Abstract
Rheumatoid arthritis-like symptoms can be initiated experimentally in naive K/BxN mice by simultaneously administering the two monoclonal antibodies 11H3 and 46H9. Both antibodies specifically recognize Glucose- 6-Phosphate Isomerase (GPI), a known auto antigen in RA patients. Amino acid sequences of the Fv parts of the antibodies were determined by translating the respective hybridoma DNA sequences and served for threedimensional structure modeling of the paratope regions. In silico docking of both Fv antibody structure models to the X-ray structures of the homodimeric murine GPI as well as to the homodimeric human GPI predicted the murine epitope of the 11H3 antibodies to comprise partial amino acid sequences QRVRSGDWKGYTGKS (aa134-148) and AAKDPSAVAK (aa232-241), generating an assembled (conformational) epitope. The 11H3 epitope on human GPI encompasses the matching partial amino acid sequences QRVRSGDWKGYTGKT (aa134-148) and AAKDPSAVAK (aa232-241). The epitope of the 46H9 antibody was determined to consist of the partial murine GPI amino acid sequence RKELQAAGKSPEDLEK (aa446-461) and the human GPI amino acid sequence RKELQAAGKSPEDLER (aa446-461), respectively, resembling consecutive (linear) epitopes. The predicted epitopes were verified by mass spectrometric epitope mapping using synthetic epitope peptides. Peptide QRVRSGDWKGYTGKS[GSMSGS] AAKDPSAAK included a small spacer sequence in between the epitope sequences, mimicking the assembled epitope for the 11H3 antibody. The peptide RKELQAAGKSPEDLEK represented the consecutive epitope for the 46H9 antibody. The determined B-cell epitopes of GPI and their interactions with the monoclonal antibodies provide a detailed structural understanding of immunological disease onset mechanisms in a mouse model of rheumatoid arthritis.
Highlights
Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease affecting about 1% of the human population and is associated with inflammation and destruction of distal joints [1,2]
For the production of Glucose-6-Phosphate Isomerase (GPI) specific monoclonal antibodies, one week prior to fusion SP2/0 myeloma fusion partner cells were expanded in Iscoves medium with 10% FCS. 96-well plates were plated with peritoneal cells obtained from C57BL/6 mice and the K/BxN mice were immunized with purified recombinant GPI after removal of Glutathione S-transferase (GST) tag
Our in silico epitope prediction started with generating 3D structures of the Fv part of the 11H3 mono-clonal antibody using amino acid sequences of the variable regions of the heavy and light chains, respectively (Supplemental Figure 1)
Summary
Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease affecting about 1% of the human population and is associated with inflammation and destruction of distal joints [1,2]. The major histological characteristics of human RA, such as leukocyte invasion, synovitis, pannus formation, and cartilage and bone destruction have been observed in the K/BxN mouse model as well [7]. Arthritis-like symptoms were observed to develop in naive Balb/c mice by the transfer of polyclonal GPI antibodies or by transferring at least two different monoclonal GPI-specific antibodies [11,12]. In the K/BxN mouse, disease onset can be triggered by simultaneously administering the two pathogenic GPI-specific monoclonal antibodies 11H3 and 46H9 [13]. GPI-antibody complexes on joint cartilage surfaces have been shown to initiate an inflammatory cascade through the alternative complement pathway [10]
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