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

蛇咬伤在世界范围内具有重要的医学意义，每年有超过500万例。临床上最相关的蛇咬毒素来自毒蛇（Viperidae）和elapids (Elapidae)，由于它们的牙齿，它们被归为前牙蛇（FFS）。蛇毒由蛋白质和多肽组成，对蛇毒的破译在解毒剂、药物的开发和对毒液的总体理解中起着重要作用。然而，目前的知识主要来源于那些与医学相关的物种。这种以人类为中心的方法是基于一个小的分类学交叉点，因为最近的研究表明，比以前认为的有毒的蛇多得多。这些非前牙colcolides （NFFC）远远超过了有牙的750种，有近2500种。几乎没有对NFFC毒液进行过研究，并显示出与经过充分研究的FFS毒液有显著差异。然而，它们的潜力几乎没有被探索或利用。本项目旨在了解NFFC毒液系统的组成、功能和生理。我将以具有后尖牙的西部猪鼻蛇（Heterodon nasicus）为例来检验这些观点。尽管它们被作为宠物饲养，这为毒液的提取以及保护和名古屋协议提供了优势，但几十年来的病例报告显示出明显的中毒迹象。症状范围从局部疼痛和大面积起泡到血小板减少。然而，这种毒液从来没有被详细研究过，也没有被现代分析方法研究过。该工作流程分为三个部分，主要考虑了蛋白质组学组成、组织中毒素的产生和毒液的生理作用。在第一部分中，使用最先进的蛇毒组学协议识别个体毒素和成分。使用高分辨率质谱法，毒素谱被整体确定（自下而上，自上而下，肽组学）。在第二部分中，通过质谱成像（MALDI imaging, Spatial Venomics）首次在NFFC中确定了西部猪鼻蛇毒腺内毒素的分布，并将其置于直接的组织学背景下。最后将检查毒液的功能，使用生物分析来表征NFFC蛇毒的外化学。它们包括血液凝固、细胞毒性、酶活性和潜在抗菌活性的测定。这将有助于对西方猪鼻蛇生物学的基本认识，并为更好地了解世界范围内NFFC蛇的毒液可塑性提供模型。

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.