Structural studies to advance understanding of IgM and its complement and receptor interactions

人类免疫系统通过各种细胞(如白细胞)和蛋白质(如抗体)提供对外来生物的保护，包括细菌、病毒和寄生虫。人类和其他动物一样，有一些特殊的抗体类型，但被称为IgM的抗体类型在进化上最保守，存在于所有有抗体的脊椎动物中。毫不奇怪，它在免疫中发挥着基本的功能，但事实上，在个人遇到入侵的有机体或外来“抗原”之前，它就已经存在于血液中，从而提供了对常见病原体的一般保护。这种循环中的IgM还在帮助清除死亡和濒临死亡的细胞方面发挥作用，这是一种基本而持续的活动；当它失败时，这些细胞及其内容物的积聚可能会导致危险的情况，并可能导致自身免疫反应，在这种反应中，免疫系统转向反对个人自身的蛋白质。免疫球蛋白还决定了健康免疫反应的进程，一旦它识别出一种抗原是外来的，它就会结合并激活防御机制，最终导致该抗原的清除。这些机制的启动涉及到与血液中其他蛋白质分子的相互作用，其中一个被称为C1q，或者在各种细胞的表面--这些后者被称为IgM的“受体”。这些受体中的一个三年前才被发现，似乎可以增强免疫反应，而我们将研究的另一个可能起到抑制作用。免疫反应调节中的正反馈和负反馈对健康至关重要：过度反应(例如过敏反应，尽管是由另一种抗体引起的，或由IgM引发的自身免疫反应)与反应不足一样危险。人们对这些相互作用知之甚少，也几乎对它们激活防御机制的方式一无所知；这个项目的目的是通过研究这些分子的结构来促进我们对免疫反应中这些关键步骤的理解。IgM分子结构的一个显著方面是它是“聚合的”，而且比其他类型的抗体大得多。大多数抗体是Y形的，有两个“臂”来检测和结合抗原，还有一个“茎”，通过它与其他分子和细胞上的受体结合。免疫球蛋白由五个或六个这样的Y形单位组成，这些单位紧密地连接在一起，形成一个环。然而，尽管它们的大小、普遍存在和对人类健康的基本重要性，我们对IgM的结构没有详细的了解。我们不知道这些单位是如何结合在一起的，如果我们要理解IgM是如何工作的，这样的知识是必不可少的：例如，在感觉到外来抗原之前，它是如何阻止与C1q结合的？有时我们想要控制IgM的活性，例如当它用于治疗目的时，我们想限制它激活炎症反应的能力；只有当我们知道IgM是如何工作的，以及它如何与C1q和它的受体结合时，我们才能做到这一点。这项工作将涉及生产IgM分子的关键部分，既作为单一的单体片段，也作为五聚体和六聚体。它们以前从未以足够纯的形式制成，无法研究它们的结构。我们还将生产C1q和IgM受体蛋白的IgM结合部分。为了确定这些分子的详细三维结构并了解它们是如何相互作用的，我们将使用一种涉及X射线的技术来定位结构中所有原子的位置。使用其他技术，我们将测量绑定相互作用的强度，并了解我们可能如何控制它们。因此，这些研究将揭示免疫的基本机制，并将对设计免疫球蛋白M的活性具有较长期的好处，当用于治疗时。

The human immune system provides protection against foreign organisms including bacteria, viruses and parasites through various cells such as the white blood cells, and proteins such as antibodies. In man, as in other animals, there are specialized types of antibody, but the type known as IgM is the most evolutionarily conserved and is present in all vertebrates that have antibodies. Not surprisingly, it performs essential functions in immunity, but in fact is present in the blood even before an individual encounters an invading organism or foreign "antigen", thus providing general protection against common pathogens. This circulating IgM also plays a role assisting in the clearance of dead and dying cells, an essential and continuous activity; when it fails, the build up of these cells and their contents can cause dangerous conditions and can lead to autoimmune reactions, in which the immune system turns against the individual's own proteins. IgM also determines the course of a healthy immune response, and once it has recognized an antigen as foreign, it binds and then activates defence mechanisms that will eventually lead to the clearance of the antigen. The initiation of these mechanisms involves interactions with other protein molecules in the blood, one of which is called C1q, or on the surfaces of various cells - these latter molecules are called "receptors" for IgM. One of these receptors was only identified three years ago and appears to enhance the immune response, while another that we will study may play an inhibitory role. Positive and negative feedback in the regulation of an immune respose is critical for health: over-reaction (allergic hypersensitivity for example, although caused by another type of antibody, or an auto-immune reaction initiated by IgM) is just as dangerous as under-reaction. Very little is known about any of these interactions and virtually nothing about the way in which they activate the defence mechanisms; the aim of this project is to advance our understanding of these key steps in the immune response through studying the structures of these molecules.One remarkable aspect of the structure of the IgM molecule is that it is "polymeric", and much larger than the other types of antibody. Most antibodies are Y-shaped, with two "arms" to detect and bind to antigens, and a "stem" through which they bind to other molecules and to receptors on cells. IgM consists of either five or six of these Y-shaped units linked closely together in a ring. Yet despite their large size, ubiquitous occurrence and fundamental importance to human health, we have no detailed knowledge of the structure of IgM. We don't know how the units fit together, and such knowledge is essential if we are to understand how IgM works: how for example does it prevent binding to C1q until it senses a foreign antigen? Sometimes we would like to control the activities of IgM, such as when it is administered for therapeutic purposes and we want to restrict its ability to activate inflammatory reactions; we will only be able to do this if we know how IgM works and how it binds to C1q and to its receptors.The work will involve producing the key parts of the IgM molecule, both as the single "monomeric" pieces and as the pentamer and hexamer. These have never been made before in a pure enough form to study their structure. We shall also produce the IgM-binding parts of the C1q and IgM receptor proteins. To determine the detailed three-dimensional structures of these molecules and to see how they interact with each other, we shall employ a technique that involves X-rays to locate the positions of all of the atoms in the structure. With other techniques we will measure the strengths of the binding interactions and understand how we might control them. These studies will thus reveal fundamental mechanisms of immunity and will have longer-term benefits for engineering the activities of IgM when used therapeutically.