Unravelling the mechanism of complement activation via the lectin pathway

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.

The overall aim of this project is to elucidate the molecular mechanism of complement activation through the lectin pathway by employing a multidisciplinary structural strategy to determine the solution structures of (1) the MASP dimer, (2) MBL-MASP complexes and (3) mutant MBL multimers.

A complete set of small crystal structures of MBL and MASP fragments are now known that define atomic-resolution structures for parts of MASP and MBL. However, these small crystal structures are not able to explain how the full-sized structures of MBL, MASP and MBL-MASP are formed, nor do they provide much mechanistic insights on activation. These full-sized structures are too flexible to be crystallised, and crystallography would not be informative on their important solution properties including the conformational changes that trigger activation.

We have developed a new strategy based on scattering/ultracentrifugation/modelling to determine the solution structures of the MBL oligomers starting from small crystal structures. This revealed near-planar MBL structures in physiological buffers. We will now extend this method to determine solution structures for (1) the MASP dimer and the necessary conformational changes that take place on MASP self-activation, (2) the MBL-MASP complex to establish its assembly and explain how activation takes place, and (3) mutant forms of MBL that over-activate or do not activate the MASP dimer in order to explain the effect of the mutants on MBL function.

We are ideally positioned to complete all three major aims within a single Project Grant. The results will resolve major currently-unanswered questions about the structure of the MBL-MASP complex, and how this activates the lectin pathway, and how abnormalities in MBL lead to disease. This knowledge in turn will reveal novel targets for therapeutics aimed at inhibiting/modulating MBL function in ischaemic diseases where activation of the lectin pathway causes damage to the host.

WHO MIGHT BENEFIT?
Companies working with complement therapeutics (eg: Glaxo-Smith-Kline - UK; Alexion and Omeros - USA) will benefit from this research by enabling them to predict how to modify lectin pathway activation. Thus the function of the human complement system will be harnessed in order to combat bacterial infections through a natural route. This knowledge should notably reduce the development times for potential MBL-based therapeutic compounds in diseases that involve excessive lectin pathway activation, in particular ischemia reperfusion disorders (heart attack and stroke) caused by MBL-MASP activation on self tissues. The time scale for these developments will be medium-long term.

Common polymorphisms in mutant MBL increase susceptibility to disease in children and adults. Immunodeficient patients will benefit because the development of new MBL-based therapeutic compounds will address this unmet clinical need, especially in circumstances when the resistance of bacteria to common powerful antibiotics is on the rise. Benefits to the NHS relate to the possibility of reducing costs through the more effective control of infections. The lectin pathway is implicated in both childhood diseases and immune-compromised adult patients. Since current therapies are based on treatment of symptoms/infections (eg with antibiotics), more effective medication will be vital in terms of stretched NHS budgets.

HOW MIGHT THEY BENEFIT?
The research will fundamentally characterise the early structural events that lead to the recognition of bacterial pathogens by MBL and lectin pathway activation. This will reveal common themes between the lectin pathway and the classical pathway of complement activation. The knowledge of an accurate structural model for the MBL-MASP complex, and likewise the knowledge of how the mutant MBL structures differ from the wild-type MBL structures, will significantly facilitate an understanding of how MBL-MASP activation takes place. This improved understanding will better direct rational protein predictive analyses to elucidate how best to target the lectin pathway. It should be possible to design complement inhibitors that target MBL by blocking the multiple binding sites for MASP within the MBL multimeric structure. In cases where there is insufficient MASP activation, a mechanistic understanding of how MBL activates MASP (at both levels of structures and affinities) may lead to alternative therapeutic approaches that aim to convert MBL into a more efficient activator. This understanding will continue to improve as academic beneficiaries in the UK refine their predictive models for MBL-MASP activation by including new patient data on different MBL mutants and determine more detailed effects of solution conformation and specific protein-protein interactions in MBL and MASP.

The UK economy will benefit because this academic research will complement the UK's strength in biomedical discovery. Collaboration between complement biochemists, complement-based clinicians and protein biophysicists on medically relevant therapeutic proteins will ensure effective knowledge and skills transfer between biomedical science and the UK pharmaceutical industry. This will expand its position in the global healthcare market and attract further R&D investment from global business which recognises the UK as a good place to conduct these activities. Such retention of expertise, know-how and intellectual property will aid the UK's capacity to remain internationally competitive.

Benefits from skills training: The postdoctoral researcher (PDRA) will gain experience at the interface between protein interactions biophysics, protein crystallography, protein molecular graphics modelling and clinical immunology. This will provide a sound basis for an academic or industrial career in biomedicine. The technician to be recruited will receive training and experience in the purification/handling of proteins important in immunology