Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis

The release of damage-associated
molecular patterns (DAMPs) by
airway epithelial cells is believed to play a crucial role in the
initiation and development of chronic airway conditions such as asthma
and chronic obstructive pulmonary disease (COPD). Intriguingly, the
classic DAMP high-mobility group box-1 (HMGB1) is detected in the
culture supernatant of airway epithelial cells under basal conditions,
indicating a role for HMGB1 in the regulation of epithelial cellular
and immune homeostasis. To gain contextual insight into the potential
role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal
and complementary methods of high-resolution clear native electrophoresis,
immunoprecipitation, and pull-downs coupled to liquid chromatography–tandem
mass spectrometry (LC–MS/MS) to profile HMGB1 and its binding
partners in the culture supernatant of unstimulated airway epithelial
cells. We found that HMGB1 presents exclusively as a protein complex
under basal conditions. Moreover, protein network analysis performed
on 185 binding proteins revealed 14 that directly associate with HMGB1:
amyloid precursor protein, F-actin-capping protein subunit alpha-1
(CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin,
several members of the heat shock protein family (HSPA8, HSP90B1,
HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone
3 (H3F3B), the FACT (facilitates chromatin transcription) complex
constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein
K (HNRNPK). These studies provide a new understanding of the extracellular
functions of HMGB1 in cellular and immune homeostasis at the airway
mucosal surface and could have implications for therapeutic targeting.