Effect of amino acid substitutions in the heavy chain CDR3 of an autoantibody on its reactivity.
In this study, we applied site-directed mutagenesis to the Fab fragment of a mouse IgM (IE12) that was previously shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. Its native structure was very similar to that of a polyreactive natural IgM (ppc15-30). Indeed, they both use the same light chain and the same VH, D and JH segments. However, the N regions differ and the D is translated in two different reading frames, giving different amino acid compositions of the heavy chain CDR3 (HCDR3). Site-directed mutagenesis modified the HCDR3 of IE12 compared to that of the natural antibody and the resulting effects on its reactivity were analyzed. Because the HCDR3 of IE12 is very rich in aliphatic residues, which are hydrophobic, we replaced them with the more hydrophilic residues of the HCDR3 of the polyreactive IgM. In addition, we evaluated the impact of the proline residues in the HCDR3 of IE12 on its activity, because they are known to restrict backbone flexibility. We found that a more hydrophilic HCDR3 conferred to the IE12 Fab a polyreactive profile. Prolines seem to play an important role in this context, because when they were replaced by glycines, the resulting Fab fragments were highly polyreactive. Our results suggest that, for polyreactivity, hydrophilicity and a certain plasticity of the HCDR3 seem to be necessary. Greater flexibility of the CDR, particularly the HCDR3, might be an important characteristic for polyreactive antibodies.
Adib-Conquy
M
M
Immunocytochimie, CNRS URA 1961, Institut Pasteur, Paris, France.
Gilbert
M
M
Avrameas
S
S
D000818:Animals; D001323:Autoantibodies / Q000737:chemistry* / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D013329:Structure-Activity Relationship
D001323:Autoantibodies; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region