中华蜜蜂化学感受蛋白CSP1的功能模式分析及亚细胞定位

【目的】研究中华蜜蜂(Apis cerana cerana,简称中蜂)重组化学感受蛋白CSP1与不同化学信息素的结合功能、模式及其亚细胞定位,明确触角特异表达的CSP1蛋白功能。【方法】将克隆的中蜂CSP1构建至p ET-32a(+)载体并转入BL21(DE3)感受态细菌中,挑取单克隆菌落接种于LB培养基,培养过夜后按1%(V/V)进行转接,继续培养至OD600≈0.4左右时,加入IPTG至终浓度为1 mmol·L~(-1)后继续诱导5 h。将诱导好的CSP1大肠杆菌菌液离心弃上清,再加入细菌裂解液超声破碎,离心后上清用镍柱对CSP1重组蛋白进行亲和层析纯化,再经PBS透析液透析后,最终获得可溶的具有生物活性的CSP1重组蛋白。设定荧光分光光度计的激发波长为281 nm,测定竞争性荧光探针1-NPN与CSP1的相互作用,用Scatchard方程计算其解离常数,再计算获得CSP1与各种候选化学信息素的亲和力。以CSPMbra A6晶体结构(PDB代码:1n8v)为模板,通过同源建模和分子对接解析CSP1蛋白与化学信息素的结合模式,根据Mol Dock Score选出最佳对接模型进行作用机理分析,获得结合时配基周围的CSP1残基分布以及氢键产生情况,以此获得信息素与CSP1的结合模式。最后将CSP1免疫注射兔子获得多克隆抗体,并对中蜂工蜂触角进行低温固定、脱水和包埋后进行超薄切片,然后对样品切片进行免疫胶体金电镜定位,以解析CSP1在触角感器中的亚细胞分布。【结果】成功诱导获得可溶性的重组中蜂CSP1蛋白,利用荧光光谱分析1-NPN与CSP1的解离常数K1-NPN为2.1μmol·L~(-1),结合位点数n为0.99,表明结合时基本以1:1结合,线性相关系数为0.9933。在9种化学信息物质中,CSP1与两种蜜蜂蜂王信息素成分对羟基苯甲酸甲酯(HOB)和9-羰基-2癸烯酸(9-ODA),和植物挥发物成分3-蒈烯均具有较强的结合能力,其中与CSP1亲和力最强的对羟基苯甲酸甲酯的[IC50]和解离常数KD分别达到10.1和7.68μmol·L~(-1)。分子对接显示不同配基与CSP1的结合分别是通过与CSP1疏水腔中特定氨基酸残基(或借助于氢键)作用结合。典型的如CSP1与HOB相互作用过程中,预测主要由8个氨基酸残基贡献能量,包括4个疏水性残基(Phe30、Phe44、Leu70和Phe85),3个极性中性残基(Tyr26、Tyr27和Ser41)以及1个酸性残基(Asp40),其中Asp40中两个羧基上的氧原子分别与HOB苯环中羟基上的氧原子分别产生一个氢键。免疫电镜定位结果显示CSP1主要表达于板形感器周围附属支持细胞中,少量表达于感器内部,这与气味结合蛋白的定位存在明显区别。【结论】中蜂CSP1与两种蜂王信息素成分和某些植物花香成分具有较强的结合能力,集合了信息素结合蛋白和普通气味结合蛋白的功能和相似的作用模式,但其亚细胞定位与气味结合蛋白存在明显区别,显示化学感受类蛋白生理特征的多样性。

Functional Mode and Immunocytochemical Localization of Chemosensory Protein 1 (CSP1) in Apis cerana cerana

【Objective】The objective of this study is to research the functional mode with candidate semiochemicals and immunocytochemical localization of the recombinant chemosensory protein1 (CSP1) in Chinese honey bee, Apis cerana cerana. It has an important theoretical significance for clarifying the function of CSP1, which is specifically expressed in antennae, and enriching the chemosensory mechanism of Chinese honey bee to semiochemicals. 【Method】 CSP1was constructed into the pET-32a (+) vector and transferred into BL21 (DE3) competent E. coli. The single positive clone was inoculated on LB medium and cultured overnight (followed by 1% (v/v) transfer to the OD₆₀₀≈0.4), then added the IPTG with a final concentration of 1 mmol·L^-1 and continued to be induced for 5 h. The recombinant CSP1 protein existing in the supernatant was purified by Ni²⁺-NTA affinity chromatography. The soluble recombinant CSP1 protein with biological activity was finally obtained after dialyzed by PBS (pH 7.4). When the excitation wavelength of the fluorescence spectrophotometer was 281 nm, the interaction between the fluorescent probe 1-NPN and CSP1 was determined. The dissociation constant was calculated by Scatchard equation, and the binding affinities of CSP1 with various candidate semiochemicals were also measured. Based on the CSPMbraA6 crystal structure (PDB entry code: 1n8v) as template, the binding model of CSP1 protein and semiochemicals was analyzed by homology modeling and molecular docking. In order to obtain the binding mode of pheromone and CSP1, according to MolDock Score, the best mechanism of the docking model was analyzed, and the hydrogen bonds between the ligand and the CSP1 residues were obtained. Finally, the polyclonal antibody of CSP1 was obtained by immunizing rabbits, and the antennae of worker bee were immobilized at low temperature, dehydrated and embedded. The subcellular distribution of CSP1 on the antennal sections samples were then immunogolded by colloidal gold and finally observed with the electron microscopy. 【Result】 After the soluble recombinant CSP1 protein was successfully induced and purified, the fluorescence quenching assay was used to obtain the dissociation constant K_1-NPN between CSP1 and 1-NPN as 2.1 μmol·L^-1. The number of binding sites n was 0.99, indicating the bind ratio of CSP1 and 1-NPN was 1﹕1. In the candidate 9 semiochemicals, CSP1 and two queen pheromone components: p-hydroxybenzoic acid methyl ester (HOB) and (E)-9-Oxodec-2-enoic acid (9-ODA), and plant volatile components, 3-carene had a strong ability to bind, the [IC₅₀] and dissociation constant K_D of the most strongly bound HOB was 10.1 and 7.68 μmol·L^-1, respectively. The results of molecular docking showed that the binding of various ligands to CSP1 was due to some specific amino acid residues (or by means of the hydrogen bonds) in the hydrophobic cavity of CSP1. Typically, the interaction between CSP1 and HOB is predicted by the contribution of eight amino acid residues, including four hydrophobic residues (Phe30, Phe44, Leu70 and Phe85), three polar neutral residues (Tyr26, Tyr27 and Ser41) and one acidic residue (Asp40). The two hydrogen bonds were produced between the oxygen atoms on the two carboxyl groups in Asp40 and the oxygen atoms on the hydroxyl group in the HOB benzene ring, respectively. The results of immuno-electron microscopy showed that CSP1 was mainly expressed in the ancillary supporting cells around the sensilla placodea, while only slightly expressed in the inner area of sensilla placodea. It was significantly different from the localization of odorant binding proteins.【Conclusion】 CSP1 of A. c. cerana has a strong ability to bind two queen pheromone components and some plant floral scents, and concentrates the function of pheromone binding protein and general odorant-binding protein. Although their functional modes are similar, there are significant differences in the antennal subcellular localization between CSP1 and odorant-binding proteins, showing the physiological characteristics variation of chemoreceptive proteins.