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Molecular & Biochemical Parasitology 119 (2002) 121– 124
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Short communication
Surface expression of the conserved ribosomal protein P0 on
parasite and other cells
Subhash Singh, Alfica Sehgal, Sanjeev Waghmare, Tirtha Chakraborty,
Arunava Goswami 1, Shobhona Sharma *
Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India
Received 9 June 2001; received in revised form 6 September 2001; accepted 12 September 2001
Keywords: Ribosomal phosphoprotein P0; Mammalian cells; Toxoplasma; Yeast; Surface expression
Invasion-blocking antibodies against the ribosomal
phosphoprotein P0 of the human malarial parasite
Plasmodium falciparum (PfP0) have been identified
through a differential immunoscreen using immune and
patient sera from malaria endemic regions of Eastern
India [1]. Purified IgG from rabbit sera, raised against
different domains of the PfP0 protein, show no effect
on the intraerythrocytic stages of Plasmodium falciparum in culture, but inhibit the invasion of erythrocytes by merozoites in a concentration dependent
manner [2,3]. The same antibodies against the PfP0
protein protect mice against challenge with a virulent
strain of Plasmodium yoelii [4]. We have recently
demonstrated this protein to be present on the surface
of merozoites and gametocytes of Plasmodium [3,4].
The phosphoprotein P0 is highly conserved across eukaryotic organisms [5], and is related to the family of
phosphoproteins P1 and P2, due to the highly homologous carboxy-terminal 22 amino acid domain [6].
The ribosomal function of P0 is mediated through a
pentameric P12·P0·P22 complex, which forms the stalk
of the large ribosomal subunit at the GTPase center [7].
Abbre6iations: GST, glutathione-S-transferase; IPTG, isopropylthio-galactosidase; PfP0, Plasmodium falciparum phosphoriboprotein
P0; PfP0N, amino-terminal domain of PfP0; PfP0C, carboxy-terminal
domain of PfP0.
* Corresponding author. Tel: + 91-22-215-2971/2979 x 2570; fax:
+91-22-215-2110/2181.
E-mail address: [email protected] (S. Sharma).
1
Present address: Lecturer, Agricultural Science Unit, Indian
Statistical Institute, 203, B.T. Road, Calcutta 700 035, India.
P0 interacts with the elongation factor eEF2 [8], and is
essential for the ribosomal activity and cell viability in
yeast [9]. Through deletion analysis of the P0 protein,
the ribosomal function has been mapped to amino acid
position 185 –230 in yeast P0 [10].
Since P0 is a very conserved protein [5], we wondered
whether the surface expression of P0 is an exclusive
property of Plasmodium cells, or if it is a general
property of the P0 protein in other organisms as well.
The presence of this protein on the host cell surface will
have serious implications with the usage of this protein
in a malaria vaccine. This question also has a direct
bearing on the mechanism of translocation of this
protein to the surface, as to whether it is a unique
phenomenon for Plasmodium. Therefore, we decided to
investigate the presence of this protein on different cell
types. In this paper we report the localization of P0
protein on the surface of another Apicomplexan parasite Toxoplasma, yeast and mammalian cell lines using
mono-specific cross-reactive anti-PfP0 antibodies.
In order to test for cross-reactivity of anti-PfP0 sera
with different cell types, Western Blot analysis was
carried out with total protein extract of these cell types.
Anti-PfP0 antisera were raised in rabbits against recombinant GST-fusion proteins, PfP0N (amino acid 17–61)
and PfP0C (amino acid 61–316), and purified as described earlier [3]. Specific single band reactivities of 38,
33, 34 and 38 kD proteins were observed with Toxoplasma gondii, yeast (Saccharomyces cere6isiae, strain
EG103), Chinese Hamster Ovary (CHO) cells and human leukocyte protein preparations respectively, using
0166-6851/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 6 8 5 1 ( 0 1 ) 0 0 3 9 4 - 2
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S. Singh et al. / Molecular & Biochemical Parasitology 119 (2002) 121–124
both anti-PfP0N and anti-PfP0C antibodies (Fig. 1).
These molecular weights are the expected molecular
weights of the respective P0 proteins. Since both antiPfP0N and anti-PfP0C sera identify the same single
protein band in each of the organisms tested, these results
show that the sera are specifically recognizing the homologous P0 proteins.
Toxoplasma gondii tachyzoites, yeast spheroplasts,
CHO cells, the human cell lines K562 (an erythroleukemic cell line), U937 (a lymphoma cell line),
Daudi (a Burkitt lymphoma cell line) and SCC (an
epithelial cell line), and peripheral human blood cells
were subjected to solution immunofluorescence assay
(SIFA). Toxoplasma tachyzoites were harvested by filtering the infected HFF cell lysate through a 3 mm filter and
used for SIFA [11]. Yeast cells were grown with 1%
glucose and harvested at an OD600 value of 1– 2 ( 107
cells). Yeast spheroplasts were prepared by treatment
with zymolyase (Seikagaku Corp., Japan) [12], and used
for SIFA. CHO cells were subcultured in 35 mm2 Petri
plates and the adherent CHO cells were used for SIFA
assay after 24 h of growth. A distinct surface staining was
observed with every tachyzoite of Toxoplasma, very
similar to that shown by the surface marker SAG1 (gift
from David Roos) (Fig. 2A). Yeast spheroplasts also
showed distinct surface reactivity with anti-PfP0 sera
samples, but the number of cells that reacted were
30 – 50% of the total cell population (Fig. 2B). With CHO
cells, the number of cells found to be reactive was about
2 –10% (Fig. 2C). In some of the reactive CHO cells, the
surface reactivity could be detected at the membrane
extensions quite distinctly (Fig. 2C, right panel). Human
peripheral blood cells showed no SIFA activity (Fig. 2D).
Approximately 100,000 total blood cells were scanned for
Fig. 1. Western blot analysis of total protein extracts from different
organisms. Lane A contains: 106 cells of Toxoplamsa tachyzoites; B:
108 cells of Saccharomyces cere6isiae; C: 106 cells of CHO cells; and
D: 106 cells of human white blood cells. In each panel, the left-hand
strip was treated with preimmune serum, the middle strip with
anti-PfP0N and the right-hand strip with anti-PfP0C antisera. In each
case, the serum was used at a dilution of 1:100, except for strips of
panel A, where the sera were used at 1:200 dilution. Positions of
molecular weight standards used are shown on the left. The arrowheads indicate the P0 bands.
each antibody tested, but no reactivity was detected with
either red cells or leukocytes. Red cells do not possess any
P0 protein, as fixed red cells do not show any IFA
reactivity [2]. To test the leukocytes specifically, 105 cells
of the buffy coat samples were collected from three
donors and each sample was tested with the antibodies,
and no surface reactivity was detected in any of these
samples (data not shown). Each of these donors was
healthy local (area of low malaria incidence) individual.
Control monoclonal antibodies against glycophorin A
(Pharmingen, USA) and HLA Class I (gift from S.
Chiplunkar) reacted as expected with red cells and
leukocytes (Fig. 2D). However, even though peripheral
blood cells did not show any surface reactivity, hemopoietic cell lines K562, U937 and Daudi cells showed distinct
cell staining (Fig. 2E). The epithelial cell line SCC also
showed surface staining (Fig. 2E, d). The frequency of
cells staining for these cell lines was low, varied with cell
lines, and with different experiments, but was always
within 2–5%. For each cell type, SIFA experiments were
performed at least three times. Preimmune sera did not
show any fluorescence activity with any of these different
cell types used. The numbers and the patterns of reactivity were found to be similar irrespective of whether
anti-PfP0N or anti-PfP0C serum sample was used.
The PfP0N and PfP0C possess no overlapping amino
acid sequence. We have shown earlier that the antiPfP0N and anti-PfP0C antibodies do not cross-react with
each other [3]. On Western Blots both these sera light up
specifically only the homologous P0 protein. Since both
anti-PfP0N and anti-PfP0C antisera react to the cell
surfaces with a similar pattern, it is unlikely that the
surface reactivity recorded is due to non-specific P0-like
cross-reactive domains located on the surface of the
parasite.
About 10–15% of patients suffering from an autoimmune disorder, Systemic Lupus Erythematosus (SLE),
possess autoantibodies against the conserved carboxyterminal 22 amino acids of the P-proteins [13]. SLE serum
sample has been used for immunoprecipitation study of
P. falciparum protein extract, and distinct cross-reactivity
with PfP0 has been observed [14]. It has been reported
earlier that SLE sera do not react to the amino terminal
domain of the P-proteins [13]. We have also recently
reported that 10% of SLE patient sera samples cross-react with P. falciparum P0 protein, but only with PfP0C
and not PfP0N protein [15]. We have shown that these
PfP0C positive SLE sera samples inhibit the growth of
P. falciparum through specific reactivity with PfP0C [15].
These results confirm the conserved structures of PfP0
and human P0.
So far only one report documents the surface localization of human P0-like protein, and this has been reported
on the plasma membrane of human HepG2 and neuronal
cell lines using purified SLE sera [16]. Results presented
in this paper show that the surface expression of P0 is
S. Singh et al. / Molecular & Biochemical Parasitology 119 (2002) 121–124
123
Fig. 2. Solution immunofluoresence assay (SIFA) of different cell types using different antisera, used at 1:100 dilutions (3); followed by secondary
antibody treatment with goat-anti-rabbit IgG, or goat anti-mouse IgG, conjugated with FITC or rhodamine (Boehringer Mannheim, Germany)
diluted in RPMI 1640 medium. Panels A: Toxoplasma tachyzoites; B: yeast spheroplasts; C: CHO cells; D: human blood cells; E: human cell lines
(a: K562 cell; b: Daudi cells; c: U937 cell and d: SCC cell). In each panel ‘Br’ shows the bright field of the corresponding dark field shown next
to it. Additional dark fields are shown on the right-hand side in panels B and C. Treatment with N: anti-PfP0N antibodies; C: anti-PfP0C
antibodies; PI: Preimmune sera; and surface markers M: anti-SAG1 antibody; RM: anti-glycophorin A antibody; LM: anti-HLA Class I antibody.
In panel A, the lowest row shows Toxoplasma tachyzoites double stained with anti-PfP0N antibodies and anti-SAG1 antibodies. In panels C and
D: the arrows indicate the SIFA active subset of CHO cells and leukocytes, respectively. The bar corresponds to 5 mm.
regulated. While every Plasmodium [3] and Toxoplasma
cell showed surface staining, the frequency for surface
reactivity was 30– 50% for yeast cells, and as low as
2–10% in mammalian cell lines. Peripheral blood cells
(red cells and leukocytes) did not show any surface
reactivity. The reason for this variation in SIFA reactivity is unclear. The low frequency of positively reacting
cells is unlikely to be dead cells in the case of leukocytes,
CHO, K562 and Daudi cell lines as we routinely performed the Trypan Blue exclusion assay to assess cell
viability. We have also performed fixed sample analysis
of these cell types, and all cells show the same patterns
of reactivity with P0 and other markers such as betatubulin and ER-marker. Whether the SIFA reactive cells
belong to an early stage of apoptosis remains to be tested.
From the cross-reactivity of anti-PfP0 antibodies it is
clear that large protein domains of PfP0 are not suitable
for a malaria vaccine. For vaccination purposes, smaller
stretches of PfP0 with no cross-reactivity with the human
P0 protein need to be identified. Passive immunizations
with purified IgG obtained from immune adults, however, have been performed earlier on malaria patients
with no apparent ill effects [17,18]. The experimental
mice, which recovered completely in our passive immunization experiments, were healthy with no pathological
symptoms of SLE [4]. The observation that peripheral
blood cells do not show surface expression of P0 protein
is an encouraging observation for passive immunotherapy. However, other human tissues also need to be
examined for surface PfP0 expression. A large number
(87%) of the malaria immune adults from Eastern
-India possessed anti-PfP0 antibodies, but did not
exhibit any symptoms of SLE as documentedthrough
extensive questionnaire [1]. Indeed, it has been noted
earlier that the proportion of SLE patients in malaria
endemic areas is lower than that in areas with no malaria
[19].
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S. Singh et al. / Molecular & Biochemical Parasitology 119 (2002) 121–124
The biology of the P0 protein is complex and intriguing. It is apparent that it plays an essential role in the
ribosomes [7,8] and also exhibits apurinic/apyrimidinic
endonuclease activity in the nucleus [20]. Unlike most
other ribosomal proteins, the level of P0 mRNA and
protein are regulated, as has been shown in carcinogenic and apoptotic cells [21,22]. In flies, P0 also plays
regulatory roles [23,24]. The ribosomal P2 protein homologue, L12, of Neisseria gonorrhoeae has recently
been shown to play a role in gonococcal invasion of
human endometrial Hec1B cells [25]. How P0 protein
gets transported on the surface remains a question, as
the deduced P0 protein sequences do not possess any
canonical signal sequence or transmembrane motif. It is
possible that the P0 protein is secreted out in a complex
with other protein(s). Future studies centered on cell
biological analysis of the membrane transportation of
P0, along with attempts to map the various functions of
P0 on sub-domains of the protein, will help in understanding the diverse functions of this protein.
Acknowledgements
We are indebted to David Roos (University of Pennsylvania, Philadelphia, PA) for antibodies to SAG1
antigen, and for the methodology with Toxoplasma. We
also thank Shubha Chiplunkar (Cancer Research Institute, Mumbai) for providing us with the anti-HLA
antibodies and the human cell lines and Satyajit Mayor
(National Centre for Biological Sciences, Bangalore)
for the CHO cells.
References
[1] Lobo CA, Kar SK, Ravindran B, Kabilan L, Sharma S. Novel
proteins of Plasmodium falciparum identified by differential immunoscreening using immune and patient sera. Infect Immun
1994;62:651 – 6.
[2] Goswami A, Singh S, Redkar VD, Sharma S. Characterization
of P0, a ribosomal phosphoprotein of Plasmodium falciparum. J
Biol Chem 1997;272:12138 –43.
[3] Chatterjee S, Singh S, Sohoni R, et al. Characterization of
domains of the phosphoriboprotein P0 of Plasmodium falciparum. Mol Biochem Parasitol 2000;107:143 –54.
[4] Chatterjee S, Singh S, Sohoni R, et al. Antibodies against the
ribosomal phosphoprotein P0 of Plasmodium falciparum protect
mice against challenge with P. yoelii. Infect Immun
2000;68:4312 – 8.
[5] Goswami A, Chatterjee S, Sharma S. Cloning of a ribosomal
phosphoprotein P0 gene homologue from Plasmodium falciparum. Mol Biochem Parasitol 1996;82:117 –20.
[6] Rich BE, Steitz JA. Human acidic ribosomal phosphoproteins
P0, P1 and P2: Analysis of cDNA clones, in vitro synthesis and
assembly. Mol Cell Biol 1987;7:4065 – 74.
[7] Uchiumi T, Kominami R. Direct evidence for interaction of the
conserved GTPase domain within 28S RNA with mammalian
ribosomal acidic phosphoproteins and L12. J Biol Chem
1992;267:19179 – 85.
[8] Justice MC, Ku T, Hsu MJ, et al. Mutations in ribosomal
protein L10e confer resistance to the fungal-specific eukaryotic
elongation factor 2 inhibitor sordarin. J Biol Chem
1999;274:4869 – 75.
[9] Santos C, Ballesta JPG. Ribosomal protein P0, contrary to
phosphoproteins P1 and P2, is required for ribosome activity
and Saccharomyces cere6isiae viability. J Biol Chem
1994;269:15689 – 96.
[10] Santos C, Ballesta JPG. The highly conserved protein P0 carboxyl end is essential for ribosome activity only in the absence of
proteins P1 and P2. J Biol Chem 1995;270:20608 – 14.
[11] Roos DS, Donald GK, Morrissette NS, Moulton LC. Molecular
tools for genetic dissection of the protozoan parasite Toxoplasma
gondii. Methods Cell Biol 1994;45:27 – 63.
[12] Pringle JR, Adams AEM, Drubin DG, Haarer BK. Immunofluorescence methods for yeast. Methods Enzymol
1991;194:565 – 602.
[13] Elkon K, Skelly S, Parnassa A, Moller W, Danho W, Weissbach
H, Brot N. Identification and chemical synthesis of a ribosomal
protein antigenic determinant in systemic lupus erythematosus.
Proc Natl Acad Sci USA 1986;83:7419 – 23.
[14] Francoeur AM, Peebles CLJ, Heckman K, Lee JC, Tan EM.
Identification of ribosomal protein autoantigens. J Immunol
1985;135:2378 – 84.
[15] Singh S, Chatterjee S, Sohoni R, Badakare S, Sharma S. Sera
from lupus patients inhibit growth of Plasmodium falciparum in
culture. Autoimmunity 2001;33:253 – 63.
[16] Koren E, Reichlin MW, Kosec M, Fugate RD, Reichlin M.
Autoantibodies to the ribosomal P proteins react with a plasma
membrane-related target on human cells. J Clin Invest
1992;89:1236 – 41.
[17] Cohen S, Mcgregor IA, Carrington SP. Gammaglobulin and
acquired immunity to human malaria. Nature 1961;192:733 –7.
[18] Sabchareon A, Burnouf T, Quattara D, et al. Parasitological and
clinical response to immunoglobulin administration in P. falciparum malaria. Am J Trop Med Hyg 1991;45:297 – 308.
[19] Greenwood BM. Autoimmune disease and parasitic infections in
Nigerians. Lancet 1968;2:380 – 2.
[20] Yacoub A, Kelley MR, Deutsch WA. Drosophila ribosomal
protein P0 contains apurinic/apyrimidinic endonuclease activity.
Nucleic Acids Res 1996;24:4298 – 303.
[21] Kondoh N, Wakatsuki T, Ryo A, et al. Identification and
characterization of genes associated with human hepatocellular
carcinogenesis. Cancer Res 1999;59:4990 – 6.
[22] Brockstedt E, Rickers A, Kostka S, et al. Identification of
apoptosis-associated proteins in a human Burkitt lymphoma cell
line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by
caspase 3. J Biol Chem 1998;273:28057 – 64.
[23] Frolov MV, Birchler JA. Mutation in P0, a dual function
ribosomal protein/apurinic/apyrimidinic endonuclease, modifies
gene expression and position effect variegation in Drosophila.
Genetics 1998;150:1487 – 95.
[24] Craig TL, Denlinger DL. Sequence and transcription patterns of
60S ribosomal protein P0, a diapause-regulated AP endonulease
in the flesh fly, Sarcophaga crassipalpis. Gene 2000;255:381 –8.
[25] Spence JM, Clark VL. Role of ribosomal protein L12 in Gonococcal invasion of Hec1B cells. Infect Immun 2001;68:5002 –10.