Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor*
Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor*
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抑制性甘氨酸受体氨基末端胞外域内二硫键的映射*
DOI：
10.1074/jbc.m109.043448
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
C. Becker
中科院分区：
文献类型：
--
作者：
Nicolas Vogel;Christoph J. Kluck;Nima Melzer;S. Schwarzinger;U. Breitinger;S. Seeber;C. Becker
士的宁敏感性甘氨酸受体（GlyR）是配体门控氯离子通道，并且是半胱氨酸环（Cys-loop）神经递质受体超家族的成员，其还包括烟碱乙酰胆碱受体（nAChR）。在细胞外结构域（ECD）内，epsilon Cys环含有两个保守的半胱氨酸，假定通过超家族特异性二硫键连接。GlyR ECD携带三个额外的半胱氨酸残基，其中两个预计形成第二个GlyR特异性键。然而，GlyR的半胱氨酸的构型都没有被直接确定。基于nAChRα1 ECD的晶体结构，我们生成了人GlyRα1的模型，其中各半胱氨酸的紧密接近与Cys环和GlyR特异性二硫键的形成一致。为了鉴定天然二硫键，将GlyRα1 ECD异源表达并在氧化条件下重折叠。通过基质辅助激光解吸电离飞行时间质谱法，我们检测到的ECD的胰蛋白酶片段指示二硫键形成的半胱氨酸对，提出了建模。使用半胱氨酸和赖氨酸残基的化学修饰确认胰蛋白酶片段的身份。从圆二色性光谱明显，诱变的单个半胱氨酸没有损害在体外的ECD的重折叠，而它导致部分或完全的细胞内滞留，因此在人胚肾293细胞中的全长GlyR亚基的功能丧失。我们的研究结果表明，GlyR ECD形成Cys环和GlyR特异性二硫键。此外，半胱氨酸残基似乎对体内蛋白质成熟很重要。
The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRα1 ECD, we generated a model of the human GlyRα1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRα1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.
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人 α1 甘氨酸受体胞外域的过表达和功能表征。
影响因子：
2.9
作者：
Liu,Zhenyu;Ramanoudjame,Gomathi;Liu,Deqian;Fox,RobertO;Jayaraman,Vasanthi;Kurnikova,Maria;Cascio,Michael
通讯作者：
Cascio,Michael
影响因子：
5.6
作者：
Unwin, N
通讯作者：
Unwin, N
DOI：
10.1016/s0021-9258(18)42693-2
发表时间：
1992-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Katumi Sumikawa;Vaughn M. Gehle
通讯作者：
Katumi Sumikawa;Vaughn M. Gehle
影响因子：
2.9
作者：
Whitmore, Lee;Wallace, B. A.
通讯作者：
Wallace, B. A.