Immunoglobulin Y: an IgG-like antibody with an IgE-like structure?

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

The human immune system provides protection against foreign invading organisms including parasites, bacteria and viruses, through a variety of different cells, such as the white blood cells, and proteins, such as the antibodies. In man, as in other mammals, there are specialized types of antibodies: a type known as IgG deal principally with viruses and bacteria, while another, called IgE, is produced in defence against parasites. However, in regions of the world where parasitic infections no longer present a challenge, these IgE antibodies are instead produced in response to substances that we call allergens. People with a tendency to produce high levels of IgE are especially susceptible to allergies, such as hayfever, asthma and food allergies. We have been studying IgE antibodies in order to understand how they cause allergy, and have discovered that there is a particular part of the antibody molecule, present in IgE but not in IgG, which gives it special properties. In particular, it causes the IgE molecules to stick extremely tightly to cells and activate them when parasites invade the body. Although this is very effective against parasites, it unfortunately has the same effects, which include a sudden inflammation reaction, when allergens such as grass pollen, bee venom or particular foods, for example peanuts, enter the body. The effects of these sudden allergic reactions are well-known, and can even be fatal. One aim of our work in this field is to develop improved drugs to treat these conditions. There is however much that we have yet to learn about the human immune system, such as the 'polarization' into the IgG and IgE type responses, and why only certain molecules that we know as allergens provoke the IgE response. One way of approaching this is from an evolutionary standpoint. Birds do not have IgG or IgE, but instead have a single type of antibody called IgY. In fact, an IgY-like antibody was present in the last common ancestor of birds and mammals, 300 million years ago, and was the evolutionary predecessor of IgG and IgE. IgY combines in a single molecule the properties of both IgG and IgE, yet birds do respond differently to viruses and parasites in their immune response, despite having only one type of antibody. We propose to study IgY from chickens, since they have the best-studied non-mammalian immune system. The publication last year of the complete DNA sequence of the chicken genome has greatly enhanced these studies. In particular, we propose to isolate the molecules to which IgY binds on the surfaces of cells, and we are the first, to our knowledge, to have identified such a 'receptor' for IgY. We shall study how IgY binds to cells, and aim to discover how it combines the roles of both IgG and IgE, thus helping us to understand, and perhaps then prevent, the unwanted IgE allergic response in man. One of the outcomes of this research is therefore an improved understanding of the human immune system and its evolution, with future benefits for human medicine. However, improved understanding of the chicken immune system is not without its own benefits. Poultry meat is an important source of human disease, and the control of bacterial disease in chickens is largely based on an indiscriminate use of antibiotics; this over-use of antibiotics has severely reduced their usefulness in controlling human bacterial infections, as the emergence of strains of antibiotic-resistant 'super-bugs' has shown. A better understanding of the chicken immune system may lead to better ways of controlling bacterial infection in these commercially important animals. Chickens and other avian species can also harbour viruses that can in turn threaten man, such as the recent outbreak of avian 'flu, and improved knowledge of immunity in birds may help us to understand how this transmission process occurs.

Technical Summary

Birds, reptiles and amphibia have only one serum antibody, IgY, in contrast to mammalian IgG and IgE, and an IgY-like molecule is the evolutionary precursor of IgG and IgE. IgY takes part in opsonisation (a function associated with mammalian IgG) and in anaphylactic reactions (mediated by mammalian IgE). We propose to find out how IgY performs these two different functions. We will prepare IgY and recombinant fragments, and identify one or more of its receptors on leukocytes to investigate IgY-receptor interaction(s). We have extensively studied IgE binding to its high affinity receptor, solved the crystal structure of IgE-Fc (the receptor-binding region of the antibody), and discovered structural features of IgE that determine its characteristically slow dissociation rate from the complex; this slow off-rate confers upon IgE its unique ability to sensitise cells and trigger allergic reactions. With this experience we are ideally placed to make a comparative study of the IgY-receptor interaction, and to produce mutants to probe key features of IgY-Fc. The recent publication of the chicken genome sequence makes this an ideal time to pursue these studies. The chicken orthologues of human Th1 and Th2 cytokines have been cloned, and it has also been discovered that different infective agents can elicit production of a Th1-like or a Th2-like response. The differential response to these cytokines in the human system depends on IgG and IgE, begging the question of how the production of a single antibody IgY can lead to two different responses in the chicken. IgY-receptor interactions may well be involved in this discrimination. We have already cloned the Fc region of IgY, with 'external' glycosylation removed, to provide a homogeneous species for X-ray analysis. We have also cloned a further carbohydrate mutant, together with cysteine mutants, to investigate the effect of the conserved 'internal' glycosylation site, and inter-chain disulphide bridges, on receptor binding. Transient expression has been obtained, but stable clones need to be produced. We shall also prepare yolk and serum IgY and their Fc fragments for binding studies, as earlier work suggested that there may be functional differences with respect to anaphylaxis. We have also recently obtained two alternatively spliced full-length chicken transcripts (one with three, and one with four extracellular immunoglubulin-like domains) of a putative IgY-binding polypeptide (alpha-chain), as well as a full-length putative signalling polypeptide (gamma-chain) that contains a signalling activation motif (ITAM). Together they may constitute leukocyte IgY-Fc receptor(s), orthologue(s) of the IgG-Fc and IgE-Fc receptors, which have not yet been identified. They are cloned and ready for expression on the surface of COS cells. If IgY binding is achieved, we can then clone the soluble extracellular portion of the alpha-chain for analysis of binding kinetics, and crystallisation with our recombinant IgY-Fc. If IgY binding is not detected, we shall search the new version of the chicken genome (expected July 2005) to identify other IgG-Fc receptor orthologues, or revert to protein purification or screening a COS cell expression library, to obtain the receptor. The kinetics and affinity of the IgY-receptor interaction will be measured on cells (using purified chicken basophils and a chicken monocyte cell line) and using the BIAcore technique with the recombinant proteins. The latter will be particularly important for making precise comparisons between the different mutant versions of IgY-Fc. Together these data will provide a complete description of the IgY-receptor interaction for comparison with mammalian IgG and IgE. We have already generated most of the starting materials for this work, and are now in a very competitive position to make a contribution to this field.

Publications

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Title Aperiodic tiling pattern 
Description Production of the largest aperiodic tiling pattern, through involvement of the public in printing the tiles and adding to the pattern. 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2018 
Impact Sparked discussion about symmetry, structure and pattern in biology, which could then be related to our work, using X-ray crystallography, to study antibodies and allergens in allergic disease. 
 
Title Crystal Symmetry 
Description In collaboration with glass sculptor Dr. Shelley James, two exhibitions at the History of Science Museum, Oxford to celebrate the International Year of Crystallography. Exhibition consisted of graphic art pieces and glass sculptures representing aspects of crystallographic symmetry. 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2014 
Impact Led to invitations to participate in public engagement activities and two public lectures at the History of Science Museum, Oxford, and at the Diamond Light Source as part of the CCP$ Summer School. 
 
Title Dynamic Symmetry 
Description In collaboration with glass sculptor Dr. Shelley James, exhibition of graphic art and glass pieces to illustrate aspects of crystallographic symmetry, at the Royal Society Summer Exhibition, London. A celebration of the International Year of Crystallography. 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2014 
Impact Public engagement with crystallography; subsequent invitation by The Royal Society to give lecture at the Royal Society and run a pubic engagement activity as part of the Big Draw Event, October 2014. 
 
Title Glass sculptures by Dr. Shelley James 
Description Glass sculptures (and also multiple, smaller 3d-printed copies) of mirror symmetry-related crystals, to illustrate lectures on topic of mirror symmetry, handedness and pattern in biology. 
Type Of Art Artefact (including digital) 
Year Produced 2018 
Impact Sparked discussion about symmetry, patterns and structure in biology, which could then be related to our work using X-ray crystallography to study antibodies and allergens in allergic disease, including asthma. 
 
Description The avian IgY antibody is the evolutionary ancestor of human IgG and IgE antibodies. IgG is responsible for protective immunity whereas IgE is involved in allergy, and these antibodies have markedly different 3d structures. Our principal long-term aim is to develop new therapeutic agents for allergic disease, and in order to do this we want to understand how the 3d structure of IgE gives rise to its unique receptor-binding properties - which in turn cause allergic reactions. Since avian IgY has an IgE-like structure, yet has IgG-like properties and provides protective immunity in birds, we studied the 3d structure and receptor-binding properties of chicken IgY. We reported several "firsts": an IgY receptor, the 3d structure of the receptor-binding Fc region of IgY (the first ever non-mammalian antibody structure), the kinetics, affinity and binding stoichiometry of the IgY/receptor complex, and the location of the receptor binding site in IgY. All of these results indicated that IgY was indeed an IgG-like antibody in terms of function, despite its IgY-like structure. The 3d structure, remarkably, showed conformational features of both IgG and IgE, but it was clear that it is not only the gross structural difference between IgG and IgE (an additional pair of domains) that causes the "allergic" properties of IgE. In fact, the very location of the receptor binding site has "migrated" in the evolution from IgY to IgG and IgE - but perhaps this shift of location enabled the new allergic activities to develop. We are thus focussing more closely in our current work on the precise structural differences between IgY and IgE (individual amino acids) in the IgE receptor binding region. This work is also informed by our recent crystal structure of the IgE/receptor complex (published in Nature Structural & Molecular Biology, 18, 571-576, 2011).
Exploitation Route Commercial prospects for IgY as explained above. IgY can be produced in large quantities, relatively cheaply, and with high specificity against any desired target; it is thus being exploited as a diagnostic reagent in many different contexts, perhaps in future in a therapeutic role. Understanding IgY's structure and function (receptor interactions) will be crucial for its future development.
Sectors Agriculture/ Food and Drink,Healthcare,Manufacturing/ including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Public interest in antibody evolution
First Year Of Impact 2012
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

 
Description Atlas Project 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Interview about X-ray crystallography, crystallisation and structure analysis; reference to our work on antibodies in allergy and asthma.
Year(s) Of Engagement Activity 2018
 
Description Atlas Project Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Presentation of crystallisation process of various substances from sea water to salivary fluid. Encouraged people to think about natural variation of environmental and medically relevant substances.
Year(s) Of Engagement Activity 2018
 
Description Bush House, King's College London, The Science and Art of Mirror Symmetry. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Hands-on activity to explore symmetry in tiling patterns, mirror symmetry and aperiodic symmetry. Included one-hour lecture (repeated). All-day event in public accessible space. Sparked discussion about pattern, structure, molecular structure and the work with antibodies in allergy and asthma in which we are engaged.
Year(s) Of Engagement Activity 2018
 
Description NHS choices website 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Web article based upon our publications, explaining to the general public how understanding chicken immunity may inform drug development for allergic disease. Web article

no actual impacts realised to date
Year(s) Of Engagement Activity 2008
 
Description Royal Society Summer Science Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Hands-on activity involving tiling patterns to present principles of symmetry, crystallography and structure analysis. Discussion of antibody structure, antibody recognition and allergic disease including asthma.
Year(s) Of Engagement Activity 2017
 
Description School Visit to KCL (The Grey Coat Hospital School) 
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 Visit to labs; tour of other facilities relevant to our work in allergy and asthma; talks; time for free discussion.
Year(s) Of Engagement Activity 2017
 
Description Various radio and press interviews, newspaper articles, following publications and associated press releases 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact National press articles, e.g. "Molecular archaeology offers new way to stop allergies" (Telegraph); "Ancient molecule offers clues to human allergies"; "Chickens unlock allergy secrets" (BBC News Channel) and many others.

no actual impacts realised to date
Year(s) Of Engagement Activity 2008
URL http://www.telegraph.co.uk/science/science-news/3344402/Molecular-archaeology-offers-new-way-to-stop...