Integrative Venomics: Studies on venom composition, function and physiology of the venom gland system of non-front-fanged colubroids - using the western hognose snake (Heterodon nasicus) as an example

蛇咬伤具有世界范围内的医学意义，每年有500多万例。临床上最相关的蛇咬伤毒液起源于毒蛇(Viperidae)和眼镜蛇科(Elapidae)，由于它们的牙列，这两种蛇被归类为前牙蛇(FFS)。蛇毒由蛋白质和多肽组成，破译蛇毒在解毒剂、药物的开发和对蛇毒的总体了解中发挥着重要作用。然而，目前的知识很大程度上只起源于那些与医学相关的物种。这种以人类为中心的方法基于一个小的分类学交叉，因为最近的研究表明，比之前认为的更多的蛇是有毒的。这些非前牙猫科动物(NFFC)远远超过750种前牙，有2500种。很少有人研究过NFFC毒液，并显示出与研究得很好的FFS毒液的显著差异。然而，它们的潜力几乎没有被发掘或利用。该项目的目的是了解NFFC毒液系统的组成、功能和生理。我将以带有后牙的西方猪鼻蛇(Heterodon Nasicus)为例来研究这些点。尽管它们被当作宠物饲养，这对毒液挤奶以及保护和名古屋协议都有好处，但几十年来，病例报告一直显示出明显的毒液迹象。症状包括局部疼痛和大量水泡，以及血小板减少。然而，这种毒液从未被详细研究过，也从未用现代分析方法进行过研究。工作流程分为三个部分，考虑了蛋白质组组成、组织中毒素产生和毒液的生理效应等主要方面。在第一部分中，使用最先进的蛇静脉组学协议识别单个毒素和成分。使用高分辨率质谱仪，从整体上(自下而上、自上而下、多肽)确定毒素图谱。在第二部分中，首次通过质谱学成像(MALDI成像，空间毒液组学)确定了西方猪鼻蛇毒腺内毒素的分布，并将其置于直接的组织学背景中。最终将检查毒液功能，使用生物测定来表征NFFC蛇毒的外化学特性。它们包括凝血、细胞毒性、酶活性和潜在的抗微生物活性的分析。这将有助于对西方猪鼻蛇生物学的基本了解，并为更好地了解世界各地NFFC蛇的毒液可塑性提供了一个模型。

Snakebites are of worldwide medical significance, with over 5 million cases per year. The most clinically relevant snakebite envenomation originate from vipers (Viperidae) and elapids (Elapidae), which are grouped together as front-fanged snakes (FFS) due to their dentition. The deciphering of snake venoms, which consist of proteins and peptides, plays a major role in the development of antidotes, drugs, and the understanding of venoms in general. However, current knowledge largely originates only on those species that are medically relevant. This anthropocentric approach is based on a small taxonomic intersection, as recent work has shown that many more snakes are venomous than previously thought. These non-front-fanged colubroids (NFFC) far exceed the 750 FFS species, with >2500 species. Few NFFC venoms have ever been studied and have shown significant differences from the well-studied FFS venoms. And yet their potential has hardly been explored or exploited. The aim of this project is to gain understanding of the venom composition, function, and physiology of the venom system of NFFC. I will examine these points using the western hognose snake (Heterodon nasicus) as an example, with its posterior fangs. Although they are kept as pets, which offers advantages for venom milking as well as conservation and the Nagoya protocols, case reports have shown striking signs of envenomation for decades. Symptoms range from localized pain and massive blistering to thrombocytopenia. However, the venom has never been studied in detail or with modern analytical methods. The workflow is divided into three parts and considers the main aspects of proteomic composition, toxin production in tissues and physiological effects of the venom. In the first part, the individual toxins and components are identified using state-of-the-art Snake Venomics protocols. Using high-resolution mass spectrometry, the toxin profile is determined holistically (bottom-up, top-down, peptidomics). In the second part, the distribution of toxins within a western hognose snake venom gland is determined for the first time of a NFFC by mass spectrometry imaging (MALDI Imaging, Spatial Venomics) and put into a direct histological context. Final will examine venom function, using bioassays to characterize the exochemistry of the NFFC snake venom. They include assays for blood coagulation, cytotoxicity, enzyme activity, and potential antimicrobial activities. This will contribute to the basic understanding of western hognose snake biology and serve as a model to better understand venom plasticity in NFFC snakes worldwide.