The Role of Perireceptive Mucoidal Proteins in Defining Vomeronasal Organ Function in a Macrosmatic Mammal

越来越多的证据表明蛋白质是信息素运输的核心。然而，哺乳动物嗅觉中一个尚未解决的问题是，这些生物学上相关的气味如何穿过周围感受性嗅觉粘膜环境，与同源气味受体相互作用？亚洲象广泛的双嗅觉系统为解决两种已鉴定的信息素：z -7-十二烯乙酸酯（Z7-12:Ac）和frontalin的来源-汇分子轨迹提供了新的和有效的研究优势。最近发现的额叶苷的两种天然对映体形式在特定比例的生物活性是一个重要的哺乳动物的发现，提供了一个非常精确的化学模型。我们的国际合作成果描述了白蛋白转运体和气味结合蛋白隔离剂在信息素通过长舌鼻管进入舌鼻器官（VNO）受体之前的最后一步之前的功能作用。初步结果支持了一种粘液蛋白促进传代的假设。本文探讨了三个研究问题：(1)VNO导管黏液中是否存在一种特定的蛋白质特异性地结合Z7-12:Ac和frontalin对映体，从而运输它们？该研究将使用大量的合成信息素和创新的放射性标记和光亲和可视化类似物。(2)这种犁鼻导管蛋白（VDP）的特性是什么？它的重组形式是否保留生物活性？待分离鉴定出该蛋白并对其基因进行克隆和测序后，将有足够数量的蛋白进行重组表达。(3) VDP及其配体是否具有作为VNO导管转运体的适当生化特性（如结合动力学）和生物活性特性？在大象中，个体生化事件在空间和时间上的缓慢和分离使得整个外感受性事件序列被识别为离散的步骤。vdp -信息素复合物的体外动力学分析将与体内对离散时间化学感觉反应和行为的监测并行进行。本研究是跨学科的，整合了不同的科学学科，从分子生物学和蛋白质组学到化学感觉生物测定和行为，整合了生物化学的共同主题，提供了一个独特的机会来量化对信息素的反应，并阐明了特异性和功能性的蛋白质-配体关联和运输现象。由国际医学博士团队进行的调查。格林伍德和拉斯穆森将继续广泛影响科学和实际帮助保护的双重传统。濒危亚洲象嗅觉知识的进步在实际威慑发展中具有直接效用，从而减少人象冲突。最终，结合蛋白如何在手性水平上与信息素相互作用将有助于对脊椎动物嗅觉的基本见解。从最广泛的意义上讲，这项研究将有利于人类社会，因为它有助于保护一个与人类互动了数千年的非凡物种。

AWARD ABSTRACTMounting evidence exists for proteins being central to pheromone transport. However, an unresolved question in mammalian olfaction is how these biologically relevant odorants traverse perireceptive olfactory mucous environments to interact with cognate odorant receptors? The extensive dual olfactory system of the macrosmatic Asian elephant (Elephas maximus) offers novel and valid investigative advantages to resolve the source-to-sink molecular trail of two identified pheromones: Z-7-dodecenyl acetate (Z7-12:Ac) and frontalin. The recent discovery of two natural enantiomeric forms of frontalin required in a specific ratio for biological activity is a significant mammalian finding, providing an exquisitely precise chemical model. Our productive international collaboration has described the functional roles of an albumin transporter and an odorant-binding protein sequestrant prior to the final step when the pheromone transits through the long vomeronasal duct before contacting vomeronasal organ (VNO) receptors. Preliminary results support the hypothesis of passage facilitation by a mucous protein.Three research questions are explored: (1) Is there a particular protein in VNO ductal mucus that specifically binds Z7-12:Ac and frontalin enantiomers, consequently transporting them? The study will employ plentiful synthetic pheromones and innovative radiolabelled and photoaffinity visualization analogs. (2) What is the identity of this vomeronasal ductal protein (VDP) and does its recombinant form retain bioactivity? After the protein has been isolated and identified and its gene cloned and sequenced, sufficient amounts will be expressed recombinantly. (3) Does the VDP with its ligand have appropriate biochemical properties (e.g., binding kinetics) and bioactive attributes to be the VNO ductal transporter?In elephants, spatial and temporal slowing and separation of individual biochemical events allows entire sequences of perireceptive events to be discerned as discrete steps. In vitro kinetic analyses of VDP-pheromone complexes will be conducted in parallel with in vivo monitoring of discrete-in-time chemosensory responses and behaviors.This study is multidisciplinary and integrates diverse scientific disciplines, from molecular biology and proteomics through chemosensory bioassays and behavior, integrating the common theme of biochemistry, providing a unique opportunity to quantify responses to pheromones, and elucidating specific and functional protein-ligand associations and transport phenomena.Investigations by the international team of Drs. Greenwood and Rasmussen will continue the dual tradition of broadly impacting science and practically helping conservation. Advances in knowledge of olfaction in the endangered Asian elephant have proximate utility in practical deterrent development, thereby reducing human-elephant conflict. Ultimately, how binding proteins interact with pheromones at the level of chirality will contribute fundamental insights into vertebrate olfaction. In the broadest sense, this research will benefit human society by helping to preserve an extraordinary species that has interacted with man for thousands of years.