A histidine-rich protein from Plasmodium falciparum and its interaction with membranes

Bulletin of the World Health Organization, 55 (2-3): 191-197 (1977) A histidine-rich protein from Plasmodium falciparum and its interaction with membranes ARAXIE KILEJIAN 1 & JAMES B. JENSEN 2 In previous studies, an unusual protein with 7000 of histidine was isolated from Plasmodium lophurae. It was shown to have a possible function in the penetration of merozoites into erythrocytes. The results of the present study indicate the presence of a similar histidine-rich protein in Plasmodium falciparum. Like that of P. lophurae, the histidine-rich protein of P. falciparum is acid-soluble, but it has a slightly higher molecular weight. Intraerythrocytic stages of Plasmodium lophurae contain prominent cytoplasmic granules. The main chemical constituent of isolated granules was found to be a rather unusual protein with 73 % of histidine (1, 4). Indirect experimental evidence indicated that the protein may be also a component of the polar organelles of P. lophurae merozoites and may play a role in the penetration of merozoites into host erythrocytes (2, 3). Even though trophozoites of mammalian species of malaria do not contain cyto- plasmic granules similar to those of P. lophurae, polar organelles are a common feature of all malaria merozoites and might be expected to have consti- tuents of similar chemical nature. The purpose of this study was to test for the presence of a histidine- rich protein in merozoites of Plasmodiumfalciparum. MATERIALS AND METHODS Culture ofparasites The FVO strain of P. falciparum was cultured in Petri dishes (6). When about 6% of the blood cells had been infected, the culture medium was supple- mented with radioactive amino acids. In the first experiment, 92.5 kBq (2.5 ,uCi) of L-(3H} histidine (general label) and 7.4 kBq (0.2 ,uCi) of L-{3-"C} serine were added per millilitre of medium. For the second experiment, the labels were reversed and 14.8 kBq (0.4,uCi) of L-{ 14C} histidine (uniform label) and 185 kBq (5 ,uCi) of L-{3H} serine (general label) were added per millilitre of medium. 1 Associate Professor, Department of Parasitology, The Rockefeller University, New York, NY 10021, USA. 2Assistant Professor, Department of Parasitology, The Rockefeller University, New York, NY 10021, USA. Collection of merozoite-enriched fractions After parasites had grown in the presence of labelled substrates for 36-44 h, cultured cells were pooled and transferred to 125-ml Erlenmeyer flasks. The flasks were incubated in a shaking water bath at 37°C and gassed with moist air containing 5% of carbon dioxide. The purpose of the transfer of cultures to a shaking bath was to prevent the penetration of released merozoites into host erythro- cytes. After 3-4 h of incubation, the contents of the flasks were transferred to centrifuge tubes. Erythro- cytes infected with trophozoites, as well as normal erythrocytes, were pelleted (15 min at 900 g in an International Centrifuge PR-6000 with a No. 278 rotor). The supernatants were processed for the collection of merozoite-enriched fractions, while pel- lets were resuspended in radioactive medium in a ratio of 1: 10 and incubated for an additional 3-4 h to obtain a second batch of merozoites. The super- natants were centrifuged (5°C, 10 min, 2700 g), pellets were suspended in 1.5 ml of ice-cold non- radioactive culture medium without serum, and transferred to a microcentrifuge tube. Centrifugation for 1 min (Beckman Microfuge B) resulted in a pellet with 4 layers: a top, pinkish, membranous layer followed by a grey, a black, and a bottom red layer. The layers were separated crudely by means of step- by-step gentle suspension of each layer in a small volume of medium, and each fraction was trans- ferred to an individual microcentrifuge tube. Follow- ing centrifugation, pellets were further separated into a top and a bottom fraction and kept frozen at -70°C until used. Selected samples were fixed and processed for electron microscopy with procedures described previously (1). 3596 191 - A. KILEJIAN & J. B. JENSEN Polyacrylamide gel electrophoresis and other methods Electrophoresis was performed in slab gels 1.5 mm thick with slight modifications of the method of Laemmli (5): instead of 11 0% acrylamide for the resolving gel, an exponential gradient gel of 7-15% was used and the acrylamide concentration of the stacking gel was 4.5 %. Since the histidine-rich protein of P. lophurae is acid-soluble, aliquots of selected samples were extracted with 10% acetic acid prior to electrophoresis. For comparison with the electrophoretic pattern of infected erythrocyte membranes, a sample of normal human erythro- cyte membranes was prepared by lysing cells with 0.01 mol/litre phosphate buffer, pH 7.4. Stained gels were cut into slices 1.1 mm or 2 mm thick. The tritium and 14C in each slice were separated in a Tri Carb sample oxidizer (Pachard 306). Samples were counted in a Nuclear Chicago, Mark I, counter. RESULTS AND DISCUSSION The rationale of the experimental design for the search of a histidine-rich protein in P. falciparum had to be based on findings in P. lophurae. In view of the absence of cytoplasmic granules in trophozoites of P. falciparum, the highest concentration of a histidine-rich protein was expected to occur in mero- zoites and schizonts. If present, that protein might have been expected to represent a rather small fraction of total parasite protein and to be detectable only by sensitive methods, such as radioactivity. The selection of the particular labelled amino acids was based on the peculiar chemistry of the P. lophurae protein, which has 70% of histidine and almost no serine. It was reasoned that in most common pro- teins the ratio of histidine to serine could be expect- ed to fall within a reasonably close range, while a histidine-rich protein similar to that of P. lophurae would show an exceptionally high ratio of histidine to serine. From electron micrographs of crudely separated fractions, it appeared that merozoites and schizonts were concentrated mainly at the bottom of the grey layer and at the top of the black layer of the 2700-g pellet (Fig. 1). The selection of samples for electrophoresis was based on these findings. The electrophoretic pattern of all tested samples appeared in nearly all cases to be quite similar to that of normal host erythrocyte membranes (Fig. 2). It is not possible to conclude whether the few differences reflect alteration of membrane proteins or represent proteins derived from parasites. The histidine-rich protein of P. lophurae is acid-soluble, yet acid-extracted samples did not show the disap- pearance of any particular band. However, measure- ments of the distribution of incorporated 14C-serine and 3H-histidine across the gel representing a mero- zoite-enriched sample showed one area with a marked increase in histidine relative to serine (Fig. 3, a). Although the histidine-rich peak had the same electrophoretic mobility as albumin, the source of the differential label could not have resulted from contamination of parasite fractions with labelled serum, since the very highly concentrated albumin band of the medium showed only 5-10 counts above the background count. To exclude the possibility that this finding was an artifact of methodology, a second experiment was performed, reversing the labels of the two amino acids. The second ex- periment confirmed the initial findings (Fig. 3, b and c). The results of this experiment raise an interesting question. The gel sample of Fig. 3 b is that of a membrane-enriched fraction, yet it clearly shows an area of high incorporated histidine. On a purely speculative basis, the chemical properties of the histidine-rich protein could account for a selec- tive adsorption of this protein-which may have been released into the medium-to membranes. However, since not all the fractions analysed were pure, the localization of the histidine-rich protein in P. falciparum cannot be stated precisely. Like the protein of P. lophurae, the histidine-rich component of P. falciparum was acid-extractable. While a 2-mm gel slice from an acid-extracted sample had 140 counts/min of 14C-serine and only 15 counts/min of 3H-histidine (a ratio of 9: 1) the parallel control slice showed 150 counts/min of serine and 42 counts/min of histidine (a ratio of 3.6: 1). One apparent differ- ence between the proteins of the two species is their molecular weight. The histidine-rich area of P. falci- parum samples shows slower electrophoretic mobili- ty, and by comparison with standard proteins has a molecular weight of around 55 000. However, the SDS gel system may not give valid molecular weights of such unusual proteins, since the histidine-rich protein of P. lophurae moves as though it had a molecular weight of 45 000; yet it has a calculated molecular weight of 37 500. In conclusion, the results of this study have shown clearly that, like P. lophurae, P. falciparum synthe- sizes an acid-soluble histidine-rich protein. 192 Fig. 1. Electron micrographs of fractions isolated from P. talciparum cultures. (a) Pinkish membrane fraction. (b) Top of grey layer. (c) Bottom of grey layer. (d) Top of black layer. Bar length equals 1 ,tm. Fig. 2. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of selected fractions isolated from cultures of P. falciparum. (a) The histidine-rich protein from P. lophurae. (b) Membrane-enriched fraction after extraction with acetic acid. (c) Membrane-enriched fraction. (d) Fraction enriched with merozoites and schizonts. (e) Fraction d after acid extraction. (f) Normal human erythrocyte membranes. (g) Fraction f after acid extraction. (h) Medium. Asterisks indicate areas of difference between normal erythrocyte membranes and samples from P. falciparum cultures. 4 5 6 7 8 Centimetres 11 12 WHO 77370 Fig. 3. Distribution of radioactive histidine and serine in fractions of P. falciparum analysed by gel electrophoresis. (a) Electrophoretic pattern of a merozoite-enriched fraction from P. falciparum cultured in the presence of 14C-serine and 3H-histidine. (b) A membrane-enriched fraction from samples cultured in the presence of 3H-serine and 14C-histidine. (c) A merozoite-enriched fraction from samples cultured in the presence of 3H-serine and 14C. histidine. Ratios (A 'F10 C) "I. _5 I0 CV) .U) . 10 0 t-5 I 0C, i_ I- I- I- 101_ 51 Un I I 0) 10 v- HISTIDINE-RICH PROTEIN FROM P. FALCIPARUM 197 ACKNOWLEDGEMENTS This study was supported mainly by NIH grant Al 10640 and in part by AID contract ta-C-1373. We thank Ellen Kracauer for her excellent technical assistance. RtSUMt PROTEINE RICHE EN HISTIDINE CHEZ PLASMODIUM FALCIPARUM, ET SON INTERACTION AVEC LES MEMBRANES Au cours de travaux precedents, une proteine inha- bituelle contenant 70% d'histidine avait ete isolee de Plasmodium lophurae et il avait ete montre qu'elle jouait peut-etre un r6le dans le processus de penetration des m6rozoites dans les erythrocytes. Les resultats de la pre- sente etude indiquent qu'il existe une proteine similaire riche en histidine chez Plasmodium falciparum. Comme celle de P. lophurae, la proteine riche en histidine de P. falciparum est soluble dans les acides, mais son poids moleculaire est un peu plus eleve. REFERENCES 1. KILEJIAN, A. Journal of biological chemistry, 249: 4650-4655 (1974). 2. KILEJIAN, A. Journal ofprotozoology, 23: 272-277 (1976). 3. KILEJIAN, A. In: Van den Bossche, H., ed. Biochemistry ofparasites and host-parasite relationships. Amsterdam, Elsevier/North-Holland Biomedical Press, 1976, pp. 441-448. 4. KILEJIAN, A. ET AL. Proceedings of the National Academy of Sciences of the United States of America, 72: 3057-3059 (1975). 5. LAEMMLI, U. K. Nature, 227: 680-685 (1970). 6. TRAGER, W. & JENSEN, J. B. Science, 193: 673-675 (1976).