Structure and function in innate immunity

Lead Research Organisation: Keele University
Department Name: Research Institute for Social Sciences

Abstract

Advances in biotechnology, biology and biomedicine, and their impact on the quality of life, the economy, medicine and health care increasingly depend on the application of structural biology which provides detailed structural information of the proteins which are central to all life processes. An important aspect of health and well being in all forms of life is the ability to resist and fight infection and disease, and the molecules which make up the immune response are a very important factor in both resistance and recovery. The immune response has two major methods of defence, the innate immune response, which has been preserved and refined throughout evolution, and the antibody-based response, a relatively recent addition. The innate immune response has non-variable components, including molecules known as collectins, and others called pentraxins, which respond in a fairly general, often rapid manner to threatening microorganisms and molecules of both internal and external origin. The Complement cascade provides one route through which molecules of both innate and adaptive immunity can clear their targets. The collectins in the lung are important in resistance to respiratory disease, allergy and asthma. Those in the blood stream, along with the pentraxins and Complement, enable the destruction of molecules and microorganisms of various kinds. Recent work suggests that the pentraxin human CRP, alongside Complement component C1q, may exacerbate rather than reduce inflammation and these molecules have become targets for inactivation by designed drugs. For the collectins, biotechnology can generate parts of the molecules, including the parts that are involved in binding their targets, and these fragments often retain their defensive properties. A major focus of this research is the definition of the three-dimensional structure of pentraxins and of active fragments of collectins, both in their own right and bound to those parts of their targets which they recognise in real life. This will provide invaluable information for the further design of potential diagnostic and therapeutic agents and strategies, for a variety of diseases, by our collaborators and other investigators.

Technical Summary

The collectins, and the pentraxins CRP and SAP, recognise a wide variety of pathogens in a Ca-dependent manner as part of the innate immune response and are involved in a wide variety of immunologic functions, including defence against invading microorganisms, immune system regulation and recognition of receptors on immune effector cells. The complement system is a key mediator of inflammatory reactions and an important effector mechanism of both innate and adaptive immunity. C1q binds to antibody and to CRP and other proteins directly involved in the inflammatory response. The collectins have a similar overall structural appearance to C1q and, in common with CRP, present multiple sites for Ca-dependent ligand-binding at the cell surface. The structural study of these key components of the innate immune response by X-ray crystallography aims to provide a detailed description of ligand-binding, selectivity and specificity, and insights into the effector mechanisms which translate the binding of natural ligands to clearance through appropriate mechanisms. The structural studies on biologically active recombinant fragments of the collectins hSP-D, conglutinin and CL-46, and in the future CL-PI, will focus on the binding of a variety of carbohydrates represented in natural ligands, and the characterisation of the binding of more complex natural ligands such as H. influenzae LPS. No structural detail for conglutinin or CL-46 is currently available. The structural studies on human CRP will concentrate on its interaction with the complement system, in the form of recombinant C1q fragments, and with various ligands, including the activating H. influenzae LPS and S. pneumoniae CPS which effect C1q binding and complement activation. No structural detail for CRP-ligand complexes which effect C1q-binding and complement activation has yet been reported. Structural study of the pentraxins will also include other representative species, for example Limulus polyphemus and the dogfish shark Mustelis canis, as part of our continuing investigation of the humoral and cellular recognition properties of the pentraxins, and of the structural, functional and evolutionary relationships between them. Complementary X-ray and EM studies on Limulus pentraxins are aimed at characterisation of their cytolytic properties and of the structural detail of pore formation.

Publications

10 25 50
 
Description Diamond MX BAG (Co-I)
Amount £457,000 (GBP)
Funding ID MX310, MX6388, MX8359 
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 04/2007 
End 09/2014
 
Description Diamond SRCD time as PI
Amount £10,500 (GBP)
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 10/2009 
End 03/2010
 
Description EM time (via ASMID) as PI
Amount £70,000 (GBP)
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 06/2008 
End 06/2009
 
Description HEFCE Capital Equipment
Amount £61,108 (GBP)
Organisation Higher Education Funding Council for England 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description SRS BAG Beamtime (Co-I)
Amount £186,000 (GBP)
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 05/2006 
End 04/2008
 
Description SRS PX beamtime as PI
Amount £16,000 (GBP)
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 05/2008 
End 08/2008
 
Description SRS SRCD time (Co-I)
Amount £16,000 (GBP)
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 10/2007 
End 03/2008
 
Title EMDB 
Description 3D EM model of Limulus SAP. Electron Microscopy Data Bank accession code 6173. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact Quality of the EM model, and the techniques used to generate it, serve as a template for EM studies of pentraxins. 
URL http://emsearch.rutgers.edu/atlas/1598_summary.html
 
Title pdb 
Description Protein Data Bank coordinate and data files of a variety of hSP-D ligand-bound structures (pdb accession codes 3IKQ, 3IKR, 3IKN, and 3IKP) and Limulus SAP (pdb accession codes 3FLR, 3FLT and 3FLP). Additionally Electron Microscopy Data Bank accession code 6173. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact New understanding of the ligand-binding mechanisms of lung surfactant protein D and the structure, function and evolution of the pentraxins. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=3ikq
 
Title pdb2012 
Description Protein Data Bank coordinate and data files for hSP-D complexed with natural LPS. Open access. 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact Collaborative research program leading to preparation of a research proposal submitted to the MRC in 2013 (not funded) and amended project sent to BBSRC in 2014. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4E52
 
Title pdb2013 
Description Protein Data Bank coordinate and data files for FIBCD1, native (pdb id 4m7h) and ligand-bound (pdb id 4m7f). 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact Putative chitin receptor. Little biological data is available and the structure will provide the impetus for functional studies. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4M7H
 
Description Brunel University 
Organisation Brunel University London
Department School of Health Sciences and Social Care
Country United Kingdom 
Sector Academic/University 
PI Contribution Structural studies of collectins and recombinant fragments of complement component C1q.
Collaborator Contribution Provision of a recombinant fragment of hSP-D and of recombinant monomeric C1q globular heads.
Impact In addition to publications 19799916 and 19452596, earlier highly cited publications include 15364058 (Trends review) and 12888356 (first ligand-bound structure of SP-D).
 
Description California Davis 
Organisation University of California, Davis
Department UC Davis College of Biological Sciences
Country United States 
Sector Academic/University 
PI Contribution Protocols for pentraxin purification, and structural studies of pentraxins from Limulus and dogfish
Collaborator Contribution Provision of limulus haemolymph and dogfish serum. EM studies of limulus pentraxins.
Impact In addition to the publication 19452596 and others in preparation, our understanding of the pentraxins in terms of their evolution and their roles in the immune system has increased substantially. We have now solved the structures of a variety of pentraxins based on this collaboration. Other successful output includes a PhD thesis (Keele) specifically focusing on this collaborative agenda.
 
Description John Radcliffe Hospital 
Organisation Oxford University Hospitals NHS Foundation Trust
Country United Kingdom 
Sector Public 
PI Contribution Structural studies of the interaction of Haemophilus influenzae LPS with a variety of collectins (particularly SP-D) and pentraxins (C-reactive protein)
Collaborator Contribution Expertise, knowledge, and provision of large quantities of the LPS from haemophilus influenzae (strain Eagan)
Impact We have achieved our goal of structural definition of the binding of H. influenzae LPS strain Eagan to the biologically active recombinant fragment of hSP-D provided by our collaboration with the MRC Immunochemistry Unit. A PhD thesis on this topic has been completed and a joint paper with our collaborators is in the final draft stage. We have also received awards from Diamond/STFC for the further investigation by SRCD and EM of the interaction of CRP with LPS and C1q as part of our aim to characterise the effector mechanisms involved in ligand-binding and complement activation by CRP. A PhD thesis (EPSRC DTG supported) has been completed and a paper is in preparation.
 
Description John Radcliffe Hospital 
Organisation University of Oxford
Department Department of Paediatrics
Country United Kingdom 
Sector Academic/University 
PI Contribution Structural studies of the interaction of Haemophilus influenzae LPS with a variety of collectins (particularly SP-D) and pentraxins (C-reactive protein)
Collaborator Contribution Expertise, knowledge, and provision of large quantities of the LPS from haemophilus influenzae (strain Eagan)
Impact We have achieved our goal of structural definition of the binding of H. influenzae LPS strain Eagan to the biologically active recombinant fragment of hSP-D provided by our collaboration with the MRC Immunochemistry Unit. A PhD thesis on this topic has been completed and a joint paper with our collaborators is in the final draft stage. We have also received awards from Diamond/STFC for the further investigation by SRCD and EM of the interaction of CRP with LPS and C1q as part of our aim to characterise the effector mechanisms involved in ligand-binding and complement activation by CRP. A PhD thesis (EPSRC DTG supported) has been completed and a paper is in preparation.
 
Description MRC Immunochemistry Unit 
Organisation Medical Research Council (MRC)
Department MRC Immunochemistry Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Structural studies of collectins and other medically important innate immune proteins.
Collaborator Contribution Provision of expertise, knowledge, and recombinant protein for structural studies.
Impact In addition to publications 19799916 and 16213021, earlier highly cited publications include 15364058 (Trends review) and 12888356 (first ligand-bound structure of SP-D). PhD thesis (MRC DTA supported) submitted (Keele) on ligand-bound structures of hSP-D and a second nearing submission. The collaboration also extends to conglutinin where a successful PhD has been completed and papers are in preparation. Invited talks (Shrive) on the collectin structures include IUCr 2005 (Florence) and INTERLEC 2008 (Edinburgh).
 
Description MRC Immunochemistry Unit / Southampton 
Organisation Medical Research Council (MRC)
Department MRC Immunochemistry Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Structural studies of hSP-D - ligand complexes, with particular reference to the LPS strain Eagan provided through our collaboration with the John Radcliffe Hospital/MRC Harwell, University of Oxford.
Collaborator Contribution Clinical, biological and biochemical data as part of our joint work on characterising the interaction of hSP-D with LPS and other ligands.see above
Impact Publication defining importanc of Asp325 in hSP-D ligand-binding (19799916). A paper is nearing completion on our joint multidisciplinary study of the interaction of H. influenzae strain Eagan LPS with hSP-D. Structural studies at Keele are complemented by biological and biochemical data from Southampton (and, earlier, Oxford). A PhD thesis (Keele) is nearing completion. This will be of considerable medical and scientific impact in the study of H. influenzae and more generally to our knowledge of the interactions of the innate immune system with pathogens.
 
Description MRC Immunochemistry Unit / Southampton 
Organisation Southampton General Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution Structural studies of hSP-D - ligand complexes, with particular reference to the LPS strain Eagan provided through our collaboration with the John Radcliffe Hospital/MRC Harwell, University of Oxford.
Collaborator Contribution Clinical, biological and biochemical data as part of our joint work on characterising the interaction of hSP-D with LPS and other ligands.see above
Impact Publication defining importanc of Asp325 in hSP-D ligand-binding (19799916). A paper is nearing completion on our joint multidisciplinary study of the interaction of H. influenzae strain Eagan LPS with hSP-D. Structural studies at Keele are complemented by biological and biochemical data from Southampton (and, earlier, Oxford). A PhD thesis (Keele) is nearing completion. This will be of considerable medical and scientific impact in the study of H. influenzae and more generally to our knowledge of the interactions of the innate immune system with pathogens.
 
Description University of Southern Denmark, Odense, Denmark 
Organisation University of Southern Denmark
Department Department of Clinical Immunology
Country Denmark 
Sector Academic/University 
PI Contribution Structural studies of native and ligand bound CL46 and FIBCD1. Host to Danish doctoral student involved in production and characterisation of FIBCD1
Collaborator Contribution Knowledge, expertise, and the provision of recombinent protein for structural studies (collectin CL46, FIBCD1)
Impact Paper submitted on completed structures of FIBCD1. Paper in preparation on completed structures of CL46.