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（如白色血细胞）和蛋白质（如抗体）提供针对外来生物体（包括细菌、病毒和寄生虫）的保护。人类和其他动物一样，也有特殊类型的抗体，但IgM是进化上最保守的类型，存在于所有具有抗体的脊椎动物中。毫不奇怪，它在免疫中发挥重要作用，但事实上，甚至在个体遇到入侵生物体或外来“抗原”之前，它就存在于血液中，从而提供对常见病原体的一般保护。这种循环IgM还起到帮助清除死亡和垂死细胞的作用，这是一种必要和持续的活动;当它失败时，这些细胞及其内容物的积累可能会导致危险的情况，并可能导致自身免疫反应，其中免疫系统转而对抗个体自身的蛋白质。IgM还决定了健康免疫反应的过程，一旦它将抗原识别为外来抗原，它就会结合并激活防御机制，最终导致抗原的清除。这些机制的启动涉及与血液中其他蛋白质分子的相互作用，其中一种称为C1 q，或在各种细胞的表面上-这些后一种分子被称为IgM的“受体”。其中一种受体仅在三年前被发现，似乎可以增强免疫反应，而我们将要研究的另一种受体可能起抑制作用。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（例如过敏性超敏反应，尽管是由另一种类型的抗体引起的，或由IgM引发的自身免疫反应）与反应不足一样危险。我们对这些相互作用知之甚少，对它们激活防御机制的方式也几乎一无所知;本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“聚合物”，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”，通过它它们与其他分子和细胞上的受体结合。IgM由五个或六个这样的Y形单位紧密连接在一起组成一个环。然而，尽管它们体积大，无处不在，对人类健康至关重要，但我们对IgM的结构没有详细的了解。我们不知道这些单位是如何组合在一起的，如果我们要了解IgM是如何工作的，这些知识是必不可少的：例如，它是如何阻止与C1 q结合的，直到它感觉到外来抗原？有时我们想控制IgM的活性，例如当它用于治疗目的时，我们想限制其激活炎症反应的能力;我们只有知道IgM的作用机制，以及它如何与C1 q及其受体结合，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”片段又作为五聚体和六聚体。以前从来没有制造出足够纯净的形式来研究它们的结构。我们还将生产C1 q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如何相互作用，我们将采用一种涉及X射线的技术来定位结构中所有原子的位置。通过其他技术，我们将测量结合相互作用的强度，并了解我们如何控制它们。因此，这些研究将揭示免疫的基本机制，并将在治疗上使用时对工程IgM的活性具有长期益处。

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.