中华蜜蜂普通气味结合蛋白ASP2的气味结合功能模式分析

doi:10.3864/j.issn.0578-1752.2013.01.018

摘要/Abstract

摘要： 【目的】研究中华蜜蜂（Apis cerana cerana）体外重组普通气味结合蛋白AcerASP2与不同气味信息的结合功能和模式。【方法】通过诱导条件的优化和纯化，在获得的重组AcerASP2蛋白基础上，研究竞争性荧光探针1-NPN及不同结构气味信息与蛋白的相互作用关系，并通过同源建模和分子对接解析蛋白与气味信息的结合模式和机理。【结果】通过条件摸索获得了可溶性表达的重组中蜂ASP2蛋白，荧光光谱分析1-NPN与AcerASP2的解离常数K1-NPN为7.38 μmol•L-1，结合位点数n为1.0321。在7种气味信息中，4-烯丙基藜芦醚亲和力最强，其IC50和解离常数KD分别达到7.09和3.46 μmol•L-1。分子对接显示AcerASP2具有1个呈狭长的口袋状的疏水性结合内腔，4-烯丙基藜芦醚绝大部分位于该预测疏水性内腔中，且与Lys74产生2个氢键。【结论】中华蜜蜂AcerASP2由于特殊的空间结构而具有较强的气味信息结合能力，且易通过与疏水内腔中的赖氨酸残基产生氢键而促进结合。

关键词: 中华蜜蜂 , 普通气味结合蛋白 , 气味结合功能 , 分子对接 , 模式分析

Abstract: 【Objective】 The objective of this study is to research the binding function and mode of different odors with general odorant binding protein AcerASP2 of Chinese honeybees (Apis cerana cerana). 【Method】 With the optimization of induction conditions, the purified recombinant AcerASP2 protein was obtained, then the competitive fluorescence assay was used to determine the binding function of AcerASP2 with odors having different structures, finally the homology modeling and molecular docking were applied to elucidate the binding mode and mechanism. 【Result】After the soluble recombinant AcerASP2 protein purified, in the competitive fluorescence assay between AcerASP2 and 1-NPN, the dissociation constants K1-NPN and the number of binding sites n were 7.38 μmol•L-1 and 1.0321, respectively. In selective 7 kinds of odors, the affinity of 4-allylveratrole seemed to be the strongest, and the IC50 and dissociation constant KD were 7.09 and 3.46 μmol•L-1, respectively. The molecular docking results showed that AcerASP2 had one elongated pocket-like hydrophobic cavity, 4-allylveratrole just existed in the cavity, and two hydrogen bonds were found with Lys74 of AcerASP2. 【Conclusion】Because of the particular spatial structure of binding cavity, AcerASP2 could bind diverse odors with different structures, and the binding interaction could be easily to be promoted by the hydrogen bonds between odors with lysines in AcerASP2.

Key words: Apis cerana cerana , general odorant binding proteins , binding function , molecular docking , binding mode

李红亮, 张林雅, 庄树林, 倪翠侠, 韩宝瑜, 商晗武. 中华蜜蜂普通气味结合蛋白ASP2的气味结合功能模式分析[J]. 中国农业科学, 2013, 46(1): 154-161.

LI Hong-Liang, ZHANG Lin-Ya, ZHUANG Shu-Lin, NI Cui-Xia, HAN Bao-Yu, SHANG Han-Wu. Interpretation of Odorant Binding Function and Mode of General Odorant Binding Protein ASP2 in Chinese Honeybee (Apis cerana cerana)[J]. Scientia Agricultura Sinica, 2013, 46(1): 154-161.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.01.018

https://www.chinaagrisci.com/CN/Y2013/V46/I1/154

参考文献

[1]马德风, 梁诗魁. 中国蜜粉源植物及其利用. 北京: 中国农业出版社, 1993: 57-101.

Ma D F, Liang S K. Honey and Pollen Resource Plants in China. Beijing: China Agriculture Press, 1993: 57-101. (in Chinese)

[2]杨冠煌. 引入西方蜜蜂对中蜂的危害及生态影响. 昆虫学报, 2005, 48(3): 401-406.

Yang G H. Harm of introducing the western honeybee Apis mellifera L. to the Chinese honeybee Apis cerana F. and its ecological impact. Acta Entomologica Sinica, 2005, 48(3): 401-406. (in Chinese)

[3]Krieger J, Breer H. Olfactory reception in invertebrates. Science, 1999, 286(5440): 720-723.

[4]Pelosi P, Maida R. Odorant-binding proteins in insects. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1995, 111(3): 503-514.

[5]Pelosi P, Zhou J J, Ban L P, Calvello M. Soluble proteins in insect chemical communication. Cellular and Molecular Life Sciences, 2006, 63(14): 1658-1676.

[6]Nagnan-Le Meillour P, Huet J C, Maibeche M, Pernollet J C, Descoins C. Purification and characterization of multiple forms of odorant/pheromone binding proteins in the antennae of Mamestra brassicae (Noctuidae). Insect Biochemistry and Molecular Biology, 1996, 26(1): 59-67.

[7]Vogt R G, Prestwich G D, Lerner M R. Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects. Journal of Neurobiology, 1991, 22(1): 74-84.

[8]Forêt S, Maleszka R. Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera). Genome Research, 2006, 16(11): 1404-1413.

[9]Danty E, Arnold G, Huet J C, Huet D, Masson C, Pernollet J C. Separation, characterization and sexual heterogeneity of multiple putative odorant-binding proteins in the honeybee Apis mellifera L. (Hymenoptera: Apidea). Chemical Senses, 1998, 23(1): 83-91.

[10]Danty E, Michard-Vanhée C, Huet J C, Genecque E, Pernollet J C, Masson C. Biochemical characterization, molecular cloning and localization of a putative odorant-binding protein in the honey bee Apis mellifera L. (Hymenoptera: Apidea). FEBS Letters, 1997, 414(3): 595-598.

[11]Briand L, Nespoulous C, Huet J C, Takahashi M, Pernollet J C. Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.). European Journal of Biochemistry, 2001, 268(3): 752-760.

[12]Lescop E, Briand L, Pernollet J C, Guittet E. Structural basis of the broad specificity of a general odorant-binding protein from honeybee. Biochemistry, 2009, 48(11): 2431-2441.

[13]Lartigue A, Gruez A, Briand L, Blon F, Bezirard V, Walsh M, Pernollet J C, Tegoni M, Cambillau C. Sulfur single-wavelength anomalous diffraction crystal structure of a pheromone-binding protein from the honeybee Apis mellifera L. The Journal of Biological Chemistry, 2004, 279(6): 4459-4464.

[14]Lagarde A, Spinelli S, Tegoni M, He X, Field L, Zhou J J, Cambillau C. The crystal structure of odorant binding protein 7 from Anopheles gambiae exhibits an outstanding adaptability of its binding site. Journal of Molecular Biology, 2011, 414(3): 401-412.

[15]Kruse S W, Zhao R, Smith D P, Jones D N. Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster. Nature Structural Biology, 2003, 10(9): 694-700.

[16]李红亮, 聂文敏, 高其康, 程家安. 中华蜜蜂气味结合蛋白ASP2 cDNA的克隆及原核表达. 中国农业科学, 2008, 41(3): 933-938.

Li H L, Nie W M, Gao Q K, Cheng J A. Cloning of cDNA encoding odorant binding protein ASP2 in working bee’s antenna of Apis cerana cerana and its prokaryotic expression. Scientia Agricultura Sinica, 2008, 41(3): 933-938. (in Chinese)

[17]Li H L, Zhang Y L, Gao Q K, Cheng J A, Lou B G. Molecular identification of cDNA, immunolocalization, and expression of a putative odorant-binding protein from an Asian honeybee, Apis cerana cerana. Journal of Chemical Ecology, 2008, 34(12): 1593-1601.

[18]李红亮, 高其康, 程家安. 中华蜜蜂信息素结合蛋白ASP1 cDNA的克隆及时空表达. 昆虫学报, 2008, 51(7): 689-693.

Li H L, Gao Q K, Cheng J A. Cloning and spatio-temporal expression of cDNA encoding pheromone binding protein ASP1 in Apis cerana cerana (Hymenoptera: Apidae). Acta Entomologica Sinica, 2008, 51(7): 689-693. (in Chinese)

[19]李红亮, 倪翠侠, 姚瑞, 高其康, 商晗武. 中华蜜蜂化学感受蛋白基因Acer-CSP1克隆与表达特征分析. 昆虫学报, 2010, 53(9): 962-968.

Li H L, Ni C X, Yao R, Gao Q K, Shang H W. Molecular cloning, characterization, and expression pattern of chemosensory protein 1 gene (Acer-CSP1) in the Chinese honeybee, Apis cerana cerana (Hymenoptera: Apidae). Acta Entomologica Sinica, 2010, 53(9): 962-968. (in Chinese)

[20]Li H L, Lou B G, Cheng J A, Gao Q K. The chemosensory protein of Chinese honeybee, Apis cerana cerana: molecular cloning of cDNA, immunocytochemical localization and expression. Chinese Science Bulletin, 2007, 52(10): 1355-1364.

[21]Li H L, Zhang Y L, Wang H Y, Gao Q K, Cheng J A. Spatio-temporal expression analysis of two kinds of chemical communication-related proteins in the worker bee Apis cerana cerana Fabricius (Hymenoptera: Apidae). Chinese Journal of Agricultural Biotechnology, 2009, 6(2): 147-152.

[22]范成平, 商志才, 郭明, 俞庆森. 荧光法研究加潜沙星与牛乳铁蛋白的相互结合作用. 浙江大学学报: 理学版, 2005, 32(1): 62-65.

Fan C P, Shang Z C, Guo M, Yu Q C. Study on the interaction between gatifloxacin and bovine lactoferrin by fluorescence spectrophotometry. Journal of Zhejiang University: Science Edition, 2005, 32(1): 62-65. (in Chinese)

[23]李红亮, 张林雅, 倪翠侠, 商晗武. 中华蜜蜂化学感受蛋白AcerCSP3的配基结合功能分析. 昆虫学报, 2011, 54(3): 259-264.

Li H L, Zhang L Y, Ni C X, Shang H W. Molecular binding characterization with chemical ligands of a chemosensory protein AcerCSP3 in the Chinese honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae). Acta Entomologica Sinica, 2011, 54(3): 259-264. (in Chinese)

[24]Gong Z J, Zhou W W, Yu H Z, Mao C G, Zhang C X, Cheng J A, Zhu Z R. Cloning, expression and functional analysis of a general odorant-binding protein 2 gene of the rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). Insect Molecular Biology, 2009, 18(3): 405-417.

[25]Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics, 2006, 22(2): 195-201.

[26]Deng S, Yin J, Zhong T, Cao Y, Li K. Function and immunocytochemical localization of two novel odorant-binding proteins in olfactory sensilla of the scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae). Chemical Senses, 2012, 37(2): 141-150.

[27]Dani F R, Iovinella I, Felicioli A, Niccolini A, Calvello M A, Carucci M G, Qiao H, Pieraccini G, Turillazzi S, Moneti G, Pelosi P. Mapping the expression of soluble olfactory proteins in the honeybee. Journal of Proteome Research, 2010, 9(4): 1822-1833.