobin or synthetic substrates. Instead, discrete peptides were generated from vacuolar digestion of exogenous hemoglobin, suggesting the existence of a peptide translocator to export hemoglobin fragments to the cytoplasm for terminal catabolism to amino acids.
Section snippets
Reagents
Human hemoglobin, l-alanine-p-nitroanalide, glutathione, and acetonitrile (CH3CN) were obtained from Sigma Chemical Co. (St. Louis, MO). HIV protease substrate III was from Bachem Bioscience, (Philadelphia, PA). The Ultrasphere C18 HPLC column was from P.J. Cobert Associates (St. Louis, MO). The Superdex Peptide PC FPLC column was from Pharmacia Biotech (Uppsala, Sweden). Phenylisothiocyanate (PITC), ionate triethylamine (TEA), and Amino Acid standard H were obtained from Pierce (Rockford, IL).
Culture
Exopeptidase activity is detected in P. falciparum parasites but not in the digestive vacuole
A Plasmodium neutral aminopeptidase has been isolated but not localized 13, 14. The preferred fluorogenic substrates had N-terminal alanine or leucine residues [13], consistent with efficient cleavage of hemoglobin-derived peptides at neutral pH, since alanine and leucine constitute 25% of the residues in human hemoglobin. Considerable aminopeptidase activity was found in P. falciparum trophozoites, young schizonts, and mature schizonts but not in isolated digestive vacuoles (Table 1). When the
Discussion
Hemoglobin catabolism is a process that is essential for parasite survival and provides a set of targets for the development of novel chemotherapuetic agents, desperately needed in the face of increasing resistance to traditional therapies 5, 12, 21, 22. Previous work on hemoglobin catabolism has focused on early proteolytic events, namely, the cleavage of hemoglobin into large peptides by plasmepsin I, plasmepsin II, and/or falcipain 5, 6, 10, 11. Yet the later events surrounding the
Acknowledgements
We thank Anna Oksman for excellent technical assistance, Carolyn Moomaw and Clive Slaughter for N-terminal sequence analysis, Jiri Gut and Richard Nelson for the AlbuMAX protocol, and Susan Francis for critical review of the manuscript.
References (43)
Hemoglobin degradation in Plasmodium-infected red blood cells
Semin Cell Biol
(1993)- et al.
Incorporation of 14C-amino acids by malaria
Int J Biochem
(1970) - et al.
Sequence, expression and modeled structure of an aspartic proteinase from the human malaria parasite Plasmodium falciparum
Mol Biol Parasitol
(1994) - et al.
Isolation and characterization of a cysteine protease gene of Plasmodium falciparum
Mol Biochem Parasitol
(1992) - et al.
Localization and characterization of hemoglobin-degrading proteinases from the malarial parasite Plasmodium falciparum
Biochim Biophys Acta
(1992) - et al.
The Plasmodium digestive vacuole: Metabolic headquarters and choice drug target
Parasitol Today
(1995) - et al.
Purification and characterization of an aminopeptidase from Plasmodium falciparum
Mol Biochem Parasitol
(1984) - et al.
A simple method for displaying the hydropathic character of a protein
J Mol Biol
(1982) - et al.
Digestive activity of lysosomes
J Biol Chem
(1968) - et al.
TAP1-dependent peptide translocation in vitro is ATP dependent and peptide selective
Cell
(1993)
The ATP-binding cassette (ABC) gene family of Plasmodium falciparum
Parasitol Today
Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins
Cell
Amplification of pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum in Thailand
Mol Biochem Parasitol
Peptide transport by the multidrug resistance pump
J Biol Chem
Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters
J Biol Chem
Structure of Saccharomyces cerevisiae mating hormone a-factor: Identification of S-farnesyl cysteine as a structural component
J Biol Chem
Nucleotide binding properties of a P-glycoprotein homologue from Plasmodium falciparum
Mol Biochem Parasitol
Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth
J Protozool
Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase
EMBO J
Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway
J Clin Invest
Cited by (113)
Plasmodium's bottomless pit: properties and functions of the malaria parasite's digestive vacuole
2022, Trends in ParasitologyCitation Excerpt :Falcilysin has been localized to the DV, but also to the cytoplasm, apicoplast, and parasite periphery, perhaps indicating an involvement in additional catabolic pathways [33–35]. Incubation of native hemoglobin with DV lysate leads to the rapid generation of globin-derived oligopeptides with an average length of eight amino acids [36]. No free amino acids were detected under these conditions, suggesting that oligopeptides are exported from the DV for further processing by cytoplasmic aminopeptidases.
Malaria parasite plasmepsins: More than just plain old degradative pepsins
2020, Journal of Biological ChemistryMulti-omic characterization of the mode of action of a potent new antimalarial compound, JPC-3210, against plasmodium falciparum
2020, Molecular and Cellular ProteomicsMembrane protein carbonylation of Plasmodium falciparum infected erythrocytes under conditions of sickle cell trait and G6PD deficiency
2019, Molecular and Biochemical ParasitologyDrug targets for resistant malaria: Historic to future perspectives
2018, Biomedicine and PharmacotherapyCitation Excerpt :Therefore, a transporter that exports the peptides for terminal degradation to aminoacids in the cytoplasm must exist. Inhibition of this transporter system could be a valid target to develop antimalarial agents [53]. Proteasome is a protein complex that degrades proteins by proteolysis by breaking the peptide bond.
Protein profiling of mefloquine resistant Plasmodium falciparum using mass spectrometry-based proteomics
2015, International Journal of Mass Spectrometry