RESUMO
We have previously produced a toolkit of antibodies, comprising recombinant human antibodies of all but one of the human isotypes, directed against the polcalcin family antigen Phl p 7. In this work, we complete the toolkit of human antibody isotypes with the IgD version of the anti-Phl p 7 monoclonal antibody. We also raised a set of nanobodies against the IgD anti-Phl p 7 antibody and identify and characterize one paratope-specific nanobody. This nanobody also binds to the IgE isotype of this antibody, which shares the same idiotype, and orthosterically inhibits the interaction with Phl p 7. The 2.1 Å resolution X-ray crystal structure of the nanobody in complex with the IgD Fab is described.
RESUMO
Antibodies of the IgD isotype remain the least well characterized of the mammalian immunoglobulin isotypes. Here we report three-dimensional structures for the Fab region of IgD, based on four different crystal structures, at resolutions of 1.45-2.75 Å. These IgD Fab crystals provide the first high-resolution views of the unique Cδ1 domain. Structural comparisons identify regions of conformational diversity within the Cδ1 domain, as well as among the homologous domains of Cα1, Cγ1 and Cµ1. The IgD Fab structure also possesses a unique conformation of the upper hinge region, which may contribute to the overall disposition of the very long linker sequence between the Fab and Fc regions found in human IgD. Structural similarities observed between IgD and IgG, and differences with IgA and IgM, are consistent with predicted evolutionary relationships for the mammalian antibody isotypes.
Assuntos
Fragmentos Fab das Imunoglobulinas , Isotipos de Imunoglobulinas , Animais , Humanos , MamíferosRESUMO
The C1q and TNF related 4 (C1QTNF4) protein is a structurally unique member of the C1QTNF family, a family of secreted proteins that have structural homology with both complement C1q and the tumor necrosis factor superfamily. C1QTNF4 has been linked to the autoimmune disease systemic lupus erythematosus through genetic studies; however, its role in immunity and inflammation remains poorly defined and a cell surface receptor of C1QTNF4 has yet to be identified. Here we report identification of nucleolin as a cell surface receptor of C1QTNF4 using mass spectrometric analysis. Additionally, we present evidence that the interaction between C1QTNF4 and nucleolin is mediated by the second C1q-like domain of C1QTNF4 and the C terminus of nucleolin. We show that monocytes and B cells are target cells of C1QTNF4 and observe extensive binding to dead cells. Imaging flow cytometry experiments in monocytes show that C1QTNF4 becomes actively internalized upon cell binding. Our results suggest that nucleolin may serve as a docking molecule for C1QTNF4 and act in a context-dependent manner through coreceptors. Taken together, these findings further our understanding of C1QTNF4's function in the healthy immune system and how dysfunction may contribute to the development of systemic lupus erythematosus.
Assuntos
Linfócitos B/imunologia , Citocinas/metabolismo , Imunidade Inata/imunologia , Inflamação/imunologia , Monócitos/imunologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Linfócitos B/citologia , Linfócitos B/metabolismo , Citocinas/genética , Humanos , Inflamação/metabolismo , Inflamação/patologia , Monócitos/citologia , Monócitos/metabolismo , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Receptores de Superfície Celular/genética , NucleolinaRESUMO
Förster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (ãEFRETexpã = 0.25 ± 0.05) and those calculated from the molecular dynamics simulations (ãEFRETMDã = 0.18 ± 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors ãκ2ã = 0.17 ± 0.16 and ã|κ|ã = 0.35 ± 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time- and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Simulação de Dinâmica Molecular , Polarização de Fluorescência , Proteínas de Fluorescência Verde/genética , Microscopia de FluorescênciaRESUMO
AIMS/HYPOTHESIS: Progressive decline in functional beta cell mass is central to the development of type 2 diabetes. Elevated serum levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are associated with beta cell failure in type 2 diabetes and eNAMPT immuno-neutralisation improves glucose tolerance in mouse models of diabetes. Despite this, the effects of eNAMPT on functional beta cell mass are poorly elucidated, with some studies having separately reported beta cell-protective effects of eNAMPT. eNAMPT exists in structurally and functionally distinct monomeric and dimeric forms. Dimerisation is essential for the NAD-biosynthetic capacity of NAMPT. Monomeric eNAMPT does not possess NAD-biosynthetic capacity and may exert distinct NAD-independent effects. This study aimed to fully characterise the structure-functional effects of eNAMPT on pancreatic beta cell functional mass and to relate these to beta cell failure in type 2 diabetes. METHODS: CD-1 mice and serum from obese humans who were without diabetes, with impaired fasting glucose (IFG) or with type 2 diabetes (from the Body Fat, Surgery and Hormone [BodyFatS&H] study) or with or at risk of developing type 2 diabetes (from the VaSera trial) were used in this study. We generated recombinant wild-type and monomeric eNAMPT to explore the effects of eNAMPT on functional beta cell mass in isolated mouse and human islets. Beta cell function was determined by static and dynamic insulin secretion and intracellular calcium microfluorimetry. NAD-biosynthetic capacity of eNAMPT was assessed by colorimetric and fluorescent assays and by native mass spectrometry. Islet cell number was determined by immunohistochemical staining for insulin, glucagon and somatostatin, with islet apoptosis determined by caspase 3/7 activity. Markers of inflammation and beta cell identity were determined by quantitative reverse transcription PCR. Total, monomeric and dimeric eNAMPT and nicotinamide mononucleotide (NMN) were evaluated by ELISA, western blot and fluorometric assay using serum from non-diabetic, glucose intolerant and type 2 diabetic individuals. RESULTS: eNAMPT exerts bimodal and concentration- and structure-functional-dependent effects on beta cell functional mass. At low physiological concentrations (~1 ng/ml), as seen in serum from humans without diabetes, eNAMPT enhances beta cell function through NAD-dependent mechanisms, consistent with eNAMPT being present as a dimer. However, as eNAMPT concentrations rise to ~5 ng/ml, as in type 2 diabetes, eNAMPT begins to adopt a monomeric form and mediates beta cell dysfunction, reduced beta cell identity and number, increased alpha cell number and increased apoptosis, through NAD-independent proinflammatory mechanisms. CONCLUSIONS/INTERPRETATION: We have characterised a novel mechanism of beta cell dysfunction in type 2 diabetes. At low physiological levels, eNAMPT exists in dimer form and maintains beta cell function and identity through NAD-dependent mechanisms. However, as eNAMPT levels rise, as in type 2 diabetes, structure-functional changes occur resulting in marked elevation of monomeric eNAMPT, which induces a diabetic phenotype in pancreatic islets. Strategies to selectively target monomeric eNAMPT could represent promising therapeutic strategies for the treatment of type 2 diabetes.
Assuntos
Citocinas/sangue , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Nicotinamida Fosforribosiltransferase/sangue , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/sangue , Glucagon/metabolismo , Humanos , Immunoblotting , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina/sangue , Somatostatina/metabolismo , Relação Estrutura-AtividadeRESUMO
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
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.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Plantas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Epitopos/imunologia , Superantígenos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/imunologia , Antígenos de Plantas/química , Antígenos de Plantas/metabolismo , Basófilos/imunologia , Basófilos/fisiologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Degranulação Celular/imunologia , Reações Cruzadas/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/metabolismo , Humanos , Imunoglobulina E/química , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Superantígenos/química , Superantígenos/metabolismoRESUMO
Degranulation of mast cells and basophils, with release of agents of the allergic response, ensues when multivalent antigens bind to and cross-link the cells' receptor-bound IgE antibodies. A widely used commercial monoclonal IgE antibody, SPE-7 IgE from Sigma, was found to possess the radically anomalous property, termed "cytokinergic", of inducing basophil degranulation without the intervention of an antigen. We show here that the IgE monomer, freed of protein contaminants, is devoid of this activity, and that the source of the anomaly is a trace impurity, identified as a dissociation-resistant IgE trimer. Possible models for the formation of IgE trimers and the manner in which they cross-link cell surface receptors are suggested herein.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Basófilos/imunologia , Degranulação Celular/imunologia , Imunoglobulina E/química , Imunoglobulina E/imunologia , Multimerização Proteica , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Basófilos/metabolismo , Linhagem Celular , Humanos , Imunoglobulina E/isolamento & purificação , Imunoglobulina E/metabolismo , Camundongos , Ligação Proteica , Receptores de IgE/metabolismoRESUMO
Small inorganic assemblies of alternating ferrous/ferric iron and sulphide ions, so-called iron-sulphur (Fe-S) clusters, are possibly nature's most ancient prosthetic groups. One of the early actors in Fe-S cluster biosynthesis is a protein complex composed of a cysteine desulphurase, Nfs1, and its functional binding partner, Isd11. Although the essential function of Nfs1·Isd11 in the liberation of elemental sulphur from free cysteine is well established, little is known about its structure. Here, we provide evidence that shows Isd11 has a profound effect on the oligomeric state of Nfs1.
Assuntos
Proteínas Ferro-Enxofre/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Estrutura Quaternária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sulfurtransferases/química , Dicroísmo Circular , Modelos Moleculares , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Homologia Estrutural de ProteínaRESUMO
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.
Assuntos
Alérgenos/análise , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Imunoglobulina E/química , Fragmentos Fc das Imunoglobulinas/química , Receptores de IgE/química , Anticorpos Anti-Idiotípicos/química , Anticorpos Monoclonais Humanizados/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Imunoglobulina E/genética , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Omalizumab , Receptores de IgE/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (nâ=â10) to primary and metastatic melanoma cells compared to healthy volunteers (nâ=â10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (nâ=â21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (nâ=â1,800) compared to 2% of cultures from healthy controls (nâ=â600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.
Assuntos
Anticorpos Antineoplásicos/química , Linfócitos B/imunologia , Imunoglobulina G/química , Melanoma/imunologia , Melanoma/terapia , Anticorpos Monoclonais/química , Estudos de Casos e Controles , Linhagem Celular , Linhagem Celular Tumoral , Estudos de Coortes , Progressão da Doença , Ensaio de Imunoadsorção Enzimática/métodos , Fibroblastos/metabolismo , Humanos , Sistema Imunitário , Imunoglobulina G/sangue , Imuno-Histoquímica/métodos , Melanoma/sangue , Fatores de TempoRESUMO
The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.
Assuntos
Proteínas Culina/metabolismo , HIV-1/metabolismo , Dobramento de Proteína , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Proteínas Culina/química , Elonguina , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Imunoprecipitação , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas Supressoras da Sinalização de Citocina/química , Fatores de Transcrição/química , Ubiquitinação , Produtos do Gene vif do Vírus da Imunodeficiência Humana/químicaRESUMO
Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20-Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.
Assuntos
Autoantígenos/química , RNA não Traduzido/química , Ribonuclease P/química , Sequência de Aminoácidos , Autoantígenos/metabolismo , Dimerização , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Longo não Codificante , RNA não Traduzido/metabolismo , Ribonuclease P/metabolismoRESUMO
IgY is the principal serum antibody in birds and reptiles, and an IgY-like molecule was the evolutionary precursor of both mammalian IgG and IgE. A receptor for IgY on chicken monocytes, chicken leukocyte receptor AB1 (CHIR-AB1), lies in the avian leukocyte receptor cluster rather than the classical Fc receptor cluster where the genes for mammalian IgE and IgG receptors are found. IgG and IgE receptors bind to the lower hinge region of their respective antibodies with 1:1 stoichiometry, whereas the myeloid receptor for IgA, FcalphaRI, and the IgG homeostasis receptor, FcRn, which are found in the mammalian leukocyte receptor cluster, bind with 2:1 stoichiometry between the heavy chain constant domains 2 and 3 of each heavy chain. In this paper, the extracellular domain of CHIR-AB1 was expressed in a soluble form and shown to be a monomer that binds to IgY-Fc with 2:1 stoichiometry. The two binding sites have similar affinities: K(a)(1) = 7.22 +/- 0.22 x 10(5) m(-1) and K(a)(2) = 3.63 +/- 1.03 x 10(6) m(-1) (comparable with the values reported for IgA binding to its receptor). The affinity constants for IgY and IgY-Fc binding to immobilized CHIR-AB1 are 9.07 +/- 0.07 x 10(7) and 6.11 +/- 0.02 x 10(8) m(-1), respectively, in agreement with values obtained for IgY binding to chicken monocyte cells and comparable with reported values for human IgA binding to neutrophils. Although the binding site for CHIR-AB1 on IgY is not known, the data reported here with a monomeric receptor binding to IgY at two sites with low affinity suggest an IgA-like interaction.
Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Imunoglobulinas/química , Receptores Fc/química , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Galinhas , Humanos , Imunoglobulina E/química , Imunoglobulina E/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Imunoglobulinas/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptores Fc/metabolismoRESUMO
Antibodies directed against tumor-associated antigens are emerging as effective treatments for a number of cancers, although the mechanism(s) of action for some are unclear and still under investigation. We have previously examined a chimeric IgE antibody (MOv18 IgE), against the ovarian tumor-specific antigen, folate binding protein (FBP), and showed that it can direct human PBMC to kill ovarian cancer cells. We have developed a three-color flow cytometric assay to investigate the mechanism by which IgE receptors on U937 monocytes target and kill ovarian tumor cells. U937 monocytes express three IgE receptors, the high-affinity receptor, FcepsilonRI, the low-affinity receptor, CD23, and galectin-3, and mediate tumor cell killing in vitro by two mechanisms, cytotoxicity, and phagocytosis. Our results suggest that CD23 mediates phagocytosis, which is enhanced by upregulation of CD23 on U937 cells with IL-4, whereas FcepsilonRI mediates cytotoxicity. We show that effector : tumor cell bridging is associated with both activities. Galectin-3 does not appear to be involved in tumor cell killing. U937 cells and IgE exerted ovarian tumor cell killing in vivo in our xenograft model in nude mice. Harnessing IgE receptors to target tumor cells suggests the potential of tumor-specific IgE antibodies to activate effector cells in immunotherapy of ovarian cancer.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Imunoterapia/métodos , Monócitos/imunologia , Neoplasias Ovarianas/terapia , Fagocitose/fisiologia , Receptores de IgE/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Camundongos NusRESUMO
Abs have a paramount place in the treatment of certain, mainly lymphoid, malignancies, although tumors of nonhemopoietic origin have proved more refractory ones. We have previously shown that the efficacy of immunotherapy of solid tumors, in particular ovarian carcinoma, may be improved by the use of IgE Abs in place of the conventional IgG. An IgE Ab (MOv18 IgE) against an ovarian-tumor-specific Ag (folate binding protein), in combination with human PBMC, introduced into ovarian cancer xenograft-bearing mice, greatly exceeded the analogous IgG1 in promoting survival. In this study, we analyzed the mechanisms by which MOv18 IgE may exert its antitumor activities. Monocytes were essential IgE receptor-expressing effector cells that mediated the enhanced survival of tumor-bearing mice by MOv18 IgE and human PBMC. Monocytes mediated MOv18 IgE-dependent ovarian tumor cell killing in vitro by two distinct pathways, cytotoxicity and phagocytosis, acting respectively through the IgE receptors FcepsilonRI and CD23. We also show that human eosinophils were potent effector cells in MOv18 IgE Ab-dependent ovarian tumor cell cytotoxicity in vitro. These results demonstrate that IgE Abs can engage cell surface IgE receptors and activate effector cells against ovarian tumor cells. Our findings offer a framework for an improved immunotherapeutic strategy for combating solid tumors.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Carcinoma/tratamento farmacológico , Imunoglobulina E/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Fagocitose , Animais , Anticorpos Monoclonais Murinos , Carcinoma/imunologia , Carcinoma/terapia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia , Camundongos , Monócitos/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Receptores de IgE/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The low affinity IgE receptor, CD23, is implicated in IgE regulation and the pathogenesis of allergic disease. CD23 is a type II integral membrane protein, comprising a lectin "head," N-terminal "stalk," and C-terminal "tail" in the extracellular sequence. Endogenous proteases cleave CD23 in the stalk and the tail to release soluble fragments that either stimulate or inhibit IgE synthesis in human B cells. The molecular basis of these paradoxical activities is not understood. We have characterized three fragments of CD23, monomeric derCD23, monomeric exCD23, and oligomeric lzCD23. We show that the monomers inhibit and the oligomer stimulates IgE synthesis in human B cells after heavy chain switching to IgE. CD23 fragments could be targets for therapeutic intervention in allergic disease.
Assuntos
Linfócitos B/imunologia , Imunoglobulina E/biossíntese , Receptores de IgE/imunologia , Linfócitos B/metabolismo , Dimerização , Humanos , Hipersensibilidade , Fragmentos de Peptídeos , Receptores de IgE/metabolismo , SolubilidadeRESUMO
The gene PA4866 from Pseudomonas aeruginosa is documented in the Pseudomonas genome database as encoding a 172 amino acid hypothetical acetyltransferase. We and others have described the 3D structure of this protein (termed pita) [Davies et al. (2005) Proteins: Struct., Funct., Bioinf. 61, 677-679; Nocek et al., unpublished results], and structures have also been reported for homologues from Agrobacterium tumefaciens (Rajashankar et al., unpublished results) and Bacillus subtilis [Badger et al. (2005) Proteins: Struct., Funct., Bioinf. 60, 787-796]. Pita homologues are found in a large number of bacterial genomes, and while the majority of these have been assigned putative phosphinothricin acetyltransferase activity, their true function is unknown. In this paper we report that pita has no activity toward phosphinothricin. Instead, we demonstrate that pita acts as an acetyltransferase using the glutamate analogues l-methionine sulfoximine and l-methionine sulfone as substrates, with Km(app) values of 1.3 +/- 0.21 and 1.3 +/- 0.13 mM and kcat(app) values of 505 +/- 43 and 610 +/- 23 s-1 for l-methionine sulfoximine and l-methionine sulfone, respectively. A high-resolution (1.55 A) crystal structure of pita in complex with one of these substrates (l-methionine sulfoximine) has been solved, revealing the mode of its interaction with the enzyme. Comparison with the apoenzyme structure has also revealed how certain active site residues undergo a conformational change upon substrate binding. To investigate the role of pita in P. aeruginosa, a mutant strain, Depp4, in which pita was inactivated through an in-frame deletion, was constructed by allelic exchange. Growth of strain Depp4 in the absence of glutamine was inhibited by l-methionine sulfoximine, suggesting a role for pita in protecting glutamine synthetase from inhibition.
Assuntos
Acetiltransferases/química , Aminobutiratos/química , Metionina Sulfoximina/química , Pseudomonas aeruginosa/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Aminobutiratos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Glutamato-Amônia Ligase/metabolismo , Cinética , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Metionina Sulfoximina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Estereoisomerismo , Especificidade por SubstratoRESUMO
Dribble (DBE) is an essential protein in Drosophila that belongs to the evolutionarily conserved Krr1p protein family. Proteins in this family are localised in the cell nucleolus and are important for the processing of ribosomal RNAs. However, little is known about their structural and biophysical properties. We have expressed and purified full-length DBE protein from Escherichia coli. Consistent with the native role of DBE in RNA processing, recombinant DBE was shown to bind RNA homo-polymers in vitro. By bioinformatics, size-exclusion chromatography, equilibrium sedimentation analysis, controlled proteolysis, and a variety of spectroscopic techniques, we have found that DBE is a monomeric protein in solution containing both alpha- and beta-structures. Moreover, the structure of DBE is expanded and significantly disordered (approximately 45% disordered). Natively disordered proteins are thought to provide a disproportionately large surface area and structural plasticity for nucleic acid binding. We therefore propose that the presence of structural disorder is an important feature of DBE that facilitates the protein to interact with RNAs in the nucleolus.
Assuntos
Proteínas de Drosophila/química , Proteínas Nucleares/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Biofísica , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Conformação Proteica , RNA/química , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Solventes/químicaRESUMO
The low-affinity immunoglobulin E (IgE) receptor, CD23 (FcepsilonRII), binds both IgE and CD21 and, through these interactions, regulates the synthesis of IgE, the antibody isotype that mediates the allergic response. We have determined the three-dimensional structure of the C-type lectin domain of CD23 in solution by nuclear magnetic resonance spectroscopy. An analysis of concentration-dependent chemical shift perturbations have allowed us to identify the residues engaged in self-association to the trimeric state, whereas ligand-induced changes have defined the binding sites for IgE and CD21. The results further reveal that CD23 can bind both ligands simultaneously. Despite the C-type lectin domain structure, none of the interactions require calcium. We also find that IgE and CD23 can interact to form high molecular mass multimeric complexes. The interactions that we have described provide a solution to the paradox that CD23 is involved in both up- and down-regulation of IgE and provide a structural basis for the development of inhibitors of allergic disease.