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

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.

Lupus antiribosomal P antisera contain antibodies to a small fragment of 28S rRNA located in the proposed ribosomal GTPase center.

The ribosomal P proteins are necessary for GTPase activity during protein synthesis. In addition to antibodies to the P proteins, sera from lupus patients contain anti-rRNA activity. To determine whether lupus antiribosomal sera recognize the region of 28S rRNA recently proposed to form part of the ribosomal GTPase center, an rRNA fragment corresponding to nucleotides (nt) 1922-2020 was transcribed in vitro and tested for antigenicity. 18 of 24 (75%) lupus sera containing anti-P antibodies, but only 2 of 24 (8%) lupus sera without anti-P, immunoprecipitated this rRNA fragment (p less than 0.001). The binding was specific, since no significant differences were observed between anti-P positive and negative lupus sera in binding to the RNA fragment transcribed in the antisense orientation or to a control region of rRNA. The majority of sera tested protected a rRNA fragment of approximately 68 nucleotides. To evaluate the fine specificity of the anti-28S antibodies, deletions and site-directed mutations were made in the RNA fragment. The anti-28S antisera required nt 1944-1955 for recognition and were remarkably sensitive to destabilizing as well as nondestabilizing mutations in the stems of the RNA fragments. Detection of antiprotein and anti-RNA antibodies directed against a functionally related domain in the ribosome, together with the remarkable specificity of anti-28S antibodies, strongly suggests a direct role for this region of the ribosome in initiating and/or maintaining antiribosomal autoantibody production.

Antiribosomal antibodies in systemic lupus erythematosus.

ARA occur in approximately 10% of randomly selected SLE patients but in up to 40% of patients with active disease. Anti-P antibodies appear to be a highly specific diagnostic marker for SLE because they are rarely detected in other multisystem autoimmune disorders. ARA are most frequently directed against the P proteins, and the shared conserved C-terminus of the P proteins is immunodominant in almost all sera tested. Anti-P antibodies increase in titer in patients with active disease and have been reported to be detected more frequently in patients with severe behavioral disturbances. This may be particularly true of patients with affective disorders. The clinical utility of serologic tests for anti-P in central nervous system lupus must await large, prospective studies. Other ARA antibodies have been detected in patients with SLE. These antibodies include anti-28S rRNA, anti-S10, and anti-L12. In all cases, the frequency with which these antibodies are detected is increased in sera containing anti-P. The P proteins and the 28S rRNA epitope play essential, but as yet undefined, roles in GTPase activity on the ribosome. The L12 protein is the mammalian homologue of the E. coli and yeast proteins known to bind to the 28S rRNA epitope. These findings indicate that some SLE patients produce autoantibodies against multiple components of a functionally related domain of the ribosome. This, in turn, supports the notion that the ribosome initiates and/or maintains autoantibody production. Despite these findings, attempts to induce anti-P antibodies by immunization with autologous ribosomes in the autoimmune strain of mouse, MRL, have been unsuccessful. It therefore seems likely that the ribosomal components must be altered to break tolerance or that other abnormalities of the immune system are necessary for autoantibody production.

Antiribosomal antibodies in SLE, infection, and following deliberate immunization.

ARA occur in approximately 10% of randomly selected SLE patients but in up to 40% of patients with active disease. Anti-P antibodies appear to be a highly specific diagnostic marker for SLE since they are rarely detected in other multisystem autoimmune disorders. ARA are most frequently directed against the P proteins and the shared conserved C-terminus of the P proteins is immunodominant in almost all sera tested. Anti-P antibodies increase in titer in patients with active disease and have been reported to be detected more frequently in patients with severe behavioral disturbances. This may be particularly true of patients with affective disorders. The clinical utility of serological tests for anti-P in central nervous system lupus must await large, prospective studies. Other ARA antibodies have been detected in patients with SLE. These antibodies include anti-28S rRNA, anti-S10, and anti-L12. In all cases, the frequency with which these antibodies are detected is increased in sera containing anti-P. The P proteins and the 28S rRNA epitope play essential, but as yet undefined, roles in GTPase activity on the ribosome. The L12 protein is the mammalian homologue of the E. coli and yeast proteins known to bind to the 28S rRNA epitope. These findings indicate that some SLE patients produce autoantibodies against multiple components of a functionally related domain of the ribosome. This, in turn, supports the notion that the ribosome initiates and/or maintains autoantibody production. Despite the evidence supporting an antigen driven immune response, attempts to induce anti-P antibodies by immunization with autologous ribosomes in the autoimmune strain of mouse, MRL, have been unsuccessful. It therefore seems likely that the ribosomal components must be altered in some way to break tolerance or that other abnormalities of the immune system are necessary for autoantibody production. Immunization with foreign ribosomes induce anti-P autoantibodies in mice and in apparently normal humans infected with the hemoflaggelate, T. cruzi. The ability of the P proteins to break tolerance in these situations is, most likely, explained by the provision of a T cell epitope (the foreign P protein) together with the multivalency of the P proteins on the ribosome (which activate autoreactive B cells). We therefore propose (Fig. 5) a two-signal model for autoantibody production similar to that suggested for T-B collaboration in the normal immune response and also in the GVHD model of lupus.(ABSTRACT TRUNCATED AT 400 WORDS)

Serological association of lupus autoantibodies to a limited functional domain of 28S ribosomal RNA and to the ribosomal proteins bound to the domain.

Site-specific anti-RNA antibodies were sought in 120 sera of patients with autoimmune diseases by ribonuclease-protection assay using six fragments covering 28S ribosomal RNA (rRNA) as antigens. Fifteen of 90 sera from patients with systemic lupus erythematosus (SLE), but none of 30 sera of the other autoimmune diseases, provided a 60 nucleotide fragment within a region termed the 'GTPase domain' of 28S rRNA. These sera had potency to precipitate 0.42-69.3 nmol of the RNA domain per ml serum, which was higher than 15 control sera of healthy donors. No other specific antigenic site was detected in 28S rRNA under conditions used. All of the 15 sera having this anti-RNA antibody showed reactivity to ribosomal P proteins (anti-P), and two of them contained an additional antibody to ribosomal protein L12. These results suggested a strong association of the production of these three antibodies. Since P and L12 proteins form a stable complex with the GTPase domain, this serological association may result from an immune response to epitopes clustered on a single RNA-protein complex domain in ribosomes.

A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes.

An autoantibody reactive with a conserved sequence of 28 S rRNA (anti-28 S) was identified in serum from a patient with systemic lupus erythematosus. Anti-28 S protected a unique 59-nucleotide fragment synthesized in vitro against RNase T1 digestion. RNA sequence analysis revealed that it corresponded to residues 1944-2002 in human 28 S rRNA and 1767-1825 in mouse 28 S rRNA. These sequences are identical and highly conserved throughout all known eukaryotic 28 S rRNAs. In addition, this fragment is homologous to residues 1052-1110 of Escherichia coli 23 S rRNA that lies within the GTP hydrolysis center of the 50 S ribosomal subunit. Anti-28 S and its Fab fragments strongly inhibited poly(U)-directed polyphenylalanine synthesis, but had no effect on ribosomal peptidyltransferase activity. This effect resulted from inhibition of the binding of elongation factors EF-1 alpha and EF-2 to ribosomes and of the associated GTP hydrolysis. The inhibitory effect was almost completely suppressed by preincubation of anti-28 S with 28 S rRNA or in vitro synthesized RNA fragments containing the immunoreactive region. These results show that the immunoreactive conserved region of 28 S rRNA participates in the interaction of ribosomes with the two elongation factors in protein synthesis.

A functional site of the GTPase-associated center within 28S ribosomal RNA probed with an anti-RNA autoantibody.

An anti-RNA autoantibody (anti-28S) was employed to identify structural and functional elements characteristic of a domain termed the 'GTPase center' in eukaryotic 28S ribosomal RNA. This antibody, an inhibitor of ribosome-associated GTP hydrolysis, has a unique property: it binds to the RNA domain of eukaryotes but not to that of prokaryotes. The antibody binding occurred in the presence of Mg2+ and protected from chemical modification three conserved bases (U1958, G1960 and A1990) and the base G1959 which is replaced by A in prokaryotic 23S rRNA (A1067 in Escherichia coli). In vitro substitution of G1959 to A drastically weakened the antibody binding, and the reciprocal substitution, A1067-->G of the E.coli domain conferred the binding ability. This suggests that the G base determines the specificity of antibody binding. The G1959 was also protected by the association of ribosomes with elongation factor EF-2. The result, together with protection of E.coli base A1067 by EFG [D.Moazed, I.M. Robertson and H.F. Noller (1988) Nature, 334, 362-364], suggests that the position of G1959 in eukaryotes and A1067 in prokaryotes constitutes at least part of the factor binding site irrespective of the base replacement during evolution.

Complement-dependent cytotoxicity for negative selection at the mRNA level.

Complement-Dependent Cytotoxicity (CDC) is a common technique used for isolating and characterizing cell populations. However, the molecular events resulting from CDC-mediated cell injury remain obscure. In order to use CDC as a selection procedure for studies at the RNA level, we examined if CDC is associated with rapid degradation of RNAs from target cells without affecting the stability and viability of RNAs of non-target cells. Using a model of anti-CD3-mediated CDC, we show that T cell-specific RNAs were absent immediately after CDC. However, ribosomal RNAs and mRNAs from non-targeted cells (non-T cells) were not affected by CDC. Our results indicate that CDC is associated with rapid degradation of only target cell RNAs, validating CDC as a method for cell isolation without interfering with further studies at the RNA level.