N-linked glycosylation plays a crucial role in the secretion of HMGB1

Young Hun Kim, Man Sup Kwak, Jun Bae Park, Shin Ae Lee, Ji Eun Choi, Hyun Soo Cho, Jeon Soo Shin

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 Nglycosylated in HMGB1 by performing liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzing for Nglycan 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 (HMGB1N37Q/N134Q and HMGB1N37Q/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.

Original languageEnglish
Pages (from-to)29-38
Number of pages10
JournalJournal of cell science
Volume129
Issue number1
DOIs
Publication statusPublished - 2016 Jan 1

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HMGB1 Protein
Glycosylation
Cell Nucleus Active Transport
Tunicamycin
Ubiquitination
DNA
Mutant Proteins
Post Translational Protein Processing
Acetylation
Nuclear Proteins
Protein Sorting Signals
Tandem Mass Spectrometry
Liquid Chromatography
Endoplasmic Reticulum
Electrophoresis
Sepsis
Gels
Phosphorylation

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

Kim, Y. H., Kwak, M. S., Park, J. B., Lee, S. A., Choi, J. E., Cho, H. S., & Shin, J. S. (2016). N-linked glycosylation plays a crucial role in the secretion of HMGB1. Journal of cell science, 129(1), 29-38. https://doi.org/10.1242/jcs.176412
Kim, Young Hun ; Kwak, Man Sup ; Park, Jun Bae ; Lee, Shin Ae ; Choi, Ji Eun ; Cho, Hyun Soo ; Shin, Jeon Soo. / N-linked glycosylation plays a crucial role in the secretion of HMGB1. In: Journal of cell science. 2016 ; Vol. 129, No. 1. pp. 29-38.
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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 Nglycosylated in HMGB1 by performing liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzing for Nglycan 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 (HMGB1N37Q/N134Q and HMGB1N37Q/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.",
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Kim, YH, Kwak, MS, Park, JB, Lee, SA, Choi, JE, Cho, HS & Shin, JS 2016, 'N-linked glycosylation plays a crucial role in the secretion of HMGB1', Journal of cell science, vol. 129, no. 1, pp. 29-38. https://doi.org/10.1242/jcs.176412

N-linked glycosylation plays a crucial role in the secretion of HMGB1. / Kim, Young Hun; Kwak, Man Sup; Park, Jun Bae; Lee, Shin Ae; Choi, Ji Eun; Cho, Hyun Soo; Shin, Jeon Soo.

In: Journal of cell science, Vol. 129, No. 1, 01.01.2016, p. 29-38.

Research output: Contribution to journalArticle

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T1 - N-linked glycosylation plays a crucial role in the secretion of HMGB1

AU - Kim, Young Hun

AU - Kwak, Man Sup

AU - Park, Jun Bae

AU - Lee, Shin Ae

AU - Choi, Ji Eun

AU - Cho, Hyun Soo

AU - Shin, Jeon Soo

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