Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production

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Abstract

Intraerythrocytic malaria parasites avidly consume hemoglobin as a source of amino acids for incorporation into parasite proteins. An acidic organelle, the digestive vacuole, is the site of hemoglobin proteolysis. Early events in hemoglobin catabolism have been well studied. Two aspartic proteases, plasmepsins I and II, and a cysteine protease, falcipain, cleave hemoglobin into peptides. While it has been presumed that hemoglobin peptide fragments are degraded to individual amino acids by exopeptidase activity in the digestive vacuole, this hypothesis lacks experimental support. Incubation of human hemoglobin with P. falciparum digestive vacuole lysate generated a series of discrete peptide fragments with cleavage sites an average of 8.4 amino acids apart. No free amino acids could be detected and there was no evidence of peptide heterogeneity due to exopeptidase trimming. These sites correspond to points of cleavage previously established for plasmepsin I, plasmepsin II, and falcipain as well as some novel sites that suggest the existence of an additional endoproteinase. By colorimetric assay, P. falciparum has abundant aminopeptidase activity but this activity is not found in the digestive vacuoles and the parasite lacks detectable carboxypeptidase activity altogether. These data support a model for hemoglobin catabolism wherein small peptides are formed from cleavage of hemoglobin by the enzymes of the digestive vacuole and then are transported through the membrane of the digestive vacuole to the cytoplasm. There, exopeptidase activity converts the peptides to individual amino acids for parasite growth and maturation.

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.

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