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中心。 然而，哺乳动物嗅觉中一个尚未解决的问题是这些生物学相关的气味物质如何穿过感受器周围的嗅觉粘液环境与相关的气味受体相互作用？ 大嗅觉亚洲象（Elephas maximus）广泛的双重嗅觉系统为解决两种已鉴定的信息素Z-7-十二碳烯乙酸酯（Z7-12：Ac）和frontalin的源-汇分子踪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frontalin所需的特定比例的生物活性是一个重要的哺乳动物的发现，提供了一个精致精确的化学模型。 我们富有成效的国际合作已经描述了白蛋白转运蛋白和气味结合蛋白螯合剂的功能作用之前，最后一步，当信息素通过长犁鼻管运输前接触犁鼻器（VNO）受体。 初步结果支持了粘液蛋白易化通道的假说，并探讨了三个研究问题：（1）VNO导管粘液中是否存在特异性结合Z7-12：Ac和frontalin对映体的蛋白，从而转运它们？ 这项研究将采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？它的重组形式是否保留生物活性？ 在蛋白质被分离和鉴定并且其基因被克隆和测序之后，将重组表达足够量。 (3)VDP及其配体是否具有适当的生物化学性质（例如，结合动力学）和生物活性属性是VNO导管转运蛋白？在大象中，个体生化事件的空间和时间减慢和分离使得整个外周感受事件序列被识别为离散步骤。 VDP-pheromone复合物的体外动力学分析将与体内监测离散的化学感受反应和行为平行进行。这项研究是多学科的，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受生物测定和行为，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，Greenwood博士和Rasmussen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，结合蛋白如何与信息素在手性水平上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.