00236
Crystal structure of bispecific antibody R310 against modified protein and DNA with oxidized lipids

Department of Food and Nutritional Sciences, University of Shizuoka* Graduate School of Bioagricultural Sciences, Nagoya University, Japan**
â—‹Sohei Ito* Mitsugu Akagawa** Koji Uchida**


The aldehydes are important mediators of cell damage and can disrupt important cellular functions and can cause mutations. 4-Hydroxy-2-nonenal (HNE) is a major product of lipid peroxidation and is largely responsible for the cytopathological effects during oxidative stress. Upon reaction with protein, HNE predominantly reacts with nucleophilic amino acids and leads to the formation of stable Michael adducts.
We created monoclonal antibody (mAb) R310, which can enantioselectively recognize the HNE-histidine adducts, and demonstrated the both epitopes in vivo. To better understand the anti-HNE immune response, we undertook a detailed comparison of the primary structure of these mAbs and discovered that the presence of anti-R mAb R310 was homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. And the mAb R310 crossreacted with the dsDNA and oligo deoxyguanosine to some extent.On the basis of these findings, we evaluated the structural identity of mAb R310 with the anti-DNA mAbs by x-ray crystallographic analysis. Crystallization of mAb R310 Fab fragment with HNE-histidine adduct shows that the adduct binds to a hydrophobic pocket in the groove, consisting of six CDRs. Comparison of the structure of R310 to that of the anti-DNA Fab, which has been shown to bind oligonucleotides by making stacking interactions between tyrosine residues and the bases, revealed a common structural motif that could be a fundamental aspect of DNA recognition by Abs. The Ab primarily binds the HNE-histidine adduct by sandwiching between hydrophobic residues. The spatial distribution of these hydrophobic amino acids makes these residues to bind to DNA in the same way as the anti-DNA mAbs, allowing the bases to form sequence-specific hydrogen bonds with the protein.