Unravelling the mechanism of complement activation via the lectin pathway

我们的免疫系统对保护我们免受所有类型的细菌、真菌和病毒感染至关重要。有两种主要类型的免疫防御，称为“先天”和“适应性”(基于识别病原体的抗体)。尽管鲜为人知，但先天免疫作为第一道防线，既可以直接摧毁入侵的病原体，又可以帮助适应性免疫系统区分什么是外来细胞(细菌细胞)，什么是自身(宿主细胞)。被称为“补体”的血液蛋白质起到识别和消灭外来入侵细菌的作用。为了有效，补体需要高度特异的识别和激活机制，以便它只有在需要时才被激活，而不是开始攻击宿主组织。在“凝集素途径”中，一种名为MBL的蛋白质专门识别细菌病原体上发现的一系列糖，但不存在于我们自己的细胞中。这种结合然后触发第二种名为MASP的蛋白质的变化，使其从非活性形式转换为活性形式。这两个事件随后导致其他补体蛋白的激活，最终触发细菌细胞膜上形成复合体，导致细菌破裂，以及刺激其他免疫过程和保护功能。尽管凝集素途径是我们免疫系统的关键部分，但它是如何发挥作用的分子细节却知之甚少。例如，MBL和MASP如何相互结合目前尚不清楚，导致MASP激活的变化也是未知的。然而，对这些事件的理解对于我们理解健康的身体是如何运作的以及疾病中哪里出了问题至关重要。这些知识反过来将有助于旨在控制补体激活的治疗学的发展。例如，中风或心脏病发作后，不受控制的补体激活会破坏宿主组织。能够在这些情况下暂时阻止激活(使用药物)将是非常有益的。其次，更好地了解补体有助于免疫缺陷患者的治疗。MBL中一系列常见的基因改变会导致一系列疾病。其中包括儿童早期感染的易感性增加，成人适应性免疫无效的情况下，例如在艾滋病或癌症化疗期间。MBL已成为对抗这些威胁的一个非常好的治疗靶点。我们处于识别补体激活如何发生的理想位置。最近，我们已经建立了MBL的解决方案结构。我们采用了一种独特的方法，将来自蛋白质结晶学的详细信息与基于蛋白质散射、超速离心法和大量计算机模拟计算的新信息相结合。这表明MBL分子是扇形的，并且形成了MASP结合的几乎平坦的模板。本项目的目的将导致对凝集素途径激活的新的全面的理解：(目标1)首先，我们将应用我们的散射和结晶学技术来确定MASP的相应结构。此外，我们将确定MASP内发生的更改，使其自我激活。(目的2)我们最近确定了MBL和MASP小片段之间的复合体的结构。我们将使用这种结构来为整个MBL-MASP综合体制作测试模型。通过针对新的散射和超速离心数据测试这些模型，我们将确定它们的绑定方式。(目的3)我们将确定新的晶体结构，并使用新的散射/超速离心实验来观察MBL在遗传疾病中被修饰后结构会发生什么。特别是，我们将阐明为什么一些MBLS过度激活MASP，而另一些则不会导致MASP激活。通过这种方式，我们将能够阐明当MBL与细菌细胞结合时触发MASP激活的变化。

Our immune system is vital to protect us from all types of bacterial, fungal and viral infections. There are two major types of immune defence, called "innate" and "adaptive" (based on antibodies that recognise pathogens). Although lesser known, innate immunity serves as a first line of defence by both destroying invading pathogens directly and helping the adaptive immune system to distinguish between what is foreign (a bacterial cell) and what is self (host cell). Blood proteins called "complement" act to recognise and destroy invading foreign bacteria. In order to be effective, complement requires highly specific recognition and activation mechanisms so that it only activates when required and does not start to attack host tissues. In the "lectin pathway", a protein called MBL specifically recognises arrays of sugars found on bacterial pathogens but not present on our own cells. This binding then triggers a change in a second protein called MASP, causing it to switch from an inactive to an active form. These two events then lead to activation of other complement proteins, which ultimately trigger formation of a complex in the cell membrane of the bacterium, causing it to burst, as well as stimulating other immune processes and protective functions. Although the lectin pathway is a key part of our immune system, the molecular details of how it works are poorly understood. For example, how MBL and MASP bind to each other is presently unknown as are the changes that lead to MASP activation. However, understanding of these events is crucial for us to comprehend how the healthy body functions and what goes wrong in disease. This knowledge in turn will aid the development of therapeutics aimed at controlling complement activation when things go wrong. For example, following a stroke or heart attack, uncontrolled complement activation destroys host tissues. Being able to block activation temporarily under these circumstances (using medicines) would be highly beneficial. Secondly, a better understanding of complement could facilitate the treatment of patients with immunodeficiencies. A range of common genetic alterations in MBL lead to a broad spectrum of disorders. These include increased susceptibility to infections in early childhood, to instances in adults where adaptive immunity becomes ineffective such as during AIDS or cancer chemotherapy. MBL has become a very good therapeutic target to counteract these threats. We are ideally situated to identify how complement activation occurs. Recently, we have established the solution structure of MBL. We employed a unique approach based on combining detailed information from protein crystallography with new information based on protein scattering, ultracentrifugation and extensive computer modelling calculations. This showed that the MBL molecules are fan-shaped and form an almost flat template for MASP to bind.The aims of this project will result in a new comprehensive understanding of lectin pathway activation: (Aim 1) To start with, we will apply our scattering and crystallography techniques to determine the corresponding structure of MASP. Further, we will identify the changes that take place within the MASP, causing it to self-activate. (Aim 2) We have recently determined the structure of a complex between small fragments of MBL and MASP. We will use this structure to make test models for the entire MBL-MASP complex. By testing these models against new scattering and ultracentrifugation data, we will establish the way in which they bind. (Aim 3) We will determine new crystal structures and use new scattering/ultracentrifugation experiments to see what happens to the structure of MBL when it is modified in genetic diseases. In particular we will clarify why some MBLs over-activate MASP while others result in no MASP activation. In this way we will be able to elucidate the changes that trigger MASP activation when MBL binds to a bacterial cell.