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

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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.