Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
J Biol Chem. 2013 Feb 27;
Authors: Zandarashvili L, Sahu D, Lee K, Lee YS, Singh P, Rajarathnam K, Iwahara J
Abstract
Some extracellular proteins are initially secreted in the reduced form via a non-canonical pathway bypassing the endoplasmic reticulum, and become oxidized in the extracellular space. One such protein is high-mobility group box 1 (HMGB1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture media. In this approach, salt-free lyophilized 15N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear 1H-15N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ~17 minutes (in the human serum and saliva) to 3 hours (in prostate cancer cell culture media). Furthermore, binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment, and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules.
PMID: 23447529 [PubMed - as supplied by publisher]
More...