RESUMO
Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE.
Assuntos
Antiasmáticos/farmacologia , Imunoglobulina E/metabolismo , Modelos Moleculares , Omalizumab/farmacologia , Receptores de IgE/antagonistas & inibidores , Sítio Alostérico , Substituição de Aminoácidos , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoglobulina E/química , Imunoglobulina E/genética , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Omalizumab/química , Omalizumab/genética , Omalizumab/metabolismo , Maleabilidade , Mutação Puntual , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Receptores de IgE/química , Receptores de IgE/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Solubilidade , Ressonância de Plasmônio de SuperfícieRESUMO
The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FcεRI on mast cells and basophils, and CD23 (FcεRII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen-presenting cells, contribute to allergen uptake and presentation to the immune system. We have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for cross-linking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single FcεRI binding site, which lies at the opposite ends of the Cε3 domains. Structural comparisons with both free IgE-Fc and its FcεRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcεRI binding, but also that the converse is true. The two binding sites are allosterically linked. We demonstrate experimentally the reciprocal inhibition of CD23 and FcεRI binding in solution and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.
Assuntos
Imunoglobulina E/química , Complexos Multiproteicos/química , Receptores de IgE/química , Regulação Alostérica/imunologia , Linfócitos B/química , Linfócitos B/imunologia , Cristalografia por Raios X , Humanos , Imunoglobulina E/imunologia , Complexos Multiproteicos/imunologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de IgE/imunologia , Relação Estrutura-AtividadeRESUMO
IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcεRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcεRI, whereas IgE-CD23 interactions control IgE expression levels. We have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. We show that the CD23 and FcεRI interaction sites are at opposite ends of the Cε3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FcεRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE-FcεRI and IgE-CD23 interactions.
Assuntos
Basófilos/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Regulação Alostérica/imunologia , Basófilos/citologia , Basófilos/imunologia , Linhagem Celular , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Mastócitos/citologia , Mastócitos/imunologia , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Mapeamento de Peptídeos/métodos , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de IgE/genética , Receptores de IgE/imunologiaRESUMO
Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding.
Assuntos
Anticorpos Anti-Idiotípicos/química , Anticorpos Anti-Idiotípicos/metabolismo , Imunoglobulina E/química , Imunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Linfócitos B/imunologia , Cristalografia por Raios X , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Mastócitos/imunologia , Ligação Proteica , Conformação ProteicaRESUMO
The antibody IgE plays a central role in allergic disease mechanisms. Its effector functions are controlled through interactions between the Fc region and two principal cell surface receptors FcεRI and CD23. The interaction with FcεRI is primarily responsible for allergic sensitization and the inflammatory response, while IgE binding to CD23 is involved in the regulation of IgE synthesis and allergen transcytosis. Here we present the crystal structure of a CD23/IgE-Fc complex and conduct isothermal titration calorimetric binding studies. Two lectin-like "head" domains of CD23 bind to IgE-Fc with affinities that differ by more than an order of magnitude, but the crystal structure reveals only one head bound to one of the two identical heavy-chains in the asymmetrically bent IgE-Fc. These results highlight the subtle interplay between receptor binding sites in IgE-Fc and their affinities, the understanding of which may be exploited for therapeutic intervention in allergic disease.
Assuntos
Linfócitos B/imunologia , Sítios de Ligação/imunologia , Imunoglobulina E/imunologia , Receptores de IgE/imunologia , Alérgenos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Ligação Proteica/imunologia , Transcitose/imunologiaRESUMO
The antibody IgE plays a central role in allergic disease, functioning principally through two cell-surface receptors: FcâRI and CD23. FcâRI on mast cells and basophils mediates the immediate hypersensitivity response, whilst the interaction of IgE with CD23 on B cells regulates IgE production. Crystal structures of the lectin-like `head' domain of CD23 alone and bound to a subfragment of IgE consisting of the dimer of Câ3 and Câ4 domains (Fcâ3-4) have recently been determined, revealing flexibility in the IgE-binding site of CD23. Here, a new crystal form of the CD23-Fcâ3-4 complex with different molecular-packing constraints is reported, which together with the earlier results demonstrates that conformational variability at the interface extends additionally to the IgE Fc and the quaternary structure of its domains.
Assuntos
Imunoglobulina E/química , Fragmentos Fc das Imunoglobulinas/química , Receptores de IgE/química , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.
Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/metabolismo , Imunoglobulina E/biossíntese , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes de Fusão/biossíntese , Animais , Antígenos de Plantas/biossíntese , Antígenos de Plantas/genética , Sequência de Bases , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Primers do DNA/síntese química , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Humanos , Imunoglobulina E/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/genética , Dados de Sequência Molecular , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genéticaRESUMO
IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcÉRI and CD23. Crosslinking of FcÉRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin "head" domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding.