title: Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.
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.
authors.affiliation: Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Sato
authors.forename: M
authors.initials: M
authors.lastname: Onozuka
authors.forename: A
authors.initials: A
authors.lastname: Hagiya
authors.forename: S
authors.initials: S
authors.lastname: Hoshino
authors.forename: I
authors.initials: I
authors.lastname: Narita
authors.forename: T
authors.initials: T
authors.lastname: Uchiumi
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D051379:Mice; D012339:RNA, Ribosomal, 28S / Q000276:immunology*; D012269:Ribosomal Proteins / Q000276:immunology*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D001323:Autoantibodies; D012339:RNA, Ribosomal, 28S; D012269:Ribosomal Proteins
keywords: SLE; autoantibody; monoclonal anti-P; ribosomal antigenic complex