title: N-linked glycosylation plays a crucial role in 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 endoplasmic reticulum (ER)-targeting signal peptide; hence, the mechanism of extracellular secretion is not completely understood, although HMGB1 is secreted after being subjected to post-translational modifications. Here, we identified the role of N-glycosylation of HMGB1 in extracellular secretion. We found two consensus (N37 and N134) and one non-consensus (N135) residues that were N-glycosylated in HMGB1 by performing liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzing for N-glycan composition and structure. Inhibition of N-glycosylation with tunicamycin resulted in a molecular shift of HMGB1 as assessed by gel electrophoresis. Non-glycosylated double mutant (N→Q) HMGB1 proteins (HMGB1(N37Q/N134Q) and HMGB1(N37Q/N135Q)) showed localization to the nuclei, strong binding to DNA, weak binding to the nuclear export protein CRM1 and rapid degradation by ubiquitylation. 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, and that this is important for its DNA interaction and is a prerequisite for its nucleocytoplasmic transport and extracellular secretion.
authors.affiliation: Department of Microbiology, Yonsei University College of Medicine, Seoul 120-752, Korea Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Young Hun
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Department of Microbiology, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Man Sup
authors.initials: MS
authors.lastname: Kwak
authors.affiliation: Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, Korea.
authors.forename: Jun Bae
authors.initials: JB
authors.lastname: Park
authors.affiliation: Department of Microbiology, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Shin-Ae
authors.initials: SA
authors.lastname: Lee
authors.affiliation: Department of Pediatrics, Seoul National University Boramae Hospital, Seoul National University College of Medicine, Seoul 156-707, Korea.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Choi
authors.affiliation: Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, Korea hscho8@yonsei.ac.kr jsshin6203@yuhs.ac.
authors.forename: Hyun-Soo
authors.initials: HS
authors.lastname: Cho
authors.affiliation: Department of Microbiology, Yonsei University College of Medicine, Seoul 120-752, Korea Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea Severance Biomedical Science Institute and Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul 120-752, Korea hscho8@yonsei.ac.kr jsshin6203@yuhs.ac.
authors.forename: Jeon-Soo
authors.initials: JS
authors.lastname: Shin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D024243:HMGB1 Protein / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D028884:Karyopherins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D000097606:Exportin 1 Protein
chemical_list: D004247:DNA; D024243:HMGB1 Protein; D028884:Karyopherins; D050505:Mutant Proteins; D011134:Polysaccharides; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein
keywords: DNA binding; HMGB1; N-glycosylation; Post-translational modification; Secretion