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

奖项摘要越来越多的证据表明，蛋白质是信息素运输的中心。然而，哺乳动物嗅觉中的一个悬而未决的问题是，这些生物学上相关的气味物质是如何穿越嗅觉周围粘膜环境与同源气味受体相互作用的？大型亚洲象广泛的双嗅觉系统为解决两种已识别的信息素：Z-7-十二烯基乙酸酯(Z7-12：Ac)和Frontalin从源到汇的分子轨迹提供了新的和有效的研究优势。最近发现的两种天然对映体形式的前列环素具有特定的生物活性，这是哺乳动物的一项重大发现，提供了一个精确的化学模型。我们富有成效的国际合作描述了白蛋白转运体和气味结合蛋白隔离剂在最后一步之前的功能作用，即信息素在接触犁鼻器(VNO)受体之前通过长犁鼻导管。初步结果支持粘液蛋白促进通道的假说。本文探讨了三个研究问题：(1)VNO导管粘液中是否有一种特定的蛋白能与Z7-12：Ac和Frontalin对映体特异结合，从而转运它们？这项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)犁鼻导管蛋白(VDP)的特性是什么？它的重组形式是否具有生物活性？在分离和鉴定该蛋白并对其基因进行克隆和测序后，将有足够的量进行重组表达。(3)VDP及其配体是否具有适当的生化特性(例如，结合动力学)和生物活性特性，以成为VNO导管转运体？在大象中，单个生化事件的空间和时间减慢和分离允许将周围感受事件的整个序列识别为离散的步骤。VDP信息素复合体的体外动力学分析将与体内离散时间化学传感反应和行为的监测并行进行。这项研究是多学科的，整合了从分子生物学和蛋白质组学到化学传感生物分析和行为的不同科学学科，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，并阐明了特定和功能的蛋白质-配体结合和运输现象。格林伍德和拉斯穆森博士的国际团队的研究将继续广泛影响科学和实际帮助保护的双重传统。濒临灭绝的亚洲象嗅觉知识的进步在实际威慑发展方面具有近乎实用的作用，从而减少了人与象之间的冲突。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。从最广泛的意义上说，这项研究将有助于保护一个与人类互动了数千年的非凡物种，从而使人类社会受益。

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.