Monitoring the glycosylation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors using specific antibodies reveals a novel regulatory mechanism of N-glycosylation occupancy by molecular chaperones in mice

Ryosuke Midorikawa, Daisuke Takakura, Jyoji Morise, Yoshihiko Wakazono, Nana Kawasaki, Shogo Oka, Kogo Takamiya

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

In the mammalian nervous system, protein N-glycosylation plays an important role in neuronal physiology. In this study, we performed a comprehensive N-glycosylation analysis of mouse GluA1, one of the major subunits of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate type glutamate receptor, which possesses six potential N-glycosylation sites in the N-terminal domain. By mass spectrometry-based analysis, we identified the N-glycoforms and semiquantitatively determined the site-specific N-glycosylation occupancy of GluA1. In addition, only the N401-glycosylation site demonstrated incomplete N-glycosylation occupancy. Therefore, we generated a peptide antibody that specifically detects the N401-glycan-free form to precisely quantify N401-glycosylation occupancy. Using this antibody, we clarified that N401 occupancy varies between cell types and increases in an age-dependent manner in mouse forebrains. To address the regulatory mechanism of N401-glycosylation, binding proteins of GluA1 around the N401 site were screened. HSP70 family proteins, including Bip, were identified as candidates. Bip has been known as a molecular chaperone that plays a key role in protein folding in the ER (endoplasmic reticulum). To examine the involvement of Bip in N401-glycosylation, the effect of Bip over-expression on N401 occupancy was evaluated in HEK293T cells, and the results demonstrated Bip increases the N401 glycan-free form by mediating selective prolongation of its protein half-life. Taken together, we propose that the N401-glycosite of GluA1 receives a unique control of modification, and we also propose a novel N-glycosylation occupancy regulatory mechanism by Bip that might be associated with α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors function in the brain. (Figure presented.).

Original languageEnglish
Pages (from-to)567-585
Number of pages19
JournalJournal of neurochemistry
Volume153
Issue number5
DOIs
Publication statusPublished - 01-06-2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cellular and Molecular Neuroscience

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