2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

Shuo Zheng, Apparao B Kummarapurugu, Daniel K Afosah, Nehru Viji Sankaranarayanan, Rio S Boothello, Umesh R Desai, Thomas Kennedy, Judith A Voynow

High mobility group box 1 (HMGB1) is an alarmin released from macrophages following infection or inflammation, and a biomarker of lung disease progression in cystic fibrosis. We reported that 2-O, 3-O desulfated heparin (ODSH) inhibits release of HMGB1 from murine macrophages triggered by neutrophil elastase (NE) both in vivo and in vitro. HMGB1 shuttles between the nucleus and the cytoplasm. When acetylated at lysine residues in the nuclear localization signal domains, HMGB1 is sequestered in the cytoplasm and is fated for secretion. In this report, we investigated the mechanism by which ODSH blocks HMGB1 secretion. We tested whether ODSH inhibits the activity of p300, a histone acetyltransferase (HAT), which has been linked to HMGB1 acetylation and release. ODSH inhibited both NE and LPS-triggered HMGB1 release from the murine macrophage cell line, RAW264.7, in a concentration-dependent manner. Fluorescein-labeled ODSH was taken up by RAW264.7 cells into the cytoplasm as well as the nucleus suggesting an intracellular site of action of ODSH for blocking HMGB1 release. ODSH inhibited both RAW264.7 cell nuclear extract, human macrophage nuclear extract, and recombinant p300 HAT activity in vitro resulting in the failure to acetylate HMGB1. In silico molecular modeling predicted that of the numerous possible ODSH sequences, a small number preferentially recognize a specific binding site on p300. Fluorescence binding studies showed that ODSH bound p300 tightly (KD ~1 nM) in a highly co-operative manner. These results suggest that ODSH inhibited HMGB1 release, at least in part, by direct molecular inhibition of p300 HAT activity.