Molecular analysis of complement activation via the classical pathway

Lead Research Organisation: University of Leicester
Department Name: Infection Immunity and Inflammation

Abstract

The human immune system uses two main weapons to counter invading pathogens: adaptive immunity, which includes the production of antibodies that develop over time to target invading bacteria; and innate immunity, which is ever present and recognizes conserved parts of bacteria, such as their cell walls. The complement cascade provides a bridge between innate and adaptive immunity: it destroys bacteria directly via innate processes and helps to direct and stimulate an effective adaptive response. Because of its central role, complement dysfunction is associated with a range of human diseases, including severe bacterial infections, kidney disease and autoimmune diseases such as lupus and rheumatoid arthritis. One of the main pathways of complement activation is known as the classical pathway, in which complement proteins bind to bacteria and set off a chain of reactions directed at killing them. In this grant proposal, we aim to develop a thorough understanding this pathway by studying the way in which the components interact with each other to initiate the reaction cascade. We have already established methods for producing key elements using recombinant DNA technology so that their mode of action can be investigated in a systematic way. As well as providing novel information that will help us to understand how complement works, this project will also foster the development of novel reagents which can be used to change complement activity. For example, inhibitors of complement could be used to prevent tissue damage in conditions where complement activates at the wrong time or place. Similarly, modified components can be used to bind and neutralize specific targets such as antibiotic resistant bacteria or diseased cells.

Technical Summary

The classical pathway of complement activation provides a frontline defence against invading pathogens. It is a central effector of natural protection, one of two current research priorities of the MRC. It neutralises invading cells directly via antibody-dependent and -independent mechanism and elicits a range of cellular responses designed to protect the host and eliminate foreign material, including inflammation, phagocytosis and stimulation of an effective adaptive immune response. Despite its important roles, the mechanisms underlying the activation and regulation of this branch of the immune system are relatively poorly understood. The goal of the proposed work is to understand how molecular interactions control complement activation via C1, the initiating complex of the classical pathway. Biochemical and structural approaches are specifically designed to identify detailed binding and structural information. The results of this study are expected to be of significance to both basic and applied applications. Understanding the fundamental issues of recognition in complement activation has general practical applications in protein-protein interactions and protein engineering and lays the foundations for the development of therapeutic agents designed to prevent complement-mediated host damage for use in the treatment of inflammatory diseases, such as rheumatoid arthritis and in ischemia-reperfusion disorders. As part of this project, short constrained peptide mimics will be developed, synthesized and characterized. These will serve as specific inhibitors of C1q-C1rs interactions, providing a novel set of reagents to dissect the complex biological processes that these proteins mediate. We also plan to use our knowledge of complement interactions to engineer novel specificities into the framework of C1qs, MBLs and ficolins, thereby altering target specificity whilst retaining the ability to activate complement and stimulate the host?s immune system. By targeting bacterial cells and endogenous epitopes associated with disease, these immunologically active reagents will offer a novel approach to tackle disease.

Publications

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Almitairi JOM (2018) Structure of the C1r-C1s interaction of the C1 complex of complement activation. in Proceedings of the National Academy of Sciences of the United States of America

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Huang J (2013) Macromol. Rapid Commun. 19/2013 in Macromolecular Rapid Communications

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Schwaeble WJ (2011) Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury. in Proceedings of the National Academy of Sciences of the United States of America

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Sim RB (2011) Surface properties: Immune attack on nanoparticles. in Nature nanotechnology

 
Description MRC project grant
Amount £532,558 (GBP)
Funding ID MR/K011715/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 12/2015
 
Title recombinant C1q heads 
Description We have made a recombinant form (produced in E. coli) of the globular heads of complement component C1q. This fragment is normally composed of three separate polypeptide chains and according to our knowledge has never be produced before. It will enable us to perform structure/function analysis of C1q interactions 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Ongoing research 
 
Description Structure function analysis of C1q-IgM interactions 
Organisation King's College London
Department Randall Division of Cell & Molecular Biophysics
Country United Kingdom 
Sector Academic/University 
PI Contribution Production of recombinant C1q for structure function analysis with IgM
Collaborator Contribution characterising the interaction of IgM with C1q
Impact Currently in preliminary stages of research
Start Year 2012
 
Description Structure function analysis of MBL-MASP interaction 
Organisation University College London
Department Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Production of MBL./MASPs for structural analysis by SAXS
Collaborator Contribution SAXS of MBL-MASP complexes
Impact Paper in Journal of Biological Chemistry 22167201
Start Year 2009
 
Description Patterson Institute for Cancer Research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact 40 academics attended day session of talks/seminars. I presented our recent work on the structure of the C1 complex of complement activation

N/A
Year(s) Of Engagement Activity 2013
 
Description Randall Institiute KCL 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Seminar at the Randall Insstitute KCL, London for postgraduates and academics

Collaboration with Brian Sutton (KCL) on C1q-IgM interactions
Year(s) Of Engagement Activity 2011
 
Description UCL 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Seminar at the Institute of Structural and Molecular Biology UCL to post graduate students and academics

Collaboration with Steve Perkins on MBL-MASP structure/function
Year(s) Of Engagement Activity 2012
 
Description Work experience for School students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We hosted two students from local schools for 1 week (each year) as work experience.

Science students from a nearby school were shown how research is carried out in the University and were able to help set up and perform experiments (e.g. running gels and PCR experiments) under supervision of Researchers in the laboratory.
Year(s) Of Engagement Activity 2011,2012
 
Description Work experience for college/sixth form students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We hosted two work experience students from a local 6th form college. We explained our research and they gained experience of working in a lab, including setting up basic experiments e.g. PCR reactions

The college are keen to continue this arrangement in the future so that students can experience working in a research laboratory. We plan to host at least two 6th form students each year.
Year(s) Of Engagement Activity 2013