Characterization of P0, a Ribosomal Phosphoprotein ofPlasmodium falciparum

Abstract

A cDNA expression clone of the human malarial parasite Plasmodium falciparum, λPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone λPf4 also immunoprecipitated the same size protein from [35S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth ofP. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites. A cDNA expression clone of the human malarial parasite Plasmodium falciparum, λPf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the35S-labeled as well as 32P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone λPf4 also immunoprecipitated the same size protein from [35S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth ofP. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites. It has been documented that people living in malaria-endemic areas acquire immunity to Plasmodium falciparum after repeated infections. The nature of this immunity is poorly understood at the molecular level. It is apparent from studies involving passive transfer of IgG from immune adults to the non-immune subjects that circulating antibodies do play an important role (1Cohen S. Mcgregor I.A. Carrington S.P. Nature. 1961; 192: 733-737Google Scholar, 2Bouharoun-Tayoun H. Attanath P. Sabchareon A. Chongsuphajaisiddhi T. Druihle P. J. Exp. Med. 1990; 172: 1633-1641Google Scholar). The specificity of these protective antibodies is as yet unknown. It has been shown that the antibodies present in immune adults recognize domains that are conserved in different strains of P. falciparum (3Marsh K. Howard R.J. Science. 1986; 231: 150-153Google Scholar). However, it has also been documented that many malarial antigens possess repetitive protein domains, which evoke a strong antibody response. Many of these antibodies are non-protective, and the corresponding malarial epitopes are postulated to be immune-evasive or smokescreen domains (4Anders R.F. Parasite Immunol. ( Oxf .). 1986; 8: 529-539Google Scholar, 5Schofield L. Parasitol. Today. 1991; 7: 99-105Google Scholar). Thus, to search for pan-specific and possibly protective antibodies, a differential immunoscreening of an erythrocytic stage-specific cDNA expression library of P. falciparum was carried out using malaria-immune and acute patient sera. This resulted in the identification of several novel cDNA clones, which reacted exclusively and yet extensively with immune sera samples (6Lobo C.A. Kar S.K. Ravindran B. Kabilan L. Sharma S. Infect. Immun. 1994; 62: 651-656Google Scholar). The clone λPf4, which was reactive to the largest number of immune sera (80 out of 92), has been cloned and sequenced recently (7Goswami A. Chatterjee S. Sharma S. Mol. Biochem. Parasitol. 1996; 82: 117-120Google Scholar). This was found to be the P. falciparum gene homologue of the ribosomal phosphoprotein P0 (PfP0). 1The abbreviations used are: PfP0, P. falciparum P0 protein; GST, glutathioneS-transferase; PfP0-N, recombinant fusion protein of GST with the 46-amino acid peptide from the amino-terminal domain of P0 protein; kb, kilobase(s); IFA, indirect immunofluorescence assay; bp, base pair(s). Ribosomal phosphoprotein P0 is considered to be related to the family of the acidic ribosomal phosphoproteins P1 and P2, because of the highly homologous carboxyl-terminal domain (8Rich B.E. Steitz J.A. Mol. Cell. Biol. 1987; 7: 4065-4074Google Scholar). Antibodies against this domain coprecipitate all three P proteins (9Towbin H. Ramjoue H.-P. Kuster H. Liverani D. Gordon J. J. Biol. Chem. 1982; 257: 12709-12715Google Scholar). P0 could be cross-linked to P1 and P2 protein in Artemia salina ribosomes (10Uchiumi T. Wahha A.J. Traut R.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5580-5584Google Scholar), and these data, along with that from yeast cells (11Saenz-Robles M.T. Remacha M. Vilella M.D. Zinker S. Ballesta J.P.G. Biochim. Biophys. Acta. 1990; 1050: 51-55Google Scholar), strongly indicate the existence of a (P1)2·P0·(P2)2 protein complex in the eukaryotic ribosomes. This complex has been compared with the bacterial complex L10·(L7/L12)2, which forms the stalk of the large subunit at the GTPase domain along with the 23 S ribosomal RNA (12Beauclerk A.A.D. Cundliffe E. Dijk J. J. Biol. Chem. 1984; 259: 6559-6563Google Scholar, 13Egebjerg J. Douthwaite S.D. Liljas A. Garrett R.A. J. Mol. Biol. 1990; 213: 275-288Google Scholar). It has been documented that P0 protein is absolutely required for the ribosomal activity and cell viability in yeast (14Santos C. Ballesta J.P.G. J. Biol. Chem. 1994; 269: 15689-15696Google Scholar). The conserved carboxyl-terminal domain of the P proteins is very antigenic and found to be the main antigenic target for sera reactivity of about 10–15% of patients of the autoimmune disorder systemic lupus erythematosus (15Elkon K. Skelley S. Parnassa A. Moller W. Waleed D. Weissbach H. Nathan B. Proc. Natl. Acad. Sci. U . S . A. 1986; 38: 7419-7423Google Scholar). Antibodies to this domain have also been detected in patients of suffering from diseases caused by protozoan parasites such as Chagas' heart disease (16Levin M.J. Vazquez M. Kaplan D. Schijman A.G. Parasitol. Today. 1993; 9: 381-384Google Scholar) and leishmaniasis (17Skeiky Y.A.W. Benson D.R. Elwasila M. Badaro R. Burns Jr., J.M. Reed S.G. Infect. Immun. 1994; 62: 1643-1651Google Scholar). In this paper we report the characterization of this protein from P. falciparum, and we show for the first time that P0 is indeed a phosphoprotein. We also show that antibodies raised against the amino-terminal domain of this protein inhibitsP. falciparum growth in vitro. All reagents, unless otherwise specified, were purchased from Sigma. [α-32P]dATP, [32P]orthophosphoric acid were provided by the Board of Radiation Technologies, India. [35S]Methionine and [35S]cysteine were purchased from Amersham International (Buckinghamshire, England). Asexual stages of FCR3 (Gambia) and the FCK2 (India) strains of P. falciparum were cultured in vitro at 37 °C in the presence of human erythrocytes of serological type O+ in complete medium (RPMI 1640 medium containing 28 mm NaHCO3, 25 mm HEPES, and supplemented with either 10% human serum or 0.5% Albumax (Life Technologies, Inc.) and 80 μg/ml Gentamycin sulfate) in sterile Petri dishes using the candle-jar method or sealed flasks flushed with 5% O2, 5% CO2, and 90% N2 gas mixture (18Rathod P.K. Leffers N.P. Young R.D. Antimicrob. Agents Chemother. 1992; 36: 704-711Google Scholar). For stage-specific RNA preparation and in vitro parasite growth inhibition assays, cultures were synchronized by sorbitol treatment according to the method of Lambros and Vanderberg (19Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Google Scholar). Intracellular parasites from each of the substages were liberated from infected erythrocytes by saponin lysis (20Zuckerman A. Spira D. Hamburger J. Bull. W. H. O. 1967; 37: 431-436Google Scholar) for total RNA and genomic DNA extraction. The gametocytic stages of the parasite (NF54 strain) used for the immunofluorescence studies were kindly provided by Dr. Nirbhay Kumar, Johns Hopkins University (21Lobo C.A. Konings R.N.H. Kumar N. Mol. Biochem. Parasitol. 1994; 68: 151-154Google Scholar). Total cellular RNA was extracted using a single step method described by Chomczynski and Sacchi (22Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar). Genomic DNA was extracted from total erythrocytic stages of the parasite as described in detail elsewhere (6Lobo C.A. Kar S.K. Ravindran B. Kabilan L. Sharma S. Infect. Immun. 1994; 62: 651-656Google Scholar). Southern and Northern hybridization were performed with the radioactively labeled [α-32P]dATP 251-bp λPf4 cDNA fragment as well as the 700-bp L-4-7 (carboxyl-terminal fragment of PfP0 protein) with specific activity of 2 × 108 cpm by following the membrane manufacturer's protocol (Amersham International) in the presence of 50% formamide. Briefly, 2 μg of parasite genomic DNA cut with appropriate restriction enzymes (New England Biolabs Inc.) was electrophoresed in a 1.0% agarose gel. In the case of Northern hybridization, total RNA from the parasite was electrophoresed in formaldehyde containing 0.8% agarose gel. 0.69–9.44-kb RNA markers (Life Technologies, Inc.) were run and stained separately with ethidium bromide before transfer of the gel onto the membrane. Gels were transferred to Hybond N+ membrane (Amersham International) by capillary blotting following membrane manufacturer's protocol. Air-dried blots were then UV cross-linked in a UV cross-linker (Bio-Rad) for 2 min and pre-hybridized at 42 °C for 2–3 h. Hybridization was carried out at 42 °C for 18–20 h. Blots were washed twice at 65 °C for 15 min each with 2 × saline/sodium/phosphate/EDTA, 0.1% (w/v) SDS followed by washes with 1 × saline/sodium/phosphate/EDTA, 0.1% SDS and 0.1 × saline/sodium/phosphate/EDTA, 0.1% SDS, and exposed to Fuji x-ray film for autoradiography. The quantitation was performed using the gel-documentation system (Ultraviolet Products Inc.) as per manufacturer's protocol by estimating the volume of the signal. A GST reporter-based vector (pGEX-1) was used as an expression vector (23Smith D.B. Johnson K.S. Gene ( Amst .). 1988; 67: 31-40Google Scholar). A HindIII-EcoRI restricted 138-bp amino-terminal fragment of PfP0 (46–184 bp of GenBankTM accession number U56663) (7Goswami A. Chatterjee S. Sharma S. Mol. Biochem. Parasitol. 1996; 82: 117-120Google Scholar) was flushed at both ends with Klenow and subcloned in pGEX-1 restricted withSmaI. The Escherichia coli cells harboring the vector pGEX-1 and the recombinant containing PFP0-N insert were induced with 1 mm isopropyl-β-d-thiogalactopyranoside for 2 h at 37 °C (23Smith D.B. Johnson K.S. Gene ( Amst .). 1988; 67: 31-40Google Scholar). The total cell lysates were run on a 12% denaturing SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue to show the fusion protein (PfP0-N). The polyacrylamide gel piece containing the fusion protein band was cut out and crushed in liquid nitrogen and dissolved in phosphate-buffered saline (8 g/liter NaCl, 0.2 g/liter KCl, 1.44 g/liter Na2HPO4, 0.24 g/liter KH2PO4, pH adjusted to 7.4 with NaOH). The resulting slurry containing an estimated amount of 100 μg of protein was injected into two rabbits. Five boosts were given to each animal to generate antibodies with reasonable titer (>1000 as checked by enzyme-linked immunosorbent assay). λPf4 cDNA clones in λgt11, as well as wild type λgt11, were grown on E. coli strain Y1090 to lytic phase (6Lobo C.A. Kar S.K. Ravindran B. Kabilan L. Sharma S. Infect. Immun. 1994; 62: 651-656Google Scholar). Hybond-C membrane (Amersham International) soaked in isopropyl-β-d-thiogalactopyranoside was overlaid on these phage plates and grown for an additional 12–16 h. The membranes were washed three times with 1 × TBS-T (200 mm Tris, 50 mm NaCl, 0.05% Tween 20, pH 7.5). A pool of six reactive human immune sera (1:100 dilution) used for the differential immunoscreening (6Lobo C.A. Kar S.K. Ravindran B. Kabilan L. Sharma S. Infect. Immun. 1994; 62: 651-656Google Scholar) was first incubated at °C with membrane with the wild type the sera was incubated with several membranes containing the of recombinant Monospecific antibodies against λPf4 were with mm from the with Tris, and then with three of antibody against λPf4 was using an to the volume of the human sera with 1 × and used for cultures of P. falciparum containing were washed twice with and 1640 medium and at a of 5% in the same medium supplemented with 10% human 100 of both and were to the medium and incubated for h at 37 °C with the cells were washed and extracted as described elsewhere N. M. M. H. Mol. Biochem. Parasitol. 1991; Scholar). In the case of 1 acid was using to the washed parasites at 5% 10% in the 1640 medium supplemented with 0.5% Albumax (Life Technologies, incubated for h at 37 and the of the were the same as Monospecific human immune sera against λPf4 as well as polyclonal antibody against were used to and 32P-labeled parasite proteins N. M. M. H. Mol. Biochem. Parasitol. 1991; Scholar). The immunoprecipitated samples were run on 12% denaturing which were stained with Coomassie Blue for 1 h and with 10% for 2 were soaked with for min and washed with for In the case of the was with several of the The were then using and exposed to Fuji x-ray film for autoradiography. studies were with P. falciparum strain) as well as gametocytic stages (NF54 strain) with polyclonal antibodies against as described P. N. M. M. J. S. 1991; Scholar). Briefly, and cultures of P. falciparum were on as and for The were with for at with 100 μg/ml serum for 2–3 h at and washed at with 1 × TBS-T for min each for three were incubated with antibodies for h at at were used as the The were a using × 100 phase and All antibodies were with GST coli proteins and used at IgG from sera were by followed by with at pH E. D. A Scholar). P. falciparum growth inhibition were performed in in plates in a total volume of 1 The of IgG on parasite growth was by with parasites at a and growth h medium cells from were on The were in plates and H. Attanath P. Sabchareon A. Chongsuphajaisiddhi T. Druihle P. J. Exp. Med. 1990; 172: 1633-1641Google Scholar). In the containing the falciparum cultures were at a of of and and and and and IgG were at a of of was h by from each well and by of In each the number of and were Total was estimated as the total of the and A Southern of genomic DNA from the strain of P. falciparum, with the 251-bp λPf4 that it with the and pair band the DNA was restricted with and The gene has an and this was by the same with a 700-bp the carboxyl-terminal of the protein (7Goswami A. Chatterjee S. Sharma S. Mol. Biochem. Parasitol. 1996; 82: 117-120Google Scholar), which the same for all restriction that of it a pair band Southern analysis of genomic DNA from two P. falciparum FCR3 and was also performed with these two and restriction fragment was show that the PfP0 is by a single gene and is well conserved in different strains of the A Northern of stage-specific total RNA from the stages ofP. falciparum, with the 251-bp λPf4 a fragment in However, a about in was also in all A of the of these that the was as as the in each of the However, the was about and in in the compared with the and the The of the P0 gene is However, the gene to possess a of about in as by the size of cDNA clones (7Goswami A. Chatterjee S. Sharma S. Mol. Biochem. Parasitol. 1996; 82: 117-120Google Scholar). studies using the the same size of genomic DNA is used as a not and are the However, the presence of in the is yet to be A the expression of the protein of the amino-terminal domain of the P. falciparum P0 protein in the total E. The 46-amino acid from the 138-bp HindIII-EcoRI fragment (7Goswami A. Chatterjee S. Sharma S. Mol. Biochem. Parasitol. 1996; 82: 117-120Google Scholar) the expected Polyclonal antibodies, with a titer of were raised against and used for and growth inhibition This antibody immunoprecipitated a single 38-kDa phosphoprotein from the and 32P-labeled P. falciparum proteins antibodies such as sera and polyclonal antibodies raised against GST not show band not that the human immune sera used for the differential the PfP0 protein domain in the λPf4 expression human immune sera was affinity-purified using λPf4 expression clone and used for This also the 38-kDa P0 protein from parasite proteins immune sera affinity-purified against the phage not show reactivity with the parasite blots of the recombinant protein with immune and patient sera samples that the recombinant protein was detected only by immune sera samples and not by patient sera samples not antibodies raised against were used for studies with different stages A as well as gametocytic stages of the parasite and sera and polyclonal antibodies against GST were used as The antibodies with the antibody with all the different stages of the In of the gametocytic of domains were the of this is not The of the was to be at the as with parasite do show antibody reactivity to the on blots not on an parasite related to it was to the as by with and markers and S. It has been that the protective human immune sera vitro growth of P. falciparum only in the presence of H. Attanath P. Sabchareon A. Chongsuphajaisiddhi T. Druihle P. J. Exp. Med. 1990; 172: 1633-1641Google Scholar). However, these were performed with of antibody the growth of P. falciparum in the presence or of in vitro inhibition studies using synchronized parasites from the stages were performed The antibodies raised against completely inhibited the growth of the parasites a sera and sera raised against an P. falciparum protein inhibition in the growth of the parasites. IgG and with sera raised against GST protein growth not were performed for each and the an of these The presence of were found to the parasite the inhibition of growth of P. falciparum by the antibody was of the presence of The growth inhibition studies were by at cells for each time The was performed the because the of by the in the with a synchronized and the different substages at time show the of the substages of the parasite with with synchronized stages were the and h the different substages of the parasite were In the presence of the the parasites the and stages in about and was an in the the invasion of cells However, in the presence of the parasites from to and of cells not as the number of stages to h in The same was for the of the parasite substages of the presence or not of we the of the gene of PfP0 from the strain of P. falciparum (7Goswami A. Chatterjee S. Sharma S. Mol. Biochem. Parasitol. 1996; 82: 117-120Google Scholar). The largest cDNA clone a an of at kb, and a of it a total of at in The with this It is not the of size is a or a The are twice as in the that the expression of this gene be The molecular of the gene is The sera raised against the recombinant protein domain of the P0 gene a 38-kDa P. falciparum protein from both and 32P-labeled which with the expected size of the P0 The immune sera also the same size studies that the antibodies raised against the recognize a protein of the that it is indeed a and that antibodies against are present malaria-immune people and not in The P0 protein was first as a of the P family of proteins studies using antibodies against the conserved carboxyl-terminal (9Towbin H. Ramjoue H.-P. Kuster H. Liverani D. Gordon J. J. Biol. Chem. 1982; 257: 12709-12715Google Scholar, K. Skelley S. Parnassa A. Moller W. Waleed D. Weissbach H. Nathan B. Proc. Natl. Acad. Sci. U . S . A. 1986; 38: 7419-7423Google Scholar). studies have been performed to that P1 and P2 are phosphoproteins T. Remacha M. Ballesta J.P.G. J. Biol. Chem. 1993; Scholar), has been that P0 is a cultures of yeast cells a large number of S. J. Biol. Chem. Scholar), of which the protein has been to be the P0 protein (9Towbin H. Ramjoue H.-P. Kuster H. Liverani D. Gordon J. J. Biol. Chem. 1982; 257: 12709-12715Google Scholar). Thus, for the first studies using antibodies, we are that P0 is indeed a phosphoprotein. against the conserved carboxyl-terminal domain of the P proteins have been found in 10–15% of patients with an autoimmune systemic lupus erythematosus (15Elkon K. Skelley S. Parnassa A. Moller W. Waleed D. Weissbach H. Nathan B. Proc. Natl. Acad. Sci. U . S . A. 1986; 38: 7419-7423Google Scholar). have been as a for the these has also been documented against P0 protein in Chagas' disease (16Levin M.J. Vazquez M. Kaplan D. Schijman A.G. Parasitol. Today. 1993; 9: 381-384Google Scholar) and leishmaniasis (17Skeiky Y.A.W. Benson D.R. Elwasila M. Badaro R. Burns Jr., J.M. Reed S.G. Infect. Immun. 1994; 62: 1643-1651Google Scholar). However, in each of these the reactivity has been against the carboxyl-terminal The systemic lupus erythematosus patient sera have been for reactivity against the amino-terminal domain of the P2 protein not show (15Elkon K. Skelley S. Parnassa A. Moller W. Waleed D. Weissbach H. Nathan B. Proc. Natl. Acad. Sci. U . S . A. 1986; 38: 7419-7423Google Scholar). The reactivity of the malaria-immune sera to P0 is of immune sera (6Lobo C.A. Kar S.K. Ravindran B. Kabilan L. Sharma S. Infect. Immun. 1994; 62: 651-656Google Scholar) and is the which is the amino-terminal domain of the PfP0 protein (7Goswami A. Chatterjee S. Sharma S. Mol. Biochem. Parasitol. 1996; 82: 117-120Google Scholar). this immune P0 in malaria-immune appears to be different from that for the systemic lupus erythematosus The of the passive of the parasites in patients with IgG of malaria-immune is not The IgG be and parasite domains, or it be to the cells and cellular of the immune system to the parasites. The has been by studies in which a pool of immune human sera inhibited the in vitro growth of P. falciparum only in the presence of H. Attanath P. Sabchareon A. Chongsuphajaisiddhi T. Druihle P. J. Exp. Med. 1990; 172: 1633-1641Google Scholar). However, these were performed with of antibody the growth of P. of the presence or of a of parasite The the inhibition of the P. falciparum growth with antibodies is given the of the PfP0 The inhibition is to be to the inhibition of falciparum ribosomal activity as the from the to the stages were in the presence of antibody a of h. The growth inhibition by IgG to at the the do indicate a in to the of P0 in the and gametocytic is a of this protein in the is yet to be the presence of an antigenic related to the carboxyl-terminal of P0 protein has been to the of and human cells E. M. R.D. M. J. 1992; Scholar). this of P0 protein be and role it play in the erythrocyte invasion by the malarial parasite to be We are to Nirbhay Kumar and for with the of and We for the FCK2 strain of P. falciparum and a for with the expression of fusion