N-linked glycosylation plays a critical role for the secretion of HMGB1

Abstract

HMGB1 protein is a delayed mediator of sepsis that is secreted to the extracellular milieu in response to various stimulants, inducing a pro-inflammatory response. HMGB1 is devoid of an ER-targeting signal peptide; extracellular secretion mechanism is not completely understood though HMGB1 is secreted after being subjected to post-translational modifications. We identified the role of HMGB1's N-glycosylation in extracellular secretion and its role. We found two consensus (Asn37 and Asn134) and one non-consensus (Asn135) residues which were N-glycosylated in HMGB1using LC-MS/MS and analyzed for N-glycan composition and structure. Inhibition of N-glycosylation with tunicamycin resulted in a molecular shift of HMGB1 by gel electrophoresis. Non-glycosylated double mutant (N→Q) HMGB1 proteins HMGB1N37Q/N134Q and HMGB1N37Q/N135Q show localization in the nuclei, strong binding to DNA supported by in silico structure modeling studies, weak binding to the nuclear export protein CRM1, and rapid degradation by ubiquitination. These mutant proteins had reduced secretion even after acetylation, phosphorylation, oxidation, and exposure to pro-inflammatory stimuli. Taken together, we propose that HMGB1 is N-glycosylated, which is important in DNA interaction, and is a prerequisite for nucleocytoplasmic transport and extracellular secretion.