Monitoring the glycosylation of AMPA (a-amino-3-hydroxy-5-methyl-4isoxazole-propionate)-type glutamate receptors using specific antibodies reveals a novel regulatory mechanism of N-glycosylation occupancy by molecular chaperones in Mice.
Monitoring the glycosylation of AMPA (a-amino-3-hydroxy-5-methyl-4isoxazole-propionate)-type glutamate receptors using specific antibodies reveals a novel regulatory mechanism of N-glycosylation occupancy by molecular chaperones in Mice.
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使用特异性抗体监测 AMPA（α-氨基-3-羟基-5-甲基-4异恶唑-丙酸酯）型谷氨酸受体的糖基化揭示了分子伴侣对 N-糖基化占据的新调节机制。
DOI：
10.1111/jnc.14964
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Takamiya K.
中科院分区：
文献类型：
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作者：
Midorikawa R;Takakura D;Morise J;Wakazono Y;Kawasaki N;Oka S;Takamiya K.
在哺乳动物神经系统中，蛋白质N-糖基化在神经元生理学中起重要作用。在这项研究中，我们对小鼠GluA 1进行了全面的N-糖基化分析，GluA 1是α-氨基-3-羟基-5-甲基-4-异恶唑-丙酸盐型谷氨酸受体的主要亚基之一，在N-末端结构域中具有6个潜在的N-糖基化位点。通过基于质谱的分析，我们鉴定了N-糖型，并半定量测定了GluA 1的位点特异性N-糖基化占有率。此外，仅N401-糖基化位点显示不完全的N-糖基化占据。因此，我们生成了一种肽抗体，可特异性检测N401-无聚糖形式，以精确定量N401-糖基化占有率。使用这种抗体，我们澄清了N401占有率在细胞类型之间存在差异，并且在小鼠前脑中以年龄依赖性方式增加。为了解决N401-糖基化的调节机制，筛选了N401位点周围的GluA 1结合蛋白。HSP 70家族蛋白，包括Bip，被鉴定为候选物。Bip是一种分子伴侣，在内质网蛋白质折叠中起着关键作用。为了检查Bip在N401-糖基化中的参与，在HEK 293 T细胞中评估了Bip过表达对N401占据的影响，结果表明Bip通过介导选择性延长其蛋白质半衰期来增加N401无聚糖形式。综上所述，我们认为GluA 1的N401-糖基受到独特的修饰控制，我们还提出了Bip的一种新的N-糖基化占据调节机制，可能与大脑中的α-氨基-3-羟基-5-甲基-4-异恶唑丙酸酯受体功能有关。
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.