Fig. 2: Ubiquitous recognition of AGEs by histones.

a Binding of DHA-modified Bt-PA to histone H2B. Recombinant histone H2B immobilized on ELISA plate was incubated with either Bt-PA or DHA-Bt-PA. The binding was detected using streptavidin-HRP. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). b HPLC analysis of DHA-modified Bt-PA capable of binding to H2B. The reaction mixture of Bt-PA and DHA was separated by reverse-phase HPLC, and each fraction was measured for the binding to H2B (middle) and anti-AGEs antibodies (bottom). The reactions were monitored with absorbance at 200–600 nm (top). The dashed square indicates the region with the binding activity to H2B and anti-AGEs antibody. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). c Binding of modified Bt-PAs to histone H2B. Bt-PA was incubated with DHA, glucose (Glc), and glucose metabolites, such as glycolaldehyde (GA), glyoxal (GO), methylglyoxal (MG), glyceraldehyde (GCA), and 3-deoxyglucosone (3-DG), and the binding to histone H2B was evaluated by solid-phase binding assay. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). d Surface plasmon resonance measurements. The interaction of H2B and either BSA or AGEs (10 µg/ml) was monitored. e Effect of NaCl on binding of AGEs to H2B. H2B immobilized on ELISA plate was incubated with Bt-AGEs (5 μg/ml) under the indicated concentrations of NaCl. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Dunnett’s test (two-sided), relative to AGEs treatment under 0.15 M NaCl. f Binding of acylated proteins to the recombinant histone H2B. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Dunnett’s test (two-sided), relative to the no-treatment control. g Involvement of H2B N-terminal tail region and its electrostatic property in the binding to AGEs. MBP-fusion H2B full length (FL), N-terminal (N-term. (1-35)), N-terminal deletion mutant (ΔN35), acetylated Nterm. (1-35), or N-terminal scrambled fragments (scrambled 1, 2, and 3) were subjected to the solid phase binding assay. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Tukey–Kramer tests (two-sided), n.s. not significant (p > 0.05).