RESUMEN
An allergic reaction is rapidly generated when allergens bind and cross-link IgE bound to its receptor FcεRI on effector cells, resulting in cell degranulation and release of proinflammatory mediators. The extent of effector cell activation is linked to allergen affinity, oligomeric state, valency, and spacing of IgE-binding epitopes on the allergen. Whereas most of these observations come from studies using synthetic allergens, in this study we have used Timothy grass pollen allergen Phl p 7 and birch pollen allergen Bet v 4 to study these effects. Despite the high homology of these polcalcin family allergens, Phl p 7 and Bet v 4 display different binding characteristics toward two human patient-derived polcalcin-specific IgE Abs. We have used native polcalcin dimers and engineered multimeric allergens to test the effects of affinity and oligomeric state on IgE binding and effector cell activation. Our results indicate that polcalcin multimers are required to stimulate high levels of effector cell degranulation when using the humanized RBL-SX38 cell model and that multivalency can overcome the need for high-affinity interactions.
Asunto(s)
Alérgenos/inmunología , Afinidad de Anticuerpos , Antígenos de Plantas/inmunología , Proteínas de Unión al Calcio/inmunología , Degranulación de la Célula , Inmunoglobulina E/inmunología , Modelos Inmunológicos , Proteínas de Plantas/inmunología , Alérgenos/genética , Antígenos de Plantas/genética , Proteínas de Unión al Calcio/genética , Epítopos/genética , Epítopos/inmunología , Células HEK293 , Humanos , Proteínas de Plantas/genética , Multimerización de Proteína/genética , Multimerización de Proteína/inmunologíaRESUMEN
Antibodies classically bind antigens via their complementarity-determining regions, but an alternative mode of interaction involving V-domain framework regions has been observed for some B cell "superantigens." We report the crystal structure of an antibody employing both modes of interaction simultaneously and binding two antigen molecules. This human antibody from an allergic individual binds to the grass pollen allergen Phl p 7. Not only are two allergen molecules bound to each antibody fragment (Fab) but also each allergen molecule is bound by two Fabs: One epitope is recognized classically, the other in a superantigen-like manner. A single allergen molecule thus cross-links two identical Fabs, contrary to the one-antibody-one-epitope dogma, which dictates that a dimeric allergen at least is required for this to occur. Allergens trigger immediate hypersensitivity reactions by cross-linking receptor-bound IgE molecules on effector cells. We found that monomeric Phl p 7 induced degranulation of basophils sensitized solely with this monoclonal antibody expressed as an IgE, demonstrating that the dual specificity has functional consequences. The monomeric state of Phl p 7 and two structurally related allergens was confirmed by size-exclusion chromatography and multiangle laser light scattering, and the results were supported by degranulation studies with the related allergens, a second patient-derived allergen-specific antibody lacking the nonclassical binding site, and mutagenesis of the nonclassically recognized allergen epitope. The antibody dual reactivity and cross-linking mechanism not only have implications for understanding allergenicity and allergen potency but, importantly, also have broader relevance to antigen recognition by membrane Ig and cross-linking of the B cell receptor.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Plantas/inmunología , Proteínas de Unión al Calcio/inmunología , Epítopos/inmunología , Superantígenos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos/inmunología , Antígenos de Plantas/química , Antígenos de Plantas/metabolismo , Basófilos/inmunología , Basófilos/fisiología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Degranulación de la Célula/inmunología , Reacciones Cruzadas/inmunología , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Humanos , Inmunoglobulina E/química , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Superantígenos/química , Superantígenos/metabolismoRESUMEN
Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against pathogens and for regulation of immune responses to self-antigens. Since the bona fide IgM Fc receptor (FcµR) was identified in humans by a functional cloning strategy in 2009, the roles of FcµR in these IgM effector functions have begun to be explored. In this short essay, we describe the differences between human and mouse FcµRs in terms of their identification processes, cellular distributions and ligand binding activities with emphasis on our recent findings from the mutational analysis of human FcµR. We have identified at least three sites of human FcµR, i.e., Asn66 in the CDR2, Lys79 to Arg83 in the DE loop and Asn109 in the CDR3, responsible for its constitutive IgM-ligand binding. Results of computational structural modeling analysis are consistent with these mutational data and a model of the ligand binding, Ig-like domain of human FcµR is proposed. Serendipitously, substitution of Glu41 and Met42 in the CDR1 of human FcµR with mouse equivalents Gln and Leu, either single or more prominently in combination, enhances both the receptor expression and IgM binding. These findings would help in the future development of preventive and therapeutic interventions targeting FcµR.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Sitios de Unión , Clonación Molecular , Humanos , Inmunoglobulina M/metabolismo , Ligandos , Proteínas de la Membrana/química , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
BACKGROUND: Understanding the discrepancy between IgE sensitization and allergic reactions to peanut could facilitate diagnosis and lead to novel means of treating peanut allergy. OBJECTIVE: To identify differences in IgE and IgG4 binding to peanut peptides between peanut-allergic (PA) and peanut-sensitized but tolerant (PS) children. METHODS: PA (n = 56), PS (n = 42) and nonsensitized nonallergic (NA, n = 10) patients were studied. Synthetic overlapping 15-mer peptides of peanut allergens (Ara h 1-11) were spotted onto microarray slides, and patients' samples were tested for IgE and IgG4 binding using immunofluorescence. IgE and IgG4 levels to selected peptides were quantified using ImmunoCAP. Diagnostic model comparisons were performed using likelihood-ratio tests between each specified nominal logistic regression models. RESULTS: Seven peptides on Ara h 1, Ara h 2, and Ara h 3 were bound more by IgE of PA compared to PS patients on the microarray. IgE binding to one peptide on Ara h 5 and IgG4 binding to one Ara h 9 peptide were greater in PS than in PA patients. Using ImmunoCAP, IgE to the Ara h 2 peptides enhanced the diagnostic accuracy of Ara h 2-specific IgE. Ratios of IgG4/IgE to 4 out of the 7 peptides were higher in PS than in PA subjects. CONCLUSIONS: Ara h 2 peptide-specific IgE added diagnostic value to Ara h 2-specific IgE. Ability of peptide-specific IgG4 to surmount their IgE counterpart seems to be important in established peanut tolerance.
Asunto(s)
Antígenos de Plantas , Hipersensibilidad al Cacahuete , Albuminas 2S de Plantas , Alérgenos , Arachis , Niño , Epítopos , Humanos , Inmunoglobulina E , Hipersensibilidad al Cacahuete/diagnóstico , Proteínas de PlantasRESUMEN
IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs.
Asunto(s)
Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Animales , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Sitios de Unión , Complemento C1q/inmunología , Complemento C1q/metabolismo , Glicosilación , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina G/uso terapéutico , Modelos Moleculares , Neoplasias/inmunología , Neoplasias/terapia , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Receptores de IgG/química , Receptores de IgG/metabolismo , Relación Estructura-ActividadRESUMEN
Immunoglobulin E (IgE) is well known for its role in allergic disease, the manifestations of which are mediated through its two Fc receptors, FcεRI and CD23 (FcεRII). IgE and its interactions with these receptors are therefore potential targets for therapeutic intervention, and exciting progress has been made in this direction. Furthermore, recent structural studies of IgE-Fc, the two receptors, and of their complexes, have revealed a remarkable degree of plasticity at the IgE-CD23 interface and an even more remarkable degree of dynamic flexibility within the IgE molecule. Indeed, there is allosteric communication between the two receptor-binding sites, which we now know are located at some distance from each other in IgE-Fc (at opposite ends of the Cε3 domain). The conformational changes associated with FcεRI and CD23 binding to IgE-Fc ensure that their interactions are mutually incompatible, and it may be that this functional imperative has driven IgE to evolve such a dynamic structure. Appreciation of these new structural data has revised our view of IgE structure, shed light on the co-evolution of antibodies and their receptors, and may open up new therapeutic opportunities.
Asunto(s)
Inmunoglobulina E/química , Inmunoglobulina E/metabolismo , Modelos Moleculares , Conformación Proteica , Receptores de IgE/química , Receptores de IgE/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Humanos , Inmunoglobulina E/inmunología , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
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.
Asunto(s)
Antiasmáticos/farmacología , Inmunoglobulina E/metabolismo , Modelos Moleculares , Omalizumab/farmacología , Receptores de IgE/antagonistas & inhibidores , Sitio Alostérico , Sustitución de Aminoácidos , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoglobulina E/química , Inmunoglobulina E/genética , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/farmacología , Omalizumab/química , Omalizumab/genética , Omalizumab/metabolismo , Docilidad , Mutación Puntual , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Replegamiento Proteico , Receptores de IgE/química , Receptores de IgE/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Solubilidad , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions. METHODS: Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with "matched" heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays. RESULTS: Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE. CONCLUSIONS: SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as "superantibodies" through CDRs and framework regions to SEEs in SEE-anti-SEE IgE-FcεRI complexes.
Asunto(s)
Enterotoxinas/inmunología , Pólipos Nasales/inmunología , Rinitis/inmunología , Sinusitis/inmunología , Asma Inducida por Aspirina/inmunología , Prueba de Desgranulación de los Basófilos , Basófilos/inmunología , Separación Celular , Enfermedad Crónica , Regiones Determinantes de Complementariedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Immunoglobulins are biomolecules involved in defence against foreign substances. Flexibility is key to their functional properties in relation to antigen binding and receptor interactions. We have developed an integrative strategy combining ion mobility mass spectrometry (IM-MS) with molecular modelling to study the conformational dynamics of human IgG antibodies. Predictive models of all four human IgG subclasses were assembled and their dynamics sampled in the transition from extended to collapsed state during IM-MS. Our data imply that this collapse of IgG antibodies is related to their intrinsic structural features, including Fab arm flexibility, collapse towards the Fc region, and the length of their hinge regions. The workflow presented here provides an accurate structural representation in good agreement with the observed collision cross section for these flexible IgG molecules. These results have implications for studying other nonglobular flexible proteins.
Asunto(s)
Inmunoglobulina G/química , Gases/química , Espectrometría de Masas , Modelos Moleculares , Conformación ProteicaRESUMEN
Immunoglobulin E (IgE) is the antibody that plays a central role in the mechanisms of allergic diseases such as asthma. Interactions with its receptors, FcεRI on mast cells and CD23 on B cells, are mediated by the Fc region, a dimer of the Cε2, Cε3 and Cε4 domains. A sub-fragment lacking the Cε2 domains, Fcε3-4, also binds to both receptors, although receptor binding almost exclusively involves the Cε3 domains. This domain also contains the N-linked glycosylation site conserved in other isotypes. We report here the crystal structures of IgE-Fc and Fcε3-4 at the highest resolutions yet determined, 1.75Šand 2.0Šrespectively, revealing unprecedented detail regarding the carbohydrate and its interactions with protein domains. Analysis of the crystallographic B-factors of these, together with all earlier IgE-Fc and Fcε3-4 structures, shows that the Cε3 domains exhibit the greatest intrinsic flexibility and quaternary structural variation within IgE-Fc. Intriguingly, both well-ordered carbohydrate and disordered polypeptide can be seen within the same Cε3 domain. A simplified method for comparing the quaternary structures of the Cε3 domains in free and receptor-bound IgE-Fc structures is presented, which clearly delineates the FcεRI and CD23 bound states. Importantly, differential scanning fluorimetric analysis of IgE-Fc and Fcε3-4 identifies Cε3 as the domain most susceptible to thermally-induced unfolding, and responsible for the characteristically low melting temperature of IgE.
Asunto(s)
Inmunoglobulina E/química , Fragmentos Fc de Inmunoglobulinas/química , Receptores de IgE/química , Secuencias de Aminoácidos , Sitios de Unión , Secuencia de Carbohidratos , Cristalografía por Rayos X , Expresión Génica , Glicosilación , Humanos , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Modelos Moleculares , Transición de Fase , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Desplegamiento Proteico , Receptores de IgE/genética , Receptores de IgE/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , TemperaturaRESUMEN
Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.
Asunto(s)
Anticuerpos , Biotecnología , Ingeniería de Proteínas , Animales , Anticuerpos Monoclonales , Bacterias , Mariposas Diurnas , Humanos , RatonesRESUMEN
IgG4 is the least abundant subclass of IgG in normal human serum, but elevated IgG4 levels are triggered in response to a chronic antigenic stimulus and inflammation. Since the immune system is exposed to tumor-associated antigens over a relatively long period of time, and tumors notoriously promote inflammation, it is unsurprising that IgG4 has been implicated in certain tumor types. Despite differing from other IgG subclasses by only a few amino acids, IgG4 possesses unique structural characteristics that may be responsible for its poor effector function potency and immunomodulatory properties. We describe the unique attributes of IgG4 that may be responsible for these regulatory functions, particularly in the cancer context. We discuss the inflammatory conditions in tumors that support IgG4, the emerging and proposed mechanisms by which IgG4 may contribute to tumor-associated escape from immune surveillance and implications for cancer immunotherapy.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoglobulina G/inmunología , Neoplasias/inmunología , Escape del Tumor/inmunología , HumanosRESUMEN
Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Cadenas Pesadas de Inmunoglobulina/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genéticaRESUMEN
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.
Asunto(s)
Inmunoglobulina E/química , Complejos Multiproteicos/química , Receptores de IgE/química , Regulación Alostérica/inmunología , Linfocitos B/química , Linfocitos B/inmunología , Cristalografía por Rayos X , Humanos , Inmunoglobulina E/inmunología , Complejos Multiproteicos/inmunología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de IgE/inmunología , Relación Estructura-ActividadRESUMEN
Development of chicken-sourced antibodies offers an alternative strategy for the development of highly specific antibodies against mammalian proteins with conserved epitopes due to the phylogenetic distance between avian and mammalian species. In this study, the single-chain variable fragments (scFvs) against porcine interferon-gamma was screened and characterized from a hyperimmunized chicken phage display library. The expressed soluble scFvs exhibited highly specific recognition of porcine interferon-gamma in ELISA, Western blot, and immunofluorescence staining assays. Results of the current study indicate that it is possible to develop scFv IgY antibodies to a mammalian interferon by using Biopanning technology. Furthermore, it also confirms that monoclonal avian IgY antibody technique could be applied as a promising tool to produce immunoglobulin molecules with high specificity and affinity towards conserved mammalian epitopes or antigens.
Asunto(s)
Fragmentos de Inmunoglobulinas/inmunología , Interferón gamma/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Pollos , PorcinosRESUMEN
Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcεRI and CD23 (FcεRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcεRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca(2+)-free and Ca(2+)-bound forms, as well as the crystal structure of the Ca(2+)-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). Together with site-directed mutagenesis, the crystal structures of four Ca(2+) ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca(2+) binds at the principal and evolutionarily conserved binding site in CD23. Ca(2+) binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca(2+)-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca(2+) brings an extra degree of modulation to CD23 function.
Asunto(s)
Linfocitos B/metabolismo , Calcio/metabolismo , Inmunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Unión Competitiva , Calorimetría , Cristalografía por Rayos X , Ciclofilina A/metabolismo , Entropía , Humanos , Inmunoglobulina E/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de IgE/química , Receptores de IgE/genética , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
The neonatal Fc receptor (FcRn) mediates the transfer of IgG and albumin, also protects them from catabolism. This study characterized the expression of FcRn in different organs of neonatal and pubertal rats by reverse transcription-PCR (RT-PCR) and immunohistochemistry, demonstrates that FcRn is expressed in liver, kidney, intestine, heart, lung, spleen, skin and skeletal muscles at varying levels post-gestation from d 1 to d 63. This finding is contrary to previous studies claiming that FcRn is undetectable in most tissues after weaning. Lungs were the predominant organs for FcRn expression, whereas skin, liver and intestine are considerably less expressed organs. The expression of FcRn fluctuated in all the organs tested, and with a higher frequency before weaning compared to puberty. These findings may provide clues for the better understanding of FcRn function, and are important for determining the dosage levels for IgG and the constant region fragment (Fc)-containing therapeutic proteins whose half-life is regulated by FcRn.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/metabolismo , Pubertad/metabolismo , Receptores Fc/genética , Receptores Fc/metabolismo , Animales , Animales Recién Nacidos , Femenino , Técnicas para Inmunoenzimas , Masculino , Pubertad/genética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
CD23, the low-affinity receptor for IgE, exists in membrane and soluble forms. Soluble CD23 (sCD23) fragments are released from membrane (m)CD23 by the endogenous metalloprotease a disintegrin and metalloprotease 10. When purified tonsil B cells are incubated with IL-4 and anti-CD40 to induce class switching to IgE in vitro, mCD23 is upregulated, and sCD23 accumulates in the medium prior to IgE synthesis. We have uncoupled the effects of mCD23 cleavage and accumulation of sCD23 on IgE synthesis in this system. We show that small interfering RNA inhibition of CD23 synthesis or inhibition of mCD23 cleavage by an a disintegrin and metalloprotease 10 inhibitor, GI254023X, suppresses IL-4 and anti-CD40-stimulated IgE synthesis. Addition of a recombinant trimeric sCD23 enhances IgE synthesis in this system. This occurs even when endogenous mCD23 is protected from cleavage by GI254023X, indicating that IgE synthesis is positively controlled by sCD23. We show that recombinant trimeric sCD23 binds to cells coexpressing mIgE and mCD21 and caps these proteins on the B cell membrane. Upregulation of IgE by sCD23 occurs after class-switch recombination, and its effects are isotype-specific. These results suggest that mIgE and mCD21 cooperate in the sCD23-mediated positive regulation of IgE synthesis on cells committed to IgE synthesis. Feedback regulation may occur when the concentration of secreted IgE becomes great enough to allow binding to mCD23, thus preventing further release of sCD23. We interpret these results with the aid of a model for the upregulation of IgE by sCD23.
Asunto(s)
Linfocitos B/inmunología , Regulación de la Expresión Génica/inmunología , Genes de Inmunoglobulinas , Inmunoglobulina E/biosíntesis , Receptores de IgE/inmunología , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Linfocitos B/metabolismo , Dipéptidos/farmacología , Retroalimentación Fisiológica , Homeostasis , Humanos , Ácidos Hidroxámicos/farmacología , Cambio de Clase de Inmunoglobulina , Recubrimiento Inmunológico , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/fisiología , Inhibidores de Proteasas/farmacología , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores de Complemento 3d/inmunología , Receptores de IgE/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Solubilidad , Regulación hacia ArribaRESUMEN
IgE binding to its high affinity receptor FcεRI on mast cells and basophils is a key step in the mechanism of allergic disease and a target for therapeutic intervention. Early indications that IgE adopts a bent structure in solution have been confirmed by recent x-ray crystallographic studies of IgEFc, which further showed that the bend, contrary to expectation, is enhanced in the crystal structure of the complex with receptor. To investigate the structure of IgEFc and its conformational changes that accompany receptor binding in solution, we created a Förster resonance energy transfer (FRET) biosensor using biologically encoded fluorescent proteins fused to the N- and C-terminal IgEFc domains (Cε2 and Cε4, respectively) together with the theoretical basis for quantitating its behavior. This revealed not only that the IgEFc exists in a bent conformation in solution but also that the bend is indeed enhanced upon FcεRI binding. No change in the degree of bending was seen upon binding to the B cell receptor for IgE, CD23 (FcεRII), but in contrast, binding of the anti-IgE therapeutic antibody omalizumab decreases the extent of the bend, implying a conformational change that opposes FcεRI engagement. HomoFRET measurements further revealed that the (Cε2)(2) and (Cε4)(2) domain pairs behave as rigid units flanking the conformational change in the Cε3 domains. Finally, modeling of the accessible conformations of the two Fab arms in FcεRI-bound IgE revealed a mutual exclusion not seen in IgG and Fab orientations relative to the membrane that may predispose receptor-bound IgE to cross-linking by allergens.
Asunto(s)
Alérgenos/análisis , Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Inmunoglobulina E/química , Fragmentos Fc de Inmunoglobulinas/química , Receptores de IgE/química , Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales Humanizados/química , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Inmunoglobulina E/genética , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Omalizumab , Receptores de IgE/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
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.