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. 2015 Jan 2;179(2):236–244. doi: 10.1111/cei.12460

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

H Sato *,#, M Onozuka †,#, A Hagiya ‡, S Hoshino †, I Narita *, T Uchiumi †
PMCID: PMC4298401  PMID: 25255895

Abstract

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.

Keywords: autoantibody, monoclonal anti-P, ribosomal antigenic complex, SLE

Introduction

Systemic lupus erythematosus (SLE) is a clinically diverse autoimmune disease characterized by the expression of autoantibodies directed against various nuclear and cytoplasmic antigens 1,2. Some of these autoantibodies have been considered to play a pathogenic role in tissue injury observed in SLE. Although the molecular mechanism of autoantibody production remains poorly understood, several previous studies using SLE patients and Murphy Roths large (MRL)/lpr mice suggested that the autoimmune response and antibody production are antigen-driven 3–6. The mechanism of autoimmune development has been studied frequently using MRL/lpr mice, which produced autoantibodies with specificities similar to patients with SLE 7–9. Therefore, MRL mice and antibodies produced in these mice are useful tools for investigating the molecular basis of antigen-driven immune response in SLE patients.

An antibody to ribosomal P protein (anti-P), which is a highly specific marker for SLE in humans 10,11, is one of these autoantibodies and this antibody is also detected in the MRL mouse 9. Results obtained from previous studies have suggested that the presence of anti-P correlates frequently with lupus psychosis 12. Later studies have also shown an association between the occurrence of kidney and liver diseases in SLE patients with anti-P antibodies 13,14. An anti-P epitope was found to lie within the conserved 22-amino acid long C-terminal tail sequence, which is shared by three phosphoproteins, P0, P1 and P2 15. Recent biochemical and structural studies have characterized the structural features of the P0-P1-P2 complex in detail (see Fig. 1). Accordingly, P1 and P2 have been shown to form heterodimers via their N-terminal domains; two P1-P2 heterodimers bind to the C-terminal helices of P0 through their N-terminal domains and form a pentameric complex, P0(P1-P2)2 16,17; the C-terminal halves of P1 and P2 are unstructured and flexible, and the C-terminal tail containing the anti-P epitope extends up to 125 Å away from the N-terminal domain 18; the P0(P1-P2)2 complex binds to a specific domain of 28S rRNA 19 through the N-terminal domain of P0 20. It is noteworthy that sera obtained from some SLE patients, in addition to containing an anti-P antibody, contained an anti-RNA autoantibody (anti-28S) which binds specifically to the P0(P1-P2)2-binding site on the 28S rRNA 21–23.

Figure 1.

Figure 1

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Schematic model depicting the nature of the ribosomal antigenic complex. The N-terminal domains of P1 and P2 are shown using black and white boxes, respectively. They form heterodimers by binding to each other and two heterodimer pairs bind to the C-terminal helix region of P0 to form a pentameric complex. The C-terminal tails of all three P proteins (P0, P1 and P2) are flexible, which are indicated using dotted lines. The anti-P epitope, which is believed to be located at the C-termini of P0/P1/P2, is shown as a grey oval.

The ribosomal P0(P1-P2)2 complex is one of the autoantigens that is best characterized structurally and functionally 24–26. It has been shown previously that the C-terminal tail (wherein lies the anti-P epitope), which is present in multiple copies within this antigenic complex, is flexible and exists in extended form 18. Therefore, the P0(P1-P2)2 complex seems to be a suitable material for exploring the relationship between the structural features and antigenic stimulus in SLE. To achieve this goal, it would be necessary to establish conditions for reconstituting the antigenic P0(P1-P2)2 complex and immunize animals with this reconstituted complex for generating anti-P antibody. In this study, we have successfully reconstituted such a complex composed of human P0, phosphorylated P1 and P2, and a 28S rRNA fragment; then, using this reconstituted complex, we immunized MRL mice and eventually derived many hybridoma cells producing anti-P monoclonal antibody. We have subsequently identified two types of anti-P monoclonal antibodies (mAbs): one type required phosphorylation of the C-terminal sequence of P1/P2 to react strongly with the epitope, whereas the other type recognized the epitope irrespective of the phosphorylation state of the C-terminal sequence; both, however, required the last three C-terminal amino acid residues to be present in order for the epitope to be recognized. These results suggest that multiple copies of C-terminal sequence, particularly those three terminal amino acid residues, present in the P0(P1-P2)2 complex, could serve as the antigenic stimulus in SLE. The immunization system we used here seems to be a useful tool for investigating the underlying mechanism of anti-P production.

Materials and methods

Ribosomal P0, P1 and P2 proteins and 28S rRNA fragment

The coding sequences of human ribosomal proteins P0, P1 and P2 were amplified by polymerase chain reaction (PCR) using primers (shown in Supporting information, Table S1) and plasmids containing individual genes as templates. Amplified P0 and P1 coding sequences were cloned into the Escherichia coli expression vector pET3a (Novagen, Darmstadt, Germany), and the amplified P2 coding sequence was cloned into the E. coli expression vector pGEX-6p-1 (GE Healthcare, Amersham, UK), which allowed adding a glutatione S-transferase (GST)-tag to the N-terminal end of P2. After expressing these proteins in the E. coli BL21 strain, P0 and P1 were purified first using a CM52 (GE Healthcare, Tokyo, Japan) column, and then using a DE52 (GE Healthcare, Japan) column following an earlier protocol as described for the purification of silkworm P0 and P1 27. Conversely, P2 fused to a GST-tag at the N-terminal end was purified using a glutathione-sepharose 4B (GE Healthcare, Japan) column and the purified fusion protein was then treated with HRV-3c protease (Takara, Ohtsu, Japan) to remove the GST-tag following a protocol provided by the manufacturer. P1 and P2 were phosphorylated in vitro using casein kinase II (New England Biolabs, Ipswich, MA, USA), as described previously 16. A human ribosomal 28S RNA fragment (residues 1922–2020) was transcribed using SP6 RNA polymerase (Takara, Ohtsu, Japan) in vitro and the transcribed RNA fragment was purified as described previously 28.

Reconstitution of ribosomal antigenic complex

Three purified P proteins (namely, P0, phosphorylated P1 and phosphorylated P2) were mixed in a ratio of 1 : 3 : 3 in 7 M urea, and the P0-P1-P2 complex was reconstituted by removing the urea from this mixture by dialysis, as described previously 16. After incubating the P0-P1-P2 complex with an excess amount of 28S rRNA fragment at 37°C for 5 min, the protein-RNA complex was isolated by ultracentrifugation on a 15-30% sucrose density gradient, which was prepared in a buffer containing 20 mM Tris-HCl (pH 7·6), 5 mM MgCl2 and 100 mM KCl. The centrifugation step was carried out at 96 000 g for 20 h at 4°C using a Hitachi P28-S rotor. The fraction containing the P0-P1-P2-RNA complex was collected, concentrated and finally dialysed against the same buffer.

Immunization, hybridoma production and cloning

MRL/lpr (MRL/MpJJmsSlc-lpr/lpr) mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Four 7-8-week-old MRL/lpr female mice were immunized by injecting the reconstituted ribosomal P protein-RNA complex (20-50 μg/mouse/per round) without any adjuvant into the footpads of each mouse. Lymphocytes obtained from the inguinal lymph nodes of immunized mice were fused with myeloma cells (P3-X63-Ag8-U1) using 50% polyethylene glycol 4000 (Merck, Darmstadt, Germany). The fusion mixtures were plated onto 96-well culture plates (Costar, Corning, NY, USA). Hybridoma culture fluids were screened against the P0-P1-P2-RNA complex that was used for the immunization by a standard enzyme-linked immunosorbent assay (ELISA) 29, and the positive clones were recloned by limiting dilution. Experiments in the immunization of animals, cell fusion and hybridoma cloning described above were consigned to Oriental Yeast Co. Ltd (Tokyo, Japan). All animal experiments were performed according to the guidelines of the Ethical Committee for Animal Experiments at Oriental Yeast Co Ltd.

Specificity of antibody

Culture fluid from each hybridoma clone was analysed by an immunoblot assay for determining the specificity of the antibody. For this purpose, total 80S ribosomal proteins or isolated P1 and P2 proteins were used as the antigenic protein samples in the immunoblot assay 30. Immunoreactivity of the antibody for the rRNA fragment was assayed by immunoprecipitation using a 32P-labelled synthetic RNA fragment, as described previously 23.

Preparation of ascites, monoclonal antibodies and Fab fragments

Preparation of ascites fluids was consigned to Oriental Yeast Co. Ltd using pristine-primed BALB/c nu/nu nude mice (Charles River, Yokohama, Japan). Monoclonal antibodies were purified from the ascites fluid by affinity chromatography using protein-A sepharose (GE Healthcare, Japan). Subtyping of purified mAbs was carried out using a mouse monoclonal antibody isotyping kit (Roche, Indianapolis, IN, USA). Fab fragments were obtained by papain (Sigma, St Louis, MO, USA) digestion, followed by removal of the undigested immunoglobulin (Ig)G and Fc fragments by gel filtration using HiLoad 16/60 Superdex 75 (GE Healthcare, Japan). The purity of Fab fragments was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Native polyacrylamide gel electrophoresis

Formation of the protein complex was confirmed by performing electrophoresis on 6% polyacrylamide (acrylamide : bisacrylamide ratio 39 : 1) native gel using a buffer system containing 5 mM MgCl2, 50 mM KCl and 50 mM Tris-HCl (pH 8·0), as described previously 16. The gel was stained with Coomassie Briliant Blue.

Ribosomes, eukaryotic elongation factor 2 (eEF-2) and GTPase activity assay

Ribosomes from rat liver 31, silkworm (Bombyx mori) 32 and brine shrimp (Artemia salina) 33 were prepared as described. Elongation factor eEF-2 was purified from the pig liver following the procedure described by Iwasaki . 33. Ten μl of brine shrimp 80S ribosome (2·5 pmol) in a buffer containing 2·5 mM MgCl2, 50 mM KCl and 20 mM Tris-HCl (pH 7·6) was preincubated in the presence or absence of anti-P IgG (0-30 μg) at 37°C for 10 min. The reaction was initiated by mixing another 10 μl of the same buffer that was supplemented with 0·5 μg eEF-2 and 3 nmol [γ-32P] guanosine triphosphate (GTP). The reaction was allowed to proceed for 10 min at 37°C, and the reaction was then stopped by adding the reaction mixture to 750 μl of a solution containing 5% activated charcoal and 50 mM sodium phosphate (pH 7·0) kept on ice. Unhydrolyzed GTP, which was adsorbed onto the charcoal, was removed by centrifugation as described previously 34, and the liberated phosphate was determined by counting the radioactivity in a Tri-Carb Liquid Scintillation Counter (Perkin-Elmer Inc., Waltham, MA, USA).

Serum containing anti-P antibody

Serum from an SLE patient that contained anti-P reactivity was obtained through the Niigata University Hospital, Japan. Written informed consent was obtained from the SLE patient.

Synthetic peptides

Synthesis of peptides corresponding to 19-22 amino acids of the C-terminal sequence shared by all three P proteins (P0, P1 and P2) was carried out by the Hokkaido System Science Co. Ltd (Sapporo, Japan).

Results

Recent biochemical and structural studies have shed lights on structural features of the anti-P autoimmune target, which is composed of ribosomal proteins P0, P1 and P2. The C-terminal tails of these three proteins share a common sequence, which could serve as the anti-P epitope. As depicted in Fig. 1, these three proteins form a pentameric complex, P0(P1-P2)2, in which the five C-terminal tails could move freely over a wide area, and the N-terminal domain of P0 binds to the 28S rRNA 19. We assumed that this unique structural property of the P0(P1-P2)2 complex might provide immunogenic stimulus for the anti-P production. To test this possibility, we have reconstituted the P0(P1-P2)2-RNA complex in vitro and injected the reconstituted complex into the MRL/lpr mice to induce antibody production. For this purpose, first we expressed human P0, P1 and P2 individually in E. coli cells and purified them (Fig. 2a). Then, after treatment of P1 and P2 with casein kinase II, the phosphorylated P1 and P2 (P1P and P2P, respectively) were mixed with P0 to form the P0(P1-P2)2 complex, which was observed as a distinct band after electrophoresis on a native polyacrylamide gel (Fig. 2b, lane 5). Upon addition of the 28S rRNA fragment to this complex, the band moved faster on the gel than the complex itself (Fig. 2b, lane 6), suggesting the formation of a stable P0(P1-P2)2-RNA complex. The P0(P1-P2)2-RNA complex was subsequently purified by sucrose density gradient centrifugation and the purified complex was then injected into the MRL/lpr mice. We did not use an adjuvant for this immunization, to avoid any possible disruptive effect that the adjuvant might have on the higher order P0(P1-P2)2-RNA structure.

Figure 2.

Figure 2

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Preparation of ribosomal antigenic complex. (a) Purified ribosomal proteins (2·5 μg each) were separated by gel electrophoresis on a 17% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel was then stained with 0·4 % Coomassie Briliant Blue. Lane 1, P0; lane 2, P1 and lane 3, P2. (b) Native gel electrophoresis of protein samples: 300 pmol each of purified human P0 (lane 1), P1 (lane 2), and P2 (lane 3); reconstituted P1-P2 dimer formed following incubation of 300 pmol each of P1 and P2 (lane 4); reconstituted P0-P1-P2 complex formed following incubation of 100 pmol of P0 and 300 pmol each of P1 and P2 (lane 5); reconstituted P0-P1-P2-rRNA complex formed following incubation of 100 pmol P0, 300 pmol P1, 300 pmol P2 and 300 pmol rRNA fragment (lane 6). Conditions used for the gel electrophoresis are described in Materials and methods. The gel was stained with Coomassie Brilliant Blue.

By screening culture supernatants of hybridoma cells using ELISA, we cloned 46 hybridoma cells that produced antibodies reactive to the P0(P1-P2)2-RNA complex. Specificities of individual mAbs were analysed by an immunoblot assay using total ribosomal proteins as well as purified P0, P1 and P2 as antigens. We found that antibodies produced from 34 of the 46 hybridoma clones recognized all three purified proteins (i.e. P0, P1 and P2) on the immunoblot (Table 1), and their reaction patterns were same as that obtained using the serum from the SLE patient (Fig. 3), suggesting that each one of these clones produced a monoclonal anti-P antibody which recognized the C-terminal sequence common to all three P proteins. Two hybridoma clones produced P0-specific mAbs, whose epitopes appeared to lie in a region different from the C-terminal sequence of P0 (Supporting information, Fig. S1). None of the hybridoma clones produced any mAb that would recognize P1 or P2 specifically (Table 1). We also failed to find any hybridoma cell line that produced a mAb against the rRNA fragment (anti-28S). Taken together, these results suggest that the immunization protocol used in this study preferentially induced the production of anti-P antibodies against the C-terminal sequence common to all three P proteins.

Table 1.

Cloned hybridoma cells producing anti-ribosome antibodies

Hybridoma clone, n 46
Anti-P antibody, n (%) 34 (74)
Anti-P0 antibody, n (%) 2 (4)
Anti-P1 antibody, n (%) 0 (0)
Anti-P2 antibody, n (%) 0 (0)
Anti-rRNA antibody, n (%) 0 (0)
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Lymphocytes obtained from the inguinal lymph node of four immunized Murphy Roths large (MRL) mice were fused with myeloma cells (P3-X63-Ag8-U1). Hybridoma cells were screened against the P0-P1-P2-RNA complex used for the immunization; 46 hybridoma cells were chosen randomly and cloned and 36 of 46 cells produced either anti-P or anti P0 antibody. The remaining 10 clones lost the ability to produce any anti-ribosome antibody. n = number of clones.

Figure 3.

Figure 3

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Immunoreactivities of anti-P monoclonal antibodies (mAbs) and serum from a systemic lupus erythematosus (SLE) patient. Total ribosomal proteins isolated from rat liver (a), silkworm (b) and brine shrimp (c) (10 pmol each) were separated on a 17% sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto nitrocellulose membranes, and probed separately with monoclonal antibodies (mAbs) 9D5 and 4H11 or with the anti-P serum from the SLE patient. The bound antibodies were detected using the respective secondary antibodies conjugated with horseradish peroxidase and visualized after staining with 4-chloro-1-naphtol as the chromogenic substrate.

There are two phosphorylation sites which corresponded to Ser-102 and Ser-105 in human P2 in the C-terminal regions of P0/P1/P2 35. To test the effect of phosphorylation of P1/P2 on the reactivity of anti-P mAbs, we analysed phosphorylated and non-phosphorylated P1 and P2 by ELISA (Fig. 4a) and immunoblot (Fig. 4b). Accordingly, we found two types of anti-P mAbs: one type showed higher reactivity to the phosphorylated P1 (P1P) and P2 (P2P) than to the non-phosphorylated P1 and P2 (mAb 9D5 is a representative of this type), while the other type recognized both P1 and P2 equally, irrespective of their phosphorylation status (mAb 4H11 is a representative of this type) (Fig. 4). Isotyping of 9D5 and 4H11 revealed that their isotypes were IgG2a and IgG3, respectively, both belonging to the IgGκ class. Conversely, the serum from the SLE patient did not show any marked difference in specificity for the phosphorylated and non-phosphorylated forms of P1 and P2 (Fig. 4), suggesting that the reactivity profile of the anti-P antibody present in the serum was comparable to that of the 4H11 mAb. To compare how these two types of mAbs would react with a native form of the P0(P1-P2)2 complex on the ribosome, we treated brine shrimp ribosomes separately with 9D5 and 4H11 mAbs and then analysed their elongation factor eEF-2-dependent GTPase activity. As shown in Fig. 5, both mAbs inhibited the ribosomal GTPase activity in a dose-dependent manner, and higher inhibition of activity was observed with mAb 9D5 than with mAb 4H11.

Figure 4.

Figure 4

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Effect of phosphorylation on reactivity of anti-P monoclonal antibodies (mAbs). (a) The following protein samples (20 pmol each) were used to attach to separate wells of an enzyme-linked immunosorbent assay (ELISA) plate: P1 and P2, purified human P1 and P2 proteins, respectively; P1p and P2p, purified and phosphorylated P1 and P2 proteins, respectively; C = control (no added protein). A standard ELISA was performed using mAb 9D5, mAb 4H11 or serum from the systemic lupus erythematosus (SLE) patient as the first antibody, and then the bound antibody was detected by using an appropriate peroxidase-conjugated secondary antibody and o-phenylenediamine as the chromogenic substrate. (b) Antigen samples, the same as in (a), were analysed using the immunoblot assay described in Fig. 2.

Figure 5.

Figure 5

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Effect of monoclonal antibodies (mAbs) on ribosome activity. Brine shrimp 80S ribosome (2·5 pmol) was preincubated with increasing amounts of mAb 9D5 (•) or mAb 4H11 (•) at 37°C for 10 min, and then the guanosine triphosphate (GTP)ase activity of the ribosome was determined in the presence of eukaryotic elongation factor-2 (eEF)-2; 100% activity corresponds to 25 pmol of GTP hydrolysis per min. This experiment was repeated three times and results shown are mean values. Error bars represent ± standard error (n = 3).

To determine whether both mAbs would inhibit the ribosome function by binding to the common C-terminal sequences of P0/P1/P2 on the ribosome, we performed an inhibition-rescue experiment using the following three C-terminal synthetic peptides (see Fig. 6 for the peptide sequence): C-peptide, which contains the C-terminal 22 amino acid residues; P-peptide, which is same as the C-peptide except that the two serine residues (corresponding to Ser-102 and Ser-105 for human P2) are phosphorylated; and Δ3-peptide, which is same as the C-peptide except that the last three amino acids at the C-terminal end are missing. Addition of 9D5 or 4H11 (5 μg each) to the 80S ribosome (2·5 pmol) reduced the eEF-2-dependent GTPase activity to 39 and 38% of the control activity (activity in the absence of mAb), respectively (Fig. 6). Remarkably, the addition of 1 nmol C-peptide to the 9D5/ribosome/eEF-2 and 4H11/ribosome/eEF-2 reaction mixtures resulted in recovering their eEF-2-dependent GTPase activities to 75 and 80% of the control activity, respectively. Conversely, addition of 1 nmol P-peptide to the 9D5/ribosome/eEF-2 reaction mixture resulted in slightly increasing the recovery in GTPase activity (87% of the control activity), whereas the recovery in activity was only up to 71% of the control activity for the 4H11/ribosome/eEF-2 reaction mixture. In contrast, the Δ3-peptide was unable to rescue the inhibited GTPase activity, suggesting that the last three C-terminal amino acid residues of P0/P1/P2 play a critical role in recognizing the C-terminal sequence of P proteins by both mAb 9D5 and mAb 4H11.

Figure 6.

Figure 6

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Effect of synthetic peptides on restoring the monoclonal antibody (mAb)-induced inhibition of guanosine triphosphate (GTP)ase activity of ribosome. Three synthetic peptides, each corresponding to 19-22 amino acid residues of the C-terminus sequence common to P0, P1 and P2, are shown in the middle column. ‘Sp’ indicates that the Ser residue is phosphorylated. Five μg of monoclonal antibody (mAb) (9D5 or 4H11) was preincubated with the indicated C-terminal synthetic peptide (1000 pmol) or with no peptide at 37°C for 10 min. The mAb/peptide mixture was then used in a GTPase assay as described in Materials and methods after adding eukaryotic elongation factor-2 (eEF)-2 (0·5 μg), 80S ribosome (2·5 pmol) and [γ-32P]GTP (3 nmol); 100% activity (control) corresponds to the activity in the absence of mAb. Each data point represents average mean ± standard error (n = 3).

Discussion

The production of anti-P autoantibodies has been known to correlate with disease activity, such as in psychotic depression, nephritis and hepatitis 12–14, and also with direct pathogenic effects 36,37. It is extremely important, therefore, to elucidate the mechanism of humoral autoimmune response to the ribosomal anti-P antigen, i.e. the P0(P1-P2)2 pentameric complex. Recent progress in understanding the structural feature of the anti-P antigen (see Fig. 1) makes this complex one of the most suitable materials for studying the structural elements that might be responsible for the autoimmune response. We have established conditions for in vitro reconstitution of functionally active form of eukaryotic P0(P1-P2)2 pentameric complex 16,26. In the present study, we first immunized the MRL mice with this active form of human P0(P1-P2)2-RNA complex without any added adjuvant. Using this immunization protocol, we preferentially obtained anti-P mAb, which reacted with the C-terminal region common to P0, P1 and P2 (Table 1). We did not obtain any mAb that would specifically recognize only P1, P2 or 28S rRNA. However, the possibility should not be excluded that the failure of detection of these mAbs may be due to the complex sample P0(P1-P2)2-RNA used in the first screening for mAb binding. The mAbs specific for P1, P2 and the RNA might be detected by screening with isolated P1, P2 and RNA.

The results obtained in this study were in contrast to those we obtained earlier, where after immunizing New Zealand black/white (NZB/W) mice with a mixture of P1 and P2 isolated from the brine shrimp ribosome, together with complete adjuvant, we obtained several P1- and P2-specific mAbs as well as a single anti-P mAb that recognized all three P proteins, P0, P1 and P2 30. We therefore infer that the P0(P1-P2)2 pentameric complex structure may have a role in inducing the production of highly selective anti-P antibodies. Hines . 4, based on their own experimental results, suggested previously that the multivalency of P0, P1 and P2, as a consequence of the C-terminal sequence shared by all three proteins, is required for anti-P production, because they detected anti-P antibody activity in the sera of MRL mice immunized either with brine shrimp ribosomes or with P1/P2 mixture, but not in the sera of mice immunized with the ribosome core depleted of P1 and P2. They also showed that the human P2 protein alone did not induce the production of anti-P antibodies in the MRL mice. Taken together, it is likely that the C-terminal sequence common to all three P proteins, five copies of which are present in the P0(P1-P2)2 complex, could play an important role in producing anti-P antibodies efficiently, and that any additional knowledge on the state of these C-terminal tails in the complex could be helpful in providing clues for understanding the mechanism of anti-P production in SLE.

Recent biochemical and structural analyses have provided evidence that: (i) the N-terminal domains of P1 and P2 bind to each other and also to P0, and C-terminal halves of two P1-P2 dimers and the C-terminal tail of P0 are located outside the complex structure 24; (ii) the C-terminal halves of both P1 and P2 are flexible, and the anti-P epitope region present in the C-terminal tail can extend up to 125 Å away from the N-terminal dimerization domain 18; and (iii) the C-terminal sequence including the anti-P epitope, five copies of which are present in the P0(P1-P2)2 complex, binds directly to translation factors 25. These lines of evidence suggest that both structural and dynamic features of the P0(P1-P2)2 complex contribute to its biological function by efficiently catching translation factors through the C-terminal tails of P0/P1/P2 and recruiting them to the functional centre of the ribosome. These structural features of the complex should be taken into consideration because of the exceptional antigenicity observed in SLE. We infer that the structure of the P0(P1-P2)2 complex, which binds efficiently to the elongation factors, could also bind efficiently to the B cell antigen receptors on the surface of resting cells, resulting in B cell activation. Thus, five copies of the flexible C-terminal tails present in the complex could also cause clustering of B cell antigen receptors which, in turn, could initiate the B cell activation process, as suggested previously 38,39.

In the present study, we have used a phosphorylated form of the P0(P1-P2)2 complex as an antigen because all P proteins exist on the ribosome as phosphorylated derivatives 35,40, and the phosphorylation may have some influence on the antigenicity 41. Consequently, we obtained a new type of mAb (i.e. mAb 9D5) that demonstrated higher reactivity with the phosphorylated P1/P2 than with the non-phosphorylated P1/P2, and also obtained another mAb (i.e. mAb 4H11) that reacted with the phosphorylated and non-phosphorylated forms of P1/P2 equally. The mAb 9D5 inhibited the ribosomal EF-2-dependent GTPase activity more strongly than the mAb 4H11. The stronger inhibitory effect of 9D5 implies that the P0(P1-P2)2 complex exists in a phosphorylated and functional state on the ribosome. It is, however, unlikely that the phosphorylation is crucial for recognition by 9D5, because 9D5 could also react with the non-phosphorylated P1/P2 (Fig. 4) and non-phosphorylated C-terminal peptide (Fig. 6). It is more likely that the three most C-terminal amino acid residues, Leu-Phe-Asp, are crucial for the binding of 9D5 as well as the binding of 4H11, because Δ3-peptide lacking these three amino acids failed to rescue the ribosome function that was inhibited by 9D5 and 4H11. The Δ3-peptide also did not show any direct binding to the 9D5 Fab fragment, as demonstrated by native acrylamide gel electrophoresis (Supporting information, Fig. S2). These results are consistent with the results of the previous studies on the anti-P epitope, demonstrating that the six amino acids at the C-terminal end are important for the binding of anti-P 42,43. Although the exact role of phosphorylation of two Ser residues, present within the C-terminal sequence shared by all three P-proteins, in enhancing the reactivity of mAb 9D5 with P1 and P2 is not known, it is possible that the phosphorylation may affect the local conformation, presumably an α-helix 43, of the C-terminal epitope for mAb 9D5. The serum from the SLE patient used in the present study did not show any difference between its reactivity with the phosphorylated P1/P2 and non-phosphorylated P1/P2 (Fig. 4), suggesting that the type of antibody present in the patient's serum is similar to the 4H11 antibody. It would be interesting to determine whether or not any SLE patient produces a 9D5-type antibody and also to determine what would be the consequent clinical relevance.

In summary, we have developed an efficient immunization method for making anti-P mAbs. We used an in vitro reconstituted P0(P1-P2)2-RNA complex, where both P1 and P2 were phosphorylated, as the antigen. Because this antigenic complex could be modified by biochemical and molecular engineering methods 26, further studies using various modified complexes might help in clarifying the relationship between the antigenic stimulus for the production of anti-P autoantibody in SLE and structural features of the P0(P1-P2)2-RNA complex. In other words, these future studies would provide insights into the roles of (i) multiple copies of the C-terminal sequence present in the complex, (ii) phosphorylated residues present within the C-terminal sequence and (iii) associated rRNA in anti-P production.

Acknowledgments

We thank Drs Maki Touma, Kosuke Ito and Tomohiro Miyoshi (all from Niigata University) for their valuable comments and discussions on the present study. This work was supported by a Grant-in-Aid for Scientific Research (no. 23657087 and no. 24370073 to T. U.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Disclosure

The authors have no competing financial interests to declare.

Author contributions

H. S and M. O. carried out the experiments described in this study and helped in preparing the figures, A. H. prepared the antigen complex, S. H. performed the epitope analysis on P0, I. N. helped in acquiring the serum from the SLE patient and also helped in revising the manuscript, and T. U. designed the study and wrote the manuscript.

Supporting Information

Fig. S1. Immunoreactivity of two kinds of monoclonal antibodies (mAbs) specific to P0.

Fig. S2. Native gel electrophoretic analysis for anti-P Fab binding to synthetic peptide of the C-terminus sequence common to P0, P1 and P2.

Table S1. Oligonucleotide primers.

cei0179-0236-sd1.pdf (256.1KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Immunoreactivity of two kinds of monoclonal antibodies (mAbs) specific to P0.

Fig. S2. Native gel electrophoretic analysis for anti-P Fab binding to synthetic peptide of the C-terminus sequence common to P0, P1 and P2.

Table S1. Oligonucleotide primers.

cei0179-0236-sd1.pdf (256.1KB, pdf)

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