Determining the molecular interactions of serum amyloid P component (SAP)

The human immune system plays a vital role in protecting our bodies from disease. It is a complex combination of specialized cells and proteins that recognize and kill bacteria and viruses. It is also extremely effective in recognizing and eliminating normal cells that may be sick or damaged.

A key protein that we all have in our blood is called serum amyloid P component (SAP). A closely related protein (CRP) is better known as an inflammatory marker that is routinely tested by doctors in blood samples of patients with suspected infection or inflammation. Much research has been performed to understand the role of SAP in numerous diseases especially protein folding conditions (such as systemic amyloidosis), and amyloid-related diseases that principally affect the brain, such as Creutzfeld-Jacob disease (CJD) and Alzheimer's, however we know very little about the normal function of SAP.

A major discovery was that SAP binds to DNA - the component of our cells that contains our genetic information. However, because SAP is normally found in our blood rather than inside cells where the DNA is normally located, it is believed that SAP scavenges DNA that is released into our blood from diseased or damaged cells. This is of critical importance, because it prevents the immune system from attacking our normal DNA, which can have disastrous consequences (such as in the disease systemic lupus erythematosus (SLE)).

As well as binding to DNA, the applicant and others have shown that SAP binds to a number of important targets including RNA - another critically important molecule normally found in our cells. Other important binding targets of SAP include bacteria, the extracellular matrix (a supporting scaffold for cells and tissues) and a wide range of immune proteins and cells. The full significance of these other interactions is yet to be determined.

The aim of this project is to determine how SAP binds to DNA at the molecular level. The applicant has successfully performed preliminary experiments making initial mutants of SAP in collaboration with the Oxford Protein Production Facility (OPPF). Now, in pilot studies with colleagues in Portsmouth who are experts in DNA-protein interactions, the applicant has determined how tightly SAP binds to DNA sequences and, for the first time, to RNA. We are poised to identify the sections of SAP responsible for binding to the various targets by mutating parts of the SAP molecule.

This fundamental research work will help us understand various human conditions linked to ageing. Furthermore, by understanding more about how this protein works, we can determine whether it is a suitable drug target for diseases such as Alzheimer's, SLE and rheumatoid arthritis. Perhaps even more exciting is the possibility that by acting as a DNA scavenger, SAP prevents DNA vaccination working in humans. By understanding more about how SAP recognizes DNA, this work could significantly contribute towards the development of new and safer vaccines.

The aim of this research is to elucidate the structural and biophysical details of the interaction between the pentraxin protein serum amyloid P component (SAP) and nucleic acid ligands.

SAP is a key protein that has been proposed to form a cross-over between the innate and the adaptive immune system. Studying this protein at a biophysical and molecular level will help to clear up a number of controversies in the literature. Translational aspects of this project may be relevant to understanding why DNA vaccination does not work in humans, and to a number of ongoing attempts to deplete or enhance SAP in various human patient populations.

Building upon successful pilot experiments, both native sequence and mutant proteins will be produced using the HEK293 expression system. One particularly important stabilising mutant, E167Q, will be used to determine binding affinities to nucleic acid and known small molecule ligands using Isothermal Titration Calorimetry (ITC).

In parallel to the E167Q binding experiments, electro-mobility shift assays (EMSA's) will be performed on the native sequence protein with double stranded and single stranded DNA and RNA to determine length and sequence specificity. Size exclusion chromatography - multi-angle laser light scattering will be used to confirm the stoichiometry of the complexes formed. Once the highest affinity nucleic acid sequences have been identified, these will be studied further using X-ray crystallography if possible, but also ITC, analytical ultracentrifugation, circular dichroism, small angle X-ray scattering and surface plasmon resonance. These experiments will then be extended using site-directed mutagenesis to identify the specific residues and regions of SAP involved in binding.

Who might benefit from this research?

* Academic, clinical and industrial groups currently conducting trials involving both increasing and decreasing SAP levels.
* Researchers interested in developing DNA vaccination.
* A wide patient population if SAP is shown to play a role in the failure of DNA vaccination.
* Researchers interested in understanding the function and evolution of the immune system.
* Undergraduate (BSc) and postgraduate (MRes) students who will work in my laboratory and contribute in small ways to this project as part of their degrees.
* Those who attend conferences or workshops given by the applicant or PDRA.
* NHS Hampshire A and Ministry of Defence Research Ethics committees.

How might they benefit from this research?

Academic, clinical and industrial beneficiaries will mainly have access to this work through open access published papers and conference presentations. The applicant has a good network of collaborators and contacts with whom the work will regularly be discussed. Use of results generated by this project in pre-clinical and licensing packages may directly influence the success or failure of clinical treatments. Results may also suggest future avenues for drug development, especially regarding DNA vaccination. From a non-clinical perspective, data gained from this project will contribute to knowledge regarding the function and evolution of the immune system.

The research project will be performed within the context of a wider collaboration within the local IBBS (Institute for Biomedical and Biomolecular Science - Portsmouth) and SWSBC (South West Structural Biology Consortium) groupings. Knowledge and expertise gained during this project will contribute towards collaborations and joint grant applications on a number of other projects. Both undergraduate and postgraduate students will be exposed to world leading research as they are completing their studies. The applicant will also talk about this work in his normal public engagement activities including contributions to local BBC radio (currently once every couple of months on science related topics). Details of the work will be presented at both internal and external meetings and conferences by the applicant and PDRA (such as the annual SWSBC meeting, International Amyloid Conference and others). As an expert member of an NHS ethics committee and the Ministry of Defence ethics committee, the applicant will use experience gained on this project during the review of many other research proposals, specifically explaining the technical details of drug development projects to lay members of these committees.