Bispecific abs against modified protein and DNA with oxidized lipids

Abstract

4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R- and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. HNE reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. We have previously raised the mAbs, anti-R mAb 310 and anti-S mAb S412, that enantioselectively recognized the R-HNE-histidine and R-HNE-histidine adducts, respectively, and demonstrated the presence of both epitopes in vivo. In the present study, to further investigate the anti-HNE immune response, we analyzed the variable genes and primary structure of these Abs and found that the sequence of R310 was highly homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. An x-ray crystallographic analysis of the R310 Fab fragment showed that the R-HNE-histidine adduct binds to a hydrophobic pocket in the antigen-binding site. Despite the structural identity to the anti-DNA autoantibodies, however, R310 showed only a slight crossreactivity with the native double-stranded DNA, whereas the Ab immunoreactivity was dramatically enhanced by the treatment of the DNA with 4-oxo-2-nonenal (ONE), an analog of HNE. Moreover, the 7-(2-oxo-heptyl)-substituted 1,N2-etheno-type ONE-2'-deoxynucleoside adducts were identified as alternative epitopes of R310. Molecular mimicry between the R-HNE-histidine configurational isomers and the ONE-DNA base adducts is proposed for the dual crossreactivity.

Fig. 1.

Binding of the anti-R- and anti-S-HNE mAbs to protein-bound HNE enantiomers. (A) Covalent modification of protein by HNE. “R” represents the side chain of nucleophilic amino acids, such as cysteine, histidine, and lysine. (B) Crossreactivity of anti-R-HNE mAb R310 with the protein-bound HNE enantiomers. (C) Crossreactivity of anti-S-HNE mAb S412 with the protein-bound HNE enantiomers. •, R-HNE-modified BSA; ○, S-HNE-modified BSA; ▵, R,S-HNE-modified BSA. The Ab reactivity was measured by direct antigen ELISA.

Fig. 2.

X-ray crystallographic analysis of R310 Fab fragment. (A) The crystal structure of a recombinant R310 Fab with the R-HNE-histidine adduct. The R-HNE-histidine is shown in wire-frame model. (B) The molecular surface model of DNA-1 Fab (PDB ID code 1I8M). The oligo(dT) of DNA-1 is shown in wire-frame model. The CDRs are colored as follows: L1, yellow; L2, cyan; L3, lime; H1, violet; H2, orange; H3, aquamarine. (C) Close-up view of R310 Fab CDRs. (D) Close-up view of DNA-1 Fab CDRs. The CDRs are shown in ribbon diagram. Tyrosine and phenylalanine residues are shown as a red wire-frame model. This figure was prepared by using pymol.

Fig. 3.

Anti-DNA affinity of anti-HNE mAbs. (A) Crossreactivity of the anti-HNE mAbs R310 and S412 with calf-thymus DNA. ○, S412; •, R310. (B) Crossreactivity of the anti-HNE mAbs and anti-DNA mAb D466, obtained from 56R and CD40L double-transgenic mice, with native DNA and oligonucleoside 50-mers. The Ab reactivity was measured by direct antigen ELISA. (C) Immunoreactivity of R310 and S412 toward aldehyde-treated DNA. In A–C, affinity of the Abs was determined by a direct antigen ELISA using aldehyde-treated DNA as the absorbed antigens. A coating antigen was prepared by incubating calf-thymus DNA (1 mg/ml) with 5 mM aldehydes in 1 ml of 0.1 M sodium phosphate buffer (pH 7.4) for 24 h at 37°C. One microgram of antigen (calf-thymus DNA or oligonucleoside 50-mers) was coated per well in polystyrene plates, and Ab binding was detected. (D) Chemical structure of ONE.

Fig. 4.

Immunoreactivity of R310 toward the ONE-2′-deoxyribonucleoside adducts. (A) Chemical structures of the 1,N²-etheno-type ONE-2′-deoxyribonucleoside adducts. (B) Competitive ELISA. Competitors: ◇, N^a-acetylhistidine; ○, deoxyguanosine; ▵, deoxyadenosine; □, deoxycytidine; ♦, HNE-histidine; •, ONE-deoxyguanosine; ▴, ONE-deoxycytidine; ■, ONE-deoxyadenosine.

Fig. 5.

Molecular mimicry between HNE-histidine and ONE-2′-deoxyribonucleoside adducts. (A) The R-HNE-histidine adducts and 2′-deoxyribonucleosides are commonly composed of functional groups (ellipsoidal moieties), which may be essential for Ab recognition: hydroxyl groups, nitrogen-containing heterocyclic rings (histidine and base), and tetrahydrofuran rings. (B) Chemical structures of R-HNE-histidine and the 7-(2-oxo-heptyl)-substituted 1,N²-etheno-type ONE-2′-deoxyguanosine adduct. The alkyl (pentyl) groups (bold lines) are common in both adducts.