RESUMO
UNLABELLED: The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells (HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism. IMPORTANCE: This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Hepacivirus/fisiologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Internalização do Vírus , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , HumanosRESUMO
Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancer cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. Here we report a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. RNA interference directed toward essential cellular cofactors in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. The ability to infect cultured cells directly with HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host-pathogen interactions and cell therapy.
Assuntos
Diferenciação Celular , Hepacivirus/fisiologia , Hepatite C/virologia , Hepatócitos/virologia , Modelos Biológicos , Células-Tronco Pluripotentes/virologia , Linhagem Celular , Hepatite C/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologiaRESUMO
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.
Assuntos
Ciclofilina A/antagonistas & inibidores , Ciclosporina/farmacologia , Hepacivirus/efeitos dos fármacos , Modelos Biológicos , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/efeitos dos fármacos , Western Blotting , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Hepacivirus/fisiologia , Humanos , Imunoprecipitação , Técnicas In Vitro , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Montagem de Vírus/fisiologiaRESUMO
Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.
Assuntos
Ciclofilina A/metabolismo , Ciclosporina/farmacologia , Farmacorresistência Viral , Regulação Viral da Expressão Gênica , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Sequência de Aminoácidos , Western Blotting , Linhagem Celular , Dicroísmo Circular , Ciclofilina A/genética , Ensaio de Imunoadsorção Enzimática , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Humanos , Técnicas Imunoenzimáticas , Imunossupressores/farmacologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Conformação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , RNA Viral/genética , Replicon/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.
Assuntos
Ciclofilina A/metabolismo , Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Linhagem Celular , Ciclofilina A/genética , Ciclosporina/farmacologia , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Mutação Puntual , RNA Viral/metabolismo , Proteínas não Estruturais Virais/efeitos dos fármacosRESUMO
UNLABELLED: Hepatitis C virus (HCV) infection leads to acute and chronic liver diseases, and new classes of anti-HCV therapeutics are needed. Cyclosporine A (CsA) inhibits HCV replication and CsA derivatives that lack the immunosuppressive function are currently in clinical trials as candidate anti-HCV drugs. Here we characterize several independently derived HCV replicons with varying levels of CsA resistance due to mutations in nonstructural protein 5B (NS5B), the HCV-encoded polymerase. Mutant HCV replicons engineered with these mutations showed resistance to CsA. The mutations reside in two distinct patches in the polymerase: the template channel and one face of a concave surface behind the template channel. Mutant NS5B made by cells expressing the HCV replicon had increased ability to bind to RNA in the presence of CsA. Purified recombinant NS5B proteins containing the mutations were better at de novo initiated RNA synthesis than the wild-type control. Furthermore, the mutant proteins were able to bind RNA with approximately 8-fold higher affinity. Last, mutation near the template channel alleviated the lethal phenotype of a mutation in the concave patch, P540A. This intramolecular compensation for the HCV replicase function by amino acid changes in different domains was further confirmed in an infectious cell culture-derived virus system. CONCLUSION: An increased level of CsA resistance is associated with distinct mutations in the NS5B gene that increase RNA binding in the presence of CsA, and the intramolecular communications between residues of the thumb and the C-terminal domains are important for HCV replicase function.
Assuntos
Ciclosporina/farmacologia , Farmacorresistência Viral , Hepacivirus/enzimologia , Hepacivirus/genética , Mutação , RNA Polimerase Dependente de RNA , RNA/metabolismo , Proteínas não Estruturais Virais/genética , Hepacivirus/efeitos dos fármacosRESUMO
Cyclosporine (CsA) and its derivatives potently suppress hepatitis C virus (HCV) replication. Recently, CsA-resistant HCV replicons have been identified in vitro. We examined the dependence of the wild-type and CsA-resistant replicons on various cyclophilins for replication. A strong correlation between CsA resistance and reduced dependency on cyclophilin A (CyPA) for replication was identified. Silencing of CyPB or CyPC expression had no significant effect on replication, whereas various forms of small interfering RNA (siRNA) directed at CyPA inhibited HCV replication of wild-type but not CsA-resistant replicons. The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication, and expression of an siRNA-resistant CyPA cDNA rescued replication. In addition, an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay. Depletion of CyPA alone in the CsA-resistant replicon cells eliminated CsA resistance, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection.
Assuntos
Ciclofilina A/metabolismo , Ciclosporina/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Sequência de Bases , Linhagem Celular , Ciclofilina A/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Viral da Expressão Gênica , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Interferente Pequeno/genética , RNA Viral/genética , Replicon/genéticaRESUMO
Because HCV RNA-dependent RNA polymerase is error-prone and the viral RNA has a high turnover rate, the genetic diversity of HCV is very high both in vitro and in vivo. The mutation rate in long-term replicon cultures approaches 3.0 x 10(-3) base substitutions/site/year in this in vitro replication model. A direct consequence of the high mutation rate is the rapid emergence of drug-resistant variants, both in cell culture and in patients. Selectable replicons have been used extensively to isolate and characterize drug-resistant HCV genomes in vitro. Typically, replicon cells are plated at a low density and then subjected to a double selection by G418 and escalating dosages of a compound of choice. Here we describe an alternative screening assay that takes advantage of an HCV replicon that is amenable to live-cell sorting with a suitable flow cytometer. We also present a strategy for determining the relative contribution to the resistance by viral genome and host cells. We use selection and characterization of Cyclosporine A (CsA)-resistant replicons as a example to present the protocols, but this method can easily be adapted for the selection of replicon cells resistant to other chemical compounds as long as the compound does not fluoresce at the same wavelength as the fluorescent reporter protein in the replicon.
Assuntos
Separação Celular/métodos , Farmacorresistência Viral/genética , Citometria de Fluxo/métodos , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Replicon/genética , Animais , Ciclosporina/farmacologia , DNA Viral/metabolismo , Eletroporação , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Hepacivirus/metabolismo , Interferon-alfa/metabolismo , Mutagênese Sítio-Dirigida , RNA Viral/biossíntese , RNA Viral/genética , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Proteínas não Estruturais Virais/genéticaRESUMO
Allergy to peanuts has become a common and severe problem, especially in westernized countries. In this study, we evaluated the target and epitope specificity of the capture and detection mouse monoclonal antibodies (mAbs) used in a commercial peanut allergen detection platform. We first identified the target of these antibodies as Ara h 3 and then used an overlapping peptide array of Ara h 3 to determine the antibody-binding epitopes. Further amino acids critical for the binding via alanine substitutions at individual amino acid residues within the epitope were mapped. Finally, inhibition ELISA and inhibition immunoblotting using a recombinant Ara h 3 protein were performed to confirm these results. Surprisingly, the capture and detection mAbs showed identical binding characteristics and were presumed to represent two isolates of the same clone, a notion supported by both isoelectric focusing electrophoresis and Liquid chromatography-mass spectrometry experiments. The simultaneous binding of a pair of identical mAbs to an individual allergen such as Ara h3 is attributed to the multivalency of the analyte and has implications for developing diagnostic assays for additional multimeric allergens.
Assuntos
Anticorpos Monoclonais/química , Antígenos de Plantas/química , Arachis/química , Epitopos/análise , Hipersensibilidade a Amendoim/diagnóstico , Proteínas de Plantas/química , Proteínas de Armazenamento de Sementes/química , Alanina/química , Alanina/genética , Alanina/imunologia , Alérgenos/química , Alérgenos/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Arachis/imunologia , Ensaio de Imunoadsorção Enzimática/normas , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Hipersensibilidade a Amendoim/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Análise Serial de Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/imunologiaRESUMO
Both linear and conformational epitopes likely contribute to the allergenicity of tree nut allergens, yet, due largely to technical issues, few conformational epitopes have been characterized. Using the well studied recombinant cashew allergen, Ana o 2, an 11S globulin or legumin, we identified a murine monoclonal antibody which recognizes a conformational epitope and competes with patient IgE Ana o 2-reactive antibodies. This epitope is expressed on the large subunit of Ana o 2, but only when associated with an 11S globulin small subunit. Both Ana o 2 and the homologous soybean Gly m 6 small subunits can foster epitope expression, even when the natural N-terminal to C-terminal subunit order is reversed in chimeric molecules. The epitope, which is also expressed on native Ana o 2, is readily susceptible to destruction by physical and chemical denaturants.
Assuntos
Alérgenos/química , Epitopos Imunodominantes/química , Proteínas de Plantas/química , Conformação Proteica , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Plantas , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Mercaptoetanol/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Ligação Proteica , Desnaturação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de Aminoácidos , Dodecilsulfato de Sódio/farmacologia , Glycine max/genética , Glycine max/imunologia , Ureia/farmacologiaRESUMO
The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope.
Assuntos
Alérgenos/química , Mapeamento de Epitopos/métodos , Epitopos Imunodominantes/química , Proteínas de Plantas/química , Conformação Proteica , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Plantas , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de AminoácidosRESUMO
Allergic reactions to walnuts and hazelnuts can be serious. The 11S globulins (legumins) have been identified as important allergens in these and other nuts and seeds. Here we identify the linear IgE-binding epitopes of walnut and hazelnut 11S globulins, and generate 3D 11S globulin models to map the locations of the epitopes for comparison to other allergenic homologues. Linear IgE-epitope mapping was performed by solid-phase overlapping 15-amino acid peptides probed with IgE from pooled allergic human sera. Several walnut (Jug r 4) and hazelnut (Cor a 9) 11S globulin peptides with reactivity to patient IgE were identified. Comparative alignment with cashew (Ana o 2), peanut (Ara h 3), and soybean G1 (Gly m 6.0101) and G2 (Gly m 6.0201) allergenic homologues revealed several shared allergenic 'hot spots'. Homology modeling was performed based on the atomic structure of the soybean glycinin. Surface map comparisons between the tree nut and peanut homologues revealed structural motifs that could be important for IgE elicitation and binding and show that, contrary to predictions, the reactive epitopes are widely distributed throughout the monomeric subunits, both internally and externally, including regions occluded by quaternary subunit association. These findings reveal structural features that may be important to allergenicity and cross-reactivity of this protein class.
Assuntos
Corylus/imunologia , Globulinas/imunologia , Imunoglobulina E/sangue , Juglans/imunologia , Hipersensibilidade a Noz/imunologia , Adolescente , Adulto , Alérgenos/química , Alérgenos/imunologia , Sequência de Aminoácidos , Anacardium/química , Anacardium/imunologia , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Arachis/química , Arachis/imunologia , Criança , Pré-Escolar , Corylus/química , Corylus/metabolismo , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Globulinas/química , Globulinas/metabolismo , Humanos , Juglans/química , Juglans/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Hipersensibilidade a Noz/sangue , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Alinhamento de Sequência , Glycine max/química , Glycine max/imunologiaRESUMO
Cashew apple juice has the potential to be a natural source of vitamin C and sugar in processed foods. The juice of the cashew apple is obtained by pressing the fleshy peduncle or receptacle, which forms a rounded apple that sits above the true fruit, the cashew nut. Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. To determine if cashew apple juice contains cashew nut allergens, immunoblotting was performed using a cashew apple juice 6X concentrate that was extracted and further concentrated through dialysis, lyophilization, and resuspension. Serum IgE of individuals allergic to cashew nut bound proteins in the cashew apple juice concentrate extract. For some serum samples, IgE reactivity could be inhibited by preincubation of the serum with cashew nut extract, suggesting the presence of cashew nut-related allergens. Using monoclonal antibodies specific for cashew nut allergens, the concentrate was found to contain Ana o 1 (vicilin) and Ana o 2 (legumin). Neither IgE from cashew nut allergic sera nor the monoclonal antibodies bound any peptides in 5 kDa filtered cashew apple juice concentrate. The cashew apple juice concentrate used in these studies contains proteins with IgE-reactive epitopes, including cashew nut legumin and vicilin. No IgE-binding peptides remained after 5 kDa filtration of the concentrate.
Assuntos
Anacardium/imunologia , Frutas/imunologia , Imunoglobulina E/imunologia , Hipersensibilidade a Noz/imunologia , Nozes/imunologia , Proteínas de Plantas/imunologia , Adolescente , Adulto , Idoso , Alérgenos/análise , Anticorpos Monoclonais , Bebidas/análise , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Proteínas de Plantas/análise , Pólen/imunologiaRESUMO
Cashew nut seeds were subjected to processing including autoclaving (121 degrees C for 5, 10, 20, and 30 min), blanching (100 degrees C for 1, 4, 7, and 10 min), microwave heating (1 and 2 min each at 500 and 1000 W), dry roasting (140 degrees C for 20 and 30 min; 170 degrees C for 15 and 20 min; and 200 degrees C for 10 and 15 min), gamma-irradiation (1, 5, 10, and 25 kGy), and pH (1, 3, 5, 7, 9, 11, and 13). Proteins from unprocessed and processed cashew nut seeds were probed for stability using anti-Ana o 2 rabbit polyclonal antibodies and mouse monoclonal antibodies directed against Ana o 1, Ana o 2, and Ana o 3 as detection agents. Results indicate that Ana o 1, Ana o 2, and Ana o 3 are stable regardless of the processing method to which the nut seeds are subjected.
Assuntos
Anacardium/química , Manipulação de Alimentos , Proteínas de Plantas/imunologia , Sementes/química , Animais , Anticorpos , Anticorpos Monoclonais , Estabilidade de Medicamentos , Irradiação de Alimentos , Raios gama , Temperatura Alta , Camundongos , Micro-Ondas , Hipersensibilidade a Noz/imunologia , Proteínas de Plantas/química , Pressão , CoelhosRESUMO
BACKGROUND: Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. We have previously cloned and characterized major cashew allergens belonging to the vicilin and legumin families of seed storage proteins. OBJECTIVE: Here we set out to describe a third major cashew allergen, a 2S albumin. METHODS: The recombinant cashew 2S albumin was amplified from a cDNA library by means of PCR, sequenced, and expressed in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition immunoblotting was used to identify the corresponding native cashew nut proteins. The mass of affinity-purified native allergen was determined by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy. Patients' sera were used to probe solid-phase 2S albumin peptides to identify linear epitopes. RESULTS: The cloned allergen, designated Ana o 3, was identified as 2S albumin. MALDI-TOF mass spectroscopy of native Ana o 3 yielded a molecular mass of 12,598 d. Immunoblot analysis showed 21 (81%) of 26 sera from patients with cashew allergy were reactive. Three native Ana o 3 large-subunit isoforms with molecular weights ranging from approximately 6 to 10 kd were identified. Probing of overlapping synthetic Ana o 3 peptides with patients' sera identified 16 reactive peptides, 4 of which gave strong signals and one of which positionally overlaps linear epitopes in mustard and walnut allergenic 2S albumins. The overlapping cashew and walnut epitopes also share considerable homology. CONCLUSIONS: We conclude that this 2S albumin protein is a major allergen in cashew nut and demonstrates a possible basis for cross-reactivity with walnut 2S albumin.
Assuntos
Alérgenos/genética , Anacardium/imunologia , Proteínas de Plantas/genética , Adolescente , Adulto , Alérgenos/efeitos adversos , Alérgenos/imunologia , Sequência de Aminoácidos , Anacardium/genética , Antígenos de Plantas , Sequência de Bases , Criança , Pré-Escolar , Reações Cruzadas , Epitopos/imunologia , Feminino , Hipersensibilidade Alimentar/etiologia , Hipersensibilidade Alimentar/imunologia , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/efeitos adversos , Proteínas de Plantas/imunologia , Proteínas Recombinantes/imunologia , Alinhamento de SequênciaRESUMO
BACKGROUND: Peanut and tree nut allergies can be life-threatening, and they appear to be growing in prevalence. Jug r 1, a 2S albumin seed storage protein, was previously characterized as a major English walnut food allergen. OBJECTIVE: We sought to identify the linear IgE-binding epitopes of Jug r 1 and to determine which, if any, amino acids are necessary for this binding to occur. METHODS: Pools of sera from walnut-allergic patients and overlapping peptides synthesized on an activated cellulose membrane were used to screen for IgE-binding epitopes. Mutational analysis of the immunodominant epitope was carried out through single and multisite amino acid substitutions. Inhibition assays were performed through use of affinity-purified IgE, soluble forms of the epitope peptide, and the recombinant 2S albumin, rJug r 1. RESULTS: One immunodominant linear epitope was identified. Amino acid mutations to the epitope demonstrated that the residues RGEE, at positions 36 through 39, were minimally required for IgE binding. Probing of this epitope with sera from each of 20 patients revealed 15 of the sera to be positive. Binding of patients' IgE to the epitope was inhibited with a soluble form of the peptide; however, soluble peptide did not completely inhibit the binding of IgE to the intact rJug r 1. CONCLUSION: One major linear IgE-reactive epitope and its critical core amino acid residues have been identified. Mutation of any of these core amino acids resulted in loss of IgE binding to the epitope, and this points toward the feasibility of reducing allergenicity in genetically modified walnuts. However, strong evidence for the existence of conformational epitopes was also obtained.
Assuntos
Alérgenos/imunologia , Mapeamento de Epitopos , Hipersensibilidade Alimentar/etiologia , Nozes/imunologia , Alérgenos/química , Sequência de Aminoácidos , Humanos , Imunoglobulina E/imunologia , Dados de Sequência MolecularRESUMO
BACKGROUND: We recently cloned and described a vicilin and showed it to be a major cashew allergen. Additional IgE-reactive cashew peptides of the legumin group and 2S albumin families have also been reported. Here, we attempt to clone, express and characterize a second major cashew allergen. METHODS: A cashew cDNA library was screened with human IgE and rabbit IgG anti-cashew extract antisera, and a reactive nonvicilin clone was sequenced and expressed as a fusion protein in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition of immunoblotting was used to identify the corresponding native peptides in cashew nut extract. The identified allergen was subjected to linear epitope mapping using SPOTs solid-phase synthetic peptide technology. RESULTS: Sequence analysis showed the selected clone, designated Ana o 2, to encode for a member of the legumin family (an 11S globulin) of seed storage proteins. By IgE immunoblotting, 13 of 21 sera (62%) from cashew-allergic patients were reactive. Immunoblot inhibition data showed that the native Ana o 2 constitutes a major band at approximately 33 kD and a minor band at approximately 53 kD. Probing of overlapping synthetic peptides with pooled human cashew-allergic sera identified 22 reactive peptides, 7 of which gave strong signals. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen, Ara h 3, in position but with little sequence similarity. Greater positional overlap and identity was observed between Ana o 2 and soybean glycinin epitopes. CONCLUSIONS: We conclude that this legumin-like protein is a major allergen in cashew nut.
Assuntos
Alérgenos/imunologia , Anacardium/imunologia , Hipersensibilidade a Noz/imunologia , Nozes/imunologia , Proteínas de Plantas/imunologia , Adulto , Alérgenos/química , Alérgenos/genética , Alérgenos/metabolismo , Sequência de Aminoácidos , Anacardium/química , Antígenos de Plantas , Sequência de Bases , Clonagem Molecular , Mapeamento de Epitopos , Escherichia coli/genética , Feminino , Humanos , Immunoblotting , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Nozes/química , Nozes/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
BACKGROUND: The allergens responsible for cashew food allergy have not been well characterized. OBJECTIVES: We initiated a study to clone cDNAs encoding cashew food allergens. METHODS: A cashew cDNA library was screened with human serum for IgE-reactive clones and rabbit IgG anti-cashew extract antisera. Reactive clones were sequenced and expressed, and linear epitopes were identified by means of solid-phase overlapping peptide analysis. Immunoblot inhibition was used to identify the native peptide in cashew extract. RESULTS: Four closely related clones reactive with both human and rabbit antisera were sequenced. Sequence analysis showed that these encode members of the vicilin/sucrose-binding protein family of plant seed storage proteins. Screening of the recombinant protein with sera from 20 patients with cashew allergy and 8 cashew-tolerant patients with allergies to other tree nuts showed that 50% and 25% of sera from patients with cashew allergy and cashew-tolerant subjects, respectively, bound the recombinant protein. The corresponding native allergen protein, designated Ana o 1, was located at approximately 50 kd. Epitope mapping revealed 11 linear IgE-binding epitopes, of which 3 appear to be immunodominant. None of the epitopes were shared in common with those of the peanut vicilin allergen Ara h 1. CONCLUSION: Ana o 1, a vicilin-like protein, is a major food allergen in cashews. Cashew and peanut vicilins do not share linear epitopes.