Haemotoxic and cytotoxic snake venom metalloproteinases - production, enzymatic specificity, snakebite treatment, and biomedical use

蛇毒是由许多不同的(约20-100)毒素组成的鸡尾酒，这些毒素会造成非常不同的影响。蛇毒金属蛋白酶(SVMPs)是一类毒素，在蛇毒中含量特别丰富，通常在一种毒液中发现12种这种酶，占毒液成分的65%。这些SVMP会导致蛇咬伤患者的组织破坏、全身出血和凝血障碍，从而导致死亡或终身残疾。一些SVMP作用于多个目标，而另一些则非常具体。后者通常包含有助于靶标识别的额外蛋白结构域(例如，去整合素和富含半胱氨酸)。众所周知的SVMP靶标包括凝血因子X、凝血酶原和纤维蛋白原，所有这些都负责控制血液凝固，以及血管壁的各种成分或血小板上的受体。然而，由于SVMP很难相互分离，这些生物活性蛋白的功能表征目前受到缺乏如何将它们制备为重组蛋白的方案的阻碍。SVMPs的产生通常会对产生细胞产生毒性，从而防止这些酶的容易过度表达。为了克服生产中的这一瓶颈，我们将对SVMP蛋白及其结构域进行工程改造，并使用杆状病毒昆虫细胞表达系统来生产它们。我们的目标是生产具有前结构域的非活性SVMP酶原，该前结构域可以被移除以激活金属蛋白酶。我们还将共表达支持这些半胱氨酸蛋白和小抑制性多肽折叠的伴侣蛋白，以及中和抗体，以促进表达和细胞生存。作为基准和黄金标准，我们还将从毒液中提纯几个具有代表性的SVMP。成功生产和纯化的SVMP和去整合素结构域将被进一步表征，以利用功能分析和质谱学来确定它们的特定靶点和切割位点。接下来，我们将以纯化的SVMPs为靶点，筛选特定的纳米体(单域抗体片段)，作为开发新型蛇咬伤抗蛇毒治疗的基础。世界卫生组织宣布毒蛇咬伤是一种被忽视的热带疾病，每年有10万人死亡。提供安全、有效的抗蛇毒血清是挽救生命的关键，但目前的抗蛇毒血清是以高度免疫的马/羊的血清为基础的，存在许多缺点，包括效果差和安全性差。因此，显然需要基于毒素特异性重组抗体或抗体片段的抗蛇毒血清，而SVMP是这些治疗方法中和的关键毒素靶标。作为新的抗蛇毒血清的基础，我们将使用‘核糖体体外选择和进化’技术从合成库中选择抗SVMP纳米抗体，并测试它们与广泛的SVMP靶标的有效中和和交叉反应。最后，我们将利用SVMPs和去整合素结构域的生物医学潜力和底物特异性，为患有凝血和出血疾病的患者开发新的抗血小板药物和临床诊断工具。到目前为止，在所研究的少量SVMP中，有几种每天都被用作医院凝血测试的标准，而两种去整合素启发了用于治疗心绞痛和心脏病发作的抗血小板药物的设计。我们将使用我们的重组表达、工程和纯化毒素以及粗蛇毒来发现具有治疗血栓的药物开发所需特性的新的抗血小板毒素，同时识别能够激活凝血因子VIII和IX的毒素，以使开发出更好的医院检测方法来识别患有出血性疾病(如血友病和von Willebrand病)的患者。

Snake venoms are composed of a cocktail of many different (~20-100) toxins that cause very diverse effects. The snake venom metalloproteinases (SVMPs) are a family of toxins particularly abundant in viper venom, often with >12 of these enzymes found in a single venom and making up to 65% of the venom content. These SVMPs are responsible for causing destruction of tissue, systemic bleeding and blood clotting disorders in snakebite victims, which can result in death or lifelong disability. Some SVMPs act on multiple targets, while others are highly specific. The latter often contain additional protein domains (e.g. disintegrin and cysteine-rich) which contribute to target recognition. Well-known SVMP-targets include factor X, prothrombin and fibrinogen, all of which are responsible for controlling blood clotting, as well as various components of the walls of blood vessels or receptors on platelets. However, because SVMPs are difficult to isolate from one another, functional characterisation of these bioactive proteins is currently hampered by a lack of protocols on how to prepare them as recombinant proteins. Production of SVMPs is usually toxic to the producing cells, thereby preventing facile overexpression of these enzymes. To overcome this bottleneck in production we will engineer the SVMP proteins and their domains and use a baculovirus insect cell expression system to produce them. We aim to produce inactive SVMP zymogens with a prodomain that can be removed to activate the metalloproteinase. We will also co-express chaperones that support the folding of these cysteinerich proteins and small inhibitory peptides, as well as neutralising antibodies, to facilitate expression and cell survival. As benchmarks and gold standards, we will also purify several representative SVMPs from venom. Successfully produced and purified SVMPs and disintegrin domains will be further characterised to determine their specific targets and cleavage sites using functional assays and mass spectrometry. Next, we will use purified SVMPs as targets to select specific nanobodies (single-domain antibody fragments) as the basis for the development of new snakebite antivenom treatment. The World Health Organization declared snakebite envenoming as a neglected tropical disease with >100,000 deaths occurring annually. Provision of safe, efficient antivenom is key to life-saving treatment, yet current antivenoms are based on sera of hyper-immunised horses/sheep and have many shortcomings, including poor effectiveness and poor safety profiles. There is therefore a clear need for antivenom based on toxin-specific recombinant antibodies or antibody fragments, and SVMPs are the key toxin targets for neutralisation by these treatments. As the basis of new antivenom, we will select anti-SVMP nanobodies from a synthetic library using 'ribosome display in vitro selection and evolution' technology, and test them for their efficient neutralisation and cross-reactivity with a broad range of SVMP targets. Finally, we will harness the biomedical potential and substrate specificity of SVMPs and disintegrin domains for the development of new anti-platelet drugs and clinical diagnostic tools for people suffering from blood clotting and bleeding disorders. Of the small number of SVMPs studied to date, several are used on a daily basis as standards for hospital blood clotting tests, while two disintegrins inspired the design of anti-platelet medications that are used for treating angina and heart attacks. We will use our recombinantly expressed, engineered and purified toxins, as well as crude snake venoms, to discover new anti-platelet toxins with desirable characteristics for drug development for treating thromboses, while simultaneously identifying toxins that activate blood clotting factors VIII and IX to enable the development of better hospital tests for identifying patients suffering from bleeding disorders like haemophilia and von Willebrand's disease.