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

蛇毒是由多种不同的（约20-100种）毒素混合而成，这些毒素会产生非常不同的效果。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bb12种这种酶，占毒液含量的65%。这些svmp会导致蛇咬伤受害者的组织破坏、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，而另一些则是高度特异性的。后者通常含有额外的蛋白质结构域（如崩解素和富含半胱氨酸），有助于目标识别。众所周知的svmp靶点包括因子X、凝血酶原和纤维蛋白原，它们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其制备为重组蛋白的协议的阻碍。SVMPs的产生通常对产生细胞是有毒的，因此阻止了这些酶的易过表达。为了克服这一生产瓶颈，我们将设计SVMP蛋白及其结构域，并使用杆状病毒昆虫细胞表达系统来生产它们。我们的目标是生产无活性的SVMP酶原，该酶原可以被去除以激活金属蛋白酶。我们还将共同表达支持这些富含半胱氨酸的蛋白质和小抑制肽折叠的伴侣蛋白，以及中和抗体，以促进表达和细胞存活。作为基准和黄金标准，我们还将从毒液中纯化几个具有代表性的svmp。成功生产和纯化的SVMPs和分解素结构域将进一步表征，以确定其特定的靶标和裂解位点，使用功能分析和质谱分析。接下来，我们将以纯化的SVMPs为靶点，选择特异性纳米体（单域抗体片段）作为开发新的蛇咬伤抗蛇毒血清治疗的基础。世界卫生组织宣布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治疗的关键，但目前的抗蛇毒血清是基于高度免疫的马/羊的血清，存在许多缺点，包括有效性差和安全性差。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是这些治疗中和毒素的关键靶点。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纳米体，并测试它们与广泛的SVMP靶点的有效中和和交叉反应性。最后，我们将利用SVMPs和崩解素结构域的生物医学潜力和底物特异性，为患有凝血和出血性疾病的人开发新的抗血小板药物和临床诊断工具。在迄今为止研究的少量svmp中，有几种被用作医院凝血试验的日常标准，而两种崩解素激发了抗血小板药物的设计，用于治疗心绞痛和心脏病发作。我们将利用重组表达、工程和纯化的毒素以及粗蛇毒，发现具有理想特性的新型抗血小板毒素，用于开发治疗血栓形成的药物，同时识别激活凝血因子VIII和IX的毒素，从而开发更好的医院检测方法，用于识别血友病和血管性血友病等出血性疾病患者。

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.