Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.
Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.
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DOI：
10.3389/fphar.2018.00038
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发表时间：
2018
影响因子：
5.6
通讯作者：
Maeyama K
中科院分区：
文献类型：
--
作者：
Suzuki Y;Ogasawara T;Tanaka Y;Takeda H;Sawasaki T;Mogi M;Liu S;Maeyama K
G蛋白偶联受体（GPCRs）是分布于细胞表面的膜蛋白，可能是潜在的药物靶点。然而，体外合成GPCR可能具有挑战性。最近，一些无细胞蛋白质合成系统已被证明产生大量的膜蛋白与化学分子伴侣，包括脂质体和甘油。含有高浓度甘油的脂质体被称为甘油体，其用于新的药物递送系统。甘油体比脂质体具有更大的形态稳定性。蛋白甘油体被定义为含有膜蛋白的甘油体。人组胺H1受体（human histamine H1 receptor，HRH 1）是研究最多的GPCR之一。在本研究中，我们在小麦胚芽无细胞蛋白合成系统中结合asolectin甘油体合成了野生型HRH 1（WT-HRH 1）蛋白甘油体和D107 A-HRH 1（其中Asp 107被Ala取代）。已报道突变体HRH 1对H1拮抗剂具有低亲和力。在本研究中，在一个合成反应中合成的WT-HRH 1的量为434 ± 66.6 μg（7.75 ± 1.19 × 103 pmol）。随着放射性配体浓度的增加，[3 H]吡拉明与WT-HRH 1蛋白甘油体的特异性结合变得饱和。合成的WT-HRH 1的解离常数（Kd）和最大密度（Bmax）分别为9.76 ± 1.25 nM和21.4 ± 0.936 pmol/mg蛋白。然而，与WT-HRH 1相比，与D107 A-HRH 1的特异性结合降低，并且结合没有变得饱和。这项研究的结果强调，HRH 1合成使用小麦胚芽无细胞蛋白质合成系统结合甘油体具有结合H1拮抗剂的能力。
G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs in vitro can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol. Liposomes containing high concentrations of glycerol are known as glycerosomes, which are used in new drug delivery systems. Glycerosomes have greater morphological stability than liposomes. Proteoglycerosomes are defined as glycerosomes that contain membrane proteins. Human histamine H1 receptor (HRH1) is one of the most studied GPCRs. In this study, we synthesized wild-type HRH1 (WT-HRH1) proteoglycerosomes and D107A-HRH1, (in which Asp107 was replaced by Ala) in a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. The mutant HRH1 has been reported to have low affinity for the H1 antagonist. In this study, the amount of synthesized WT-HRH1 in one synthesis reaction was 434 ± 66.6 μg (7.75 ± 1.19 × 103pmol). The specific binding of [3H]pyrilamine to the WT-HRH1 proteoglycerosomes became saturated as the concentration of the radioligand increased. The dissociation constant (Kd) and maximum density (Bmax) of the synthesized WT-HRH1 were 9.76 ± 1.25 nM and 21.4 ± 0.936 pmol/mg protein, respectively. However, specific binding to D107A-HRH1 was reduced compared with WT-HRH1 and the binding did not become saturated. The findings of this study highlight that HRH1 synthesized using a wheat germ cell-free protein synthesis system combined with glycerosomes has the ability to bind to H1 antagonists.
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