常见尘螨水通道蛋白结构和功能比较研究

Agre and MacKinnon were awarded the 2003 Noble Prize in Chemistry for the discovery and cDNA cloning of aquaporin (AQP) in human red blood cells. Subsequently, scientists have identified more than 450 AQPs from microbes, plants, animals and human bodies, but there is no report of aquaporins in mites. However, there is presently no experimental method with sufficient spatial and time resolution to monitor permeation through aquaporins on a molecular level. House dust mites accumulate in carpets, bedding, fabrics and furniture, and many species have been demonstrated to be related to human diseases like allergic asthma, dermatitis, and pulmonary, intestinal and urinary acariasis. More than one hundred mite-species have been reported in house dust. Dermatophagoides pteronyssinus is globally distributed , Dermatophagoides farinae breeds in arid regions, Blomia tropicalis is predominant in tropical and subtropical regions, and Euroglyphus maynei is predominant in humid climates. In the present study, the gene fragment encoding AQPs will be amplified from cDNA libraries of the above four mite-species. After sequencing, the amino acid sequences are translated for thes mite aquaporins. By homology modeling, the spatial structure and protein data bank (PDB) structure file are obtained and then embedded in a palmitoyl-oleoyl- phosphatidyl-choline (POPC) lipid bilayer, solvated in water, then minimized and compared using molecular dynamics simulations. The transport of water, glycerol molecules, and other common substances through these AQPs will be simulated to obtain time-resolved, atomic-resolution models to reveal the permeation mechanism. This project will demonstrate for the first time the existence of AQPs in mites and will reveal different spatial structures and transporting abilities of AQPs among different mite species using computational simulations. Our study will provide new ideas for understanding the of geographic distribution of mite species and could provide useful information for new approches to control house dust mites.

Arge等(1988)在红细胞膜上发现了第一个水通道蛋白(Aquaporins,AQPs)，1991完成其cDNA克隆，2003年因此获得了诺贝尔化学奖。迄今，人们已从微生物、植物、动物及人体内鉴定出450余种AQPs，但尚无文献报道螨类AQPs。尘螨与过敏性疾病密切相关，其种类繁多，常见的屋尘螨为全球分布、粉尘螨孳生于干旱地区、热带无爪螨是热带和亚热带地区的优势螨种、梅氏嗜霉螨多见于潮湿地区。本研究拟分别构建此4种常见尘螨的cDNA文库，分别克隆其AQPs基因全长，证实螨类存在AQPs的假说；同源建模得此4种螨类AQPs空间结构，依次建立其动力学平衡体系后进行结构比较，分子模拟各螨种AQPs运输常见物质的过程并计算自由能谱，通过比较证实"不同螨种AQPs结构不同、运输物质能力不同并与其孳生于不同气侯环境有关"的假说。研究有望为进一步针对AQPs设计抑制剂、控制室内尘螨孳生提供科学依据。

水通道蛋白(Aquaporins，AQPs)广泛存于各种生命体，以往文献根据其保守的NPA模序(Asn-Pro-Ala，天冬酰胺–脯氨酸–丙氨酸序列)设计简并引物，扩增获得昆虫以及蜱类只有1-2种AQPs。本研究在实验室内人工培养粉尘螨、屋尘螨、热带无爪螨，分别对其进行转录组测序，功能基因注释获得疑似AQPs，然后RT-PCR验证这些疑似AQPs的存在；对的确存在的基因，进一步用cDNA末端快速扩增技术 (rapid amplification of cDNA ends，RACE)获得全长基因；以总RNA为模板，RT-PCR扩增全长基因并测序，获得了粉尘螨DerfAQP1-4 (GenBank Nos. KY231248、KY231249、KY231250、KY231251)、DerfAQP5.01、DerfAQP5.02 (KY231252、KY231253)， 屋尘螨DerpAQP1-5 (GenBank Nos. KY305293、KY305294、KY305295、KY305296、KY305298)，热带无爪螨BlotAQP1-5 (GenBank Nos. KX655540、KX655541、 KX655542、KX655543、KX655544)。用螨类AQPs与人类AQPs氨基酸序列构建分子进化树，结果显示螨类AQPs分别与人类经典水通道蛋白(classical aquaporins，AQP0、1、2、4、5、6、8)、水甘油通道蛋白(aquaglyceroporins，AQP3、7、9、10)和超级水通道蛋白(S-aquaporins，AQP11、12)聚成一簇。研究进一步采用MODELLER v9.16依次构建了螨类AQPs三维结构，用VMD软件构建其分子模拟体系、用NAMD2.9进行动力学平衡，模拟了其运输水分子的过程，DerfAQP1、DerpAQP1和BlotAQP1自由能计算结果分别为2kcal/mol、3.5kcal/mol、1.6kcal/mol，DerfAQP2和BlotAQP2自由能计算结果为1.8 kcal/mol和4kcal/mol。本研究首次证实螨类AQPs的存在，而且每种螨类至少有5种AQPs，分为三种类型；通过动力学模拟获得对水分子运输起关键作用的氨基酸残基，为进一步寻找安全有效的AQPs抑制剂、控制尘螨孳生提供了依据。