The DNA-binding protein HMGB1 is a CpG-DNA engaging factor released by macrophages in response to IFNγ. (A) Purification of HMGB1 as a CpG-DNA–binding protein. Raw264.7 cells (2.5 × 10⁶/mL) were treated with IFNγ (30 ng/mL) for 18 hours. Cell-free supernatants (50 mL) were slowly loaded (10 mL/hour) onto a single-stranded DNA-cellulose column pre-equilibrated with buffer A (20 mM Tris-Cl, pH 8.8, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol). After absorption, the column was washed sequentially with 150 mL buffer A and rinsed with 150 mL 0.1 M NaCl in buffer A. Proteins were recovered from the column by sequential elution with 0.5 and 2 M NaCl in buffer A. Fractions (1 mL) were dialyzed overnight against buffer B (5 mM KH₂PO₄/NaOH, pH 7.9, 10% glycerol). Of each fraction, 30 μL was loaded on a 10% SDS-PAGE and subsequently silver stained. Fractions 2, 3, and 4 that had peak protein content were pooled, diluted with buffer A at 0.16 M NaCl, and incubated with 0.1 mg biotinyl-1018 ODN for 30 minutes followed by incubation overnight at 4°C with 0.5 mL streptavidin-agarose beads. The beads were washed with buffer A containing 0.16 M NaCl, boiled, and loaded on a 10% SDS-PAGE, and proteins were detected by silver staining. All visible bands were excised and subjected to mass spectrometry. Several representative HMGB1 peptides are listed. (B) HMGB1 binds CpG-ODNs (1018, 1668, and D-19) preferentially over controls (1019, n-1668, and c-405). Mouse rHMGB1 (25 ng) or histone H2A (25 ng) was incubated in the absence (control) or presence of CpG-ODN-biotin (5 μg) for 60 minutes. ODNs were immunoprecipitated with streptavidin-agarose beads, washed, and subjected to immunoblot analysis (IB) with anti-HMGB1 antibody. The levels of unbound HMGB1 or H2A were estimated by collecting 5% of the supernatant from each precipitation reaction. The gray value of the pixel intensity (range, 1 to 250) of the respective protein bands is listed. Results represent 1 of 6 or 3 reproducible independent experiments for HMGB1 and H2A binding, respectively. (C) CD spectra of oligos 1018 and 1019 in the presence of increasing amounts of HMGB1. All spectra have been acquired at 20°C, 20 mM phosphate buffer, pH 7.0, 10 mM NaCl, with an initial DNA concentration of 10 μM. Traces from red to violet correspond to the spectra acquired by adding 0, 1, 2, 2.8, 4.5, or 10 μM protein to the oligo solutions. The spectra were corrected by subtracting the buffer and the protein, and compensating for dilution. The panel “HMGB1 alone” shows the spectra recorded for 0, 1, 2, 2.8, 4.5, or 10 μM protein (in the same buffer and in the absence of DNA), to show that corrections applied to the recorded spectra are neutral in the wavelength range considered here.