Unravelling the mechanism of complement activation via the lectin pathway

我们的免疫系统对保护我们免受各种细菌、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）。先天免疫虽然鲜为人知，但它作为第一道防线，既能直接摧毁入侵的病原体，又能帮助适应性免疫系统区分外来（细菌细胞）和自身（宿主细胞）。血液中被称为“补体”的蛋白质可以识别并摧毁入侵的外来细菌。为了发挥作用，补体需要高度特异性的识别和激活机制，以便它只在需要时激活，而不会开始攻击宿主组织。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糖阵列，但不存在于我们自己的细胞中。这种结合会触发另一种叫做MASP的蛋白质的变化，使其从无活性的形式转变为活性的形式。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形成复合物，导致其破裂，同时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，但其工作原理的分子细节却知之甚少。例如，MBL和MASP如何相互绑定目前尚不清楚，导致MASP激活的变化也是如此。然而，了解这些事件对于我们理解健康的身体如何运作以及疾病中的问题是至关重要的。这一知识反过来将有助于在出现问题时控制补体激活的治疗方法的发展。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。在这种情况下（使用药物）能够暂时阻断激活将是非常有益的。其次，更好地了解补体可以促进免疫缺陷患者的治疗。MBL中一系列常见的遗传改变导致广泛的疾病。其中包括儿童早期对感染的易感性增加，以及成人在艾滋病或癌症化疗期间适应性免疫失效的情况。MBL已经成为对抗这些威胁的一个很好的治疗靶点。我们处于确定补体活化如何发生的理想位置。最近，我们建立了MBL的解决方案结构。我们采用了一种独特的方法，将蛋白质晶体学的详细信息与基于蛋白质散射、超离心和广泛的计算机建模计算的新信息相结合。这表明MBL分子呈扇形，形成一个几乎平坦的模板供MASP结合。该项目的目的将导致对凝集素途径激活的新的全面理解：（目标1）首先，我们将应用我们的散射和晶体学技术来确定MASP的相应结构。此外，我们将确定在MASP中发生的导致其自激活的变化。（目的2）我们最近确定了MBL和MASP小片段之间复合物的结构。我们将使用这个结构为整个MBL-MASP复合体制作测试模型。通过针对新的散射和超离心数据测试这些模型，我们将建立它们结合的方式。（目标3）我们将确定新的晶体结构，并使用新的散射/超离心实验来观察MBL在遗传疾病中被修饰时结构会发生什么变化。特别是，我们将澄清为什么一些mbl过度激活MASP，而另一些则导致没有MASP激活。通过这种方式，我们将能够阐明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.