Mechanism of binding of mycobacterial glycolipids to mincle, a stimulatory glycan-binding receptor on macrophages

Different cells are characterised by different patterns of sugars linked to their surfaces. One mechanism used by the innate immune system, which responds directly to microbial pathogens before a specific antibody response is mounted, is to identify pathogens based on the way the sugars on their surfaces differ from those of the host. The work we propose is directed toward developing a molecular understanding of how mincle, a sugar-binding receptor on the surface of macrophages from humans and other mammals, binds to unusual sugars on the surface of mycobacteria, which are the causative agent of tuberculosis in humans, cows and other mammals. Mincle is believed to play a role in the complex interaction between mycobacteria and the host immune system that leads to development of tuberculosis.

The approaches that will be undertaken include creating small molecules that mimic portions of the cell surface of the mycobacteria to see which features are essential for binding to mincle and leading to stimulation of macrophages. The molecules will also facilitate structural analysis of how the binding occurs. Mincle from humans, cows and mice will be examined. The first two species are important because of the way that naturally occurring mycobacterial infections affect them, leading to an enormous worldwide disease burden in humans and providing a major challenge to the safety of the domestic cattle industry. Mice are used as models for study of mincle function in the whole organism, so it is important to demonstrate that mincle from mice has the same properties as the human and cow proteins. In addition to these studies of the binding of mincle to mycobacterial sugars, further studies will address the question of how mincle interacts with other proteins in the macrophage membrane so that the binding of sugar targets can be translated into stimulation of the macrophages.

This is a basic science proposal directed toward elucidating the mechanisms underlying the functions of a poorly understood receptor. However, there are clear practical goals that will benefit from the information that will be obtained, including the fact that an improved understanding of how mycobacteria can interact with the host immune system may provide insights into how this process can be altered in a way that reduces the disease burden. Most directly, this understanding will provide knowledge necessary to exploit the fact that mycobacterial surface components are able to stimulate the immune system and improve production of antibodies. Up to now, this property has not been used in human vaccine development because of the complexity and uncontrolled nature of the reaction to mycobacteria, but the proposed studies will help to define simpler molecules that can trigger the beneficial effects in more controlled ways for vaccine development in humans and animals.

Mincle is a macrophage receptor that binds several structurally distinct oligosaccharide ligands and which is able to activate cytokine secretion. The goal of this project is to develop a molecular understanding of the basis for sugar recognition and how sugar binding might lead to intracellular signalling events. Ligands for mincle that are of particular interest are glycolipids found in the outer membrane of mycobacteria. Analysis of the binding of these ligands will provide insight into the role of mincle in mycobacterial infection leading to tuberculosis in humans and in cows and will also inform attempts to exploit the stimulatory properties of mincle in development of novel adjuvants. The objectives of the project are: (1) To define the ligand-binding specificity of mincle by developing a panel of synthetic soluble analogues of trehalose dimycolate and testing these compounds in binding competition assays along with screening of arrays of synthetic and pathogen-derived glycans. (2) To investigate the mechanism of ligand binding by creating suitable targets for crystallisation of mincle-ligand complexes. (3) To characterise mincle from key animal species: mouse because it is used as a model for study of mincle function in vitro and cow because of the potential importance of mincle in mycobacterial infection leading to bovine tuberculosis. (4) To analyse the oligomeric structure of mincle and examine the molecular basis for the interaction between mincle polypeptides and the ITAM-containing FcRgamma subunits, which leads to downstream signalling.

Potential beneficiaries
As this is a basic science proposal, the immediate beneficiaries will be in the academic community as detailed in the preceding section. There is also significant potential for benefit to industry and society resulting from novel avenues to enhancing vaccine development.

Potential benefits emerging from the science
The adjuvant activity of mycobacteria has been long recognised and the predominant role of the glycolipid trehalose dimycolate, also designated as cord factor is well established. It has also been clear that the pathway to an enhanced immune response is through cytokine production by macrophages. However, the importance of mincle as the receptor linking these two has only recently been noted. Our proposal to develop a mechanistic understanding of how mincle binds to the glycolipid will lay the foundation for rational design of molecules that mimic the adjuvant activity of the mycobacterial product. Such alternative adjuvants could provide a major advance in vaccine technology, since mycobacteria themselves have long been used as highly effective adjuvants in experimental animals but have been considered too dangerous for use in humans. A molecular understanding of how mincle recognises its ligand will facilitate development of molecules that selectively invoke the stimulatory activity without unwanted side effects. In addition to their use in vaccine development, such improved stimulators of macrophages provide a molecular approach to enhancing the immune response in cancer patients, which is now achieved in a less controlled way using mycobacterial therapy.

The UK skills base
The UK has historically had a world-leading role in the investigation of the nature of cell surface glycosylation, highlighted by determination of the nature of the blood group substances, and maintains a high profile in the area of glycomics, describing the nature of the sugars present in cells and tissues. To benefit fully from the knowledge being developed in this area, an understanding of the functions of the sugars is essential, and analysis of sugar-specific receptors potentially provides the most important route to such understanding. Because of the special challenges associated with the chemistry and biochemistry of glycosylation, progress in this area is absolutely dependent on the availability of investigators skilled and knowledgeable in these areas but also able to undertake biochemical and cell biological analysis of the receptors. This research project provides an ideal multi-disciplinary training opportunity for developing such talent. The ability of UK industry to exploit knowledge of glycomics will depend on individuals with this type of training.