RESUMEN
Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.
Asunto(s)
Antígenos CD55/fisiología , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Pulmón/inmunología , Viremia/inmunología , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Antígenos CD55/química , Antígenos CD55/deficiencia , Quimiotaxis de Leucocito , Activación de Complemento , Glicoproteínas Hemaglutininas del Virus de la Influenza/fisiología , Adaptación al Huésped , Especificidad del Huésped , Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H1N1 del Virus de la Influenza A/fisiología , Interferón gamma/análisis , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Ácido N-Acetilneuramínico , Neuraminidasa/fisiología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Carga Viral , Proteínas Virales/fisiología , Virulencia , Replicación Viral , Pérdida de PesoRESUMEN
The adhesion G protein-coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1-4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1-3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55-CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55-CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein-coupled receptors.
Asunto(s)
Antígenos CD/metabolismo , Antígenos CD55/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Antígenos CD/química , Antígenos CD55/química , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores Acoplados a Proteínas G/químicaRESUMEN
CD55 is a major regulator of the complement system, a complex network of proteins that cooperate to clear tissue and blood pathogens from the organism. Indeed, overexpression of CD55 is associated with many diseases and is connected to the resistance mechanisms exhibited by several cancers towards immunotherapy approaches. High level of CD55 expression on tumour cells renders it a good target for both imaging and immunotherapy. Indeed, a conceivable approach to tackle disease is to interfere with CD55-mediated complement regulation with the use of CD55-targeting antibodies. However, the large size and poor tissue penetration together with to the high costs of antibodies often limits their widespread therapeutic use. Here, we employed bioinformatic and chemical approaches to design and synthesize molecules of small dimensions able to mimic a CD55 blocking antibody. As a result, a bicyclic peptide, named as miniAB55, proved to bind CD55 with nanomolar affinity. This molecule represents an attracting chemical scaffold for CD55-directed diagnostic tools in diseases associated with CD55 overproduction. To further support the applicative potential of miniAB55, we prove that the miniAB55 binds CD55 on the same region involved in inactivation of the complement C3 and C5 convertases, thus opening promising scenarios for the development of complement-modulating tools.
Asunto(s)
Anticuerpos/farmacología , Antígenos CD55/inmunología , Miniaturización , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Sitios de Unión de Anticuerpos/inmunología , Antígenos CD55/química , Ciclización , Humanos , Cinética , Modelos Moleculares , Simulación del Acoplamiento MolecularRESUMEN
Dysregulation of the complement system is involved in the pathogenesis of several diseases, and its inhibition has been shown to be a feasible therapeutic option. Therefore, there is an interest in the development of complement modulators to treat complement activation-related inflammatory pathologies. Mannose-binding lectin (MBL)/ficolin/collectin-associated protein-1 (MAP-1) is a regulatory molecule of the lectin pathway (LP), whereas complement receptor 1 (CD35) and decay-accelerating factor (CD55) are membrane-anchored regulators with effects on the central effector molecule C3. In this study, we developed 2 novel soluble chimeric inhibitors by fusing MAP-1 to the 3 first domains of CD35 (CD351-3) or the 4 domains of CD55 (CD551-4) to modulate the complement cascade at 2 different stages. The constructs showed biologic properties similar to those of the parent molecules. In functional complement activation assays, the constructs were very efficient in inhibiting LP activation at the level of C3 and in the formation of terminal complement complex. This activity was enhanced when coincubated with recombinant LP recognition molecules MBL and ficolin-3. Recombinant MAP-1 fusion proteins, combined with recombinant LP recognition molecules to target sites of inflammation, represent a novel and effective therapeutic approach involving the initiation and the central and terminal effector functions of the complement cascade.-Pérez-Alós, L., Bayarri-Olmos, R., Skjoedt, M.-O., Garred, P. Combining MAP-1:CD35 or MAP-1:CD55 fusion proteins with pattern-recognition molecules as novel targeted modulators of the complement cascade.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Antígenos CD55 , Activación de Complemento/efectos de los fármacos , Complemento C3 , Receptores de Complemento 3b , Receptores de Reconocimiento de Patrones , Proteínas Recombinantes de Fusión , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/farmacología , Antígenos CD55/química , Antígenos CD55/genética , Antígenos CD55/farmacología , Células CHO , Complemento C3/química , Complemento C3/metabolismo , Cricetulus , Humanos , Receptores de Complemento 3b/química , Receptores de Complemento 3b/genética , Receptores de Reconocimiento de Patrones/química , Receptores de Reconocimiento de Patrones/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Decay accelerating factor (DAF or CD55) is a cell associated C3 and C5 convertase regulator originally described in terms of protection of self-cells from systemic complement but now known to modulate adaptive T cell responses. It is expressed on all cell types. We investigated whether nonenzymatic glycation could impair its function and potentially be relevant to complications of diabetes mellitus and other conditions that result in nonenzymatic glycation including cancer, Alzheimer's disease, and aging. Immunoblots of affinity-purified DAF from erythrocytes of patients with diabetes showed pentosidine, glyoxal-AGEs, carboxymethyllysine, and argpyrimidine. HPLC/MS analyses of glucose modified DAF localized the sites of AGE modifications to K125 adjacent to K126, K127 at the junction of CCPs2-3 and spatially near R96, and R100, all identified as being critical for DAF's function. Functional analyses of glucose or ribose treated DAF protein showed profound loss of its regulatory activity. The data argue that de-regulated activation of systemic complement and de-regulated activation of T cells and leukocytes could result from non-enzymatic glycation of DAF.
Asunto(s)
Antígenos CD55/química , Diabetes Mellitus/sangre , Productos Finales de Glicación Avanzada/química , Aminoácidos/química , Arginina/análogos & derivados , Arginina/análisis , Antígenos CD55/sangre , Antígenos CD55/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Activación de Complemento , Eritrocitos/química , Glucosa/farmacología , Productos Finales de Glicación Avanzada/sangre , Humanos , Activación de Linfocitos , Lisina/análogos & derivados , Lisina/análisis , Modelos Moleculares , Ornitina/análogos & derivados , Ornitina/análisis , Conformación Proteica , Pirimidinas/análisis , Ribosa/farmacologíaRESUMEN
Regulators of complement activation (RCA) inhibit complement-induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i-iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b-binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease-related mutations and immune evasion.
Asunto(s)
Complemento C3b/química , Complemento C3b/metabolismo , Sitios de Unión , Antígenos CD55/química , Antígenos CD55/metabolismo , Activación de Complemento , Humanos , Proteína Cofactora de Membrana/química , Proteína Cofactora de Membrana/metabolismo , Dominios Proteicos , Receptores de Complemento 3b/química , Receptores de Complemento 3b/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismoRESUMEN
BACKGROUND: A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack. In the present study, we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. METHODS: Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC), was measured. RESULTS: SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59, respectively, in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7%, respectively. CONCLUSIONS: When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach.
Asunto(s)
Antígenos CD55/genética , Antígenos CD59/genética , Activación de Complemento/genética , Inactivadores del Complemento , Proteína Cofactora de Membrana/genética , Transgenes , Animales , Antígenos CD55/química , Antígenos CD59/química , Inactivadores del Complemento/administración & dosificación , Inactivadores del Complemento/química , Dependovirus , Vectores Genéticos , Humanos , Proteína Cofactora de Membrana/química , Ratones , Estructura Terciaria de Proteína/genéticaRESUMEN
Embryonic stem cell research has facilitated the generation of many cell types for the production of tissues and organs for both humans and companion animals. Because ≥30% of pet cats suffer from chronic kidney disease (CKD), xenotransplantation between pigs and cats has been studied. For a successful pig to cat xenotransplant, the immune reaction must be overcome, especially hyperacute rejection. In this study, we isolated the gene for feline decay-accelerating factor (fDAF), an inhibitor of complement proteins, and transfected a swine endothelial cell line with fDAF to "felinize" the pig cells. These fDAF-expressing cells were resistant to feline serum containing anti-pig antibodies, suggesting that felinized pig cells were resistant to hyperacute rejection. Our results suggest that a "felinized" pig kidney can be generated for the treatment of CKD in cats in the future.
Asunto(s)
Antígenos CD55/genética , Células Endoteliales/metabolismo , Trasplante Heterólogo , Secuencia de Aminoácidos , Animales , Antígenos CD55/química , Enfermedades de los Gatos/cirugía , Gatos , Línea Celular , Células Clonales/metabolismo , Clonación Molecular , Expresión Génica , Rechazo de Injerto , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ratones , Datos de Secuencia Molecular , Insuficiencia Renal Crónica/veterinaria , Porcinos , Transfección , Trasplante Heterólogo/efectos adversosRESUMEN
The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.
Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos/química , Linfoma de Burkitt/inmunología , Inmunoterapia/métodos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos CD20/química , Antígenos CD55/química , Antígenos CD59/química , Separación Celular , Clonación Molecular , Proteínas del Sistema Complemento , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Células Asesinas Naturales/citología , Macrófagos/citología , Ratones , Ratones SCID , Microscopía FluorescenteRESUMEN
Binding of C1q to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher C1q binding affinity compared with human IgG1, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of IgG1- and IgG3-driven complement activation using isotype variants of the therapeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding and similar efficiency in Fab-mediated effector mechanisms. Whereas anti-EGFR-IgG1 did not promote CML of investigated target cells, anti-EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti-EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory proteins CD55 and CD59, but not CD46. Notably, anti-EGFR-IgG3 promoted strong C1q and C3b, but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti-EGFR-IgG3 triggered C4a release on all cells but failed to induce C3a and C5a release on CD55/CD59 highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti-EGFR-IgG3-triggered CML and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification. Together, these data suggest human anti-EGFR-IgG3, although highly reactive with C1q, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway.
Asunto(s)
Antígenos CD55/genética , Vía Alternativa del Complemento/genética , Vía Alternativa del Complemento/inmunología , Citotoxicidad Inmunológica/genética , Receptores ErbB/genética , Inmunoglobulina G/genética , Animales , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/uso terapéutico , Antígenos CD55/biosíntesis , Antígenos CD55/química , Células CHO , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Convertasas de Complemento C3-C5/antagonistas & inhibidores , Convertasas de Complemento C3-C5/genética , Cricetinae , Cricetulus , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Femenino , Marcación de Gen/métodos , Humanos , Inmunoglobulina G/uso terapéutico , Técnicas de Amplificación de Ácido Nucleico/métodos , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunologíaRESUMEN
The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses.
Asunto(s)
Antígenos CD55/química , Enterovirus Humano B/ultraestructura , Modelos Moleculares , Conformación Proteica , Receptores Virales/química , Antígenos CD55/metabolismo , Línea Celular Tumoral , Microscopía por Crioelectrón , Cristalización , Cristalografía por Rayos X , Humanos , Receptores Virales/metabolismoRESUMEN
Over approximately the last 15 years the electron paramagnetic resonance (EPR) technique of double electron electron resonance (DEER) has attracted considerable attention since it allows for the precise measurement of the dipole-dipole coupling between radicals and thus can lead to distance information between pairs of radicals separated by up to ca. 8 nm. The "deadtime free" 4-pulse DEER sequence is widely used but can suffer from poor sensitivity if the electron spin-echo decays too quickly to allow collection of a sufficiently long time trace. In this paper we present a method which takes advantage of the much greater sensitivity that the 3-pulse sequence offers over the 4-pulse sequence since the measured electron spin-echo intensity (for equal sequence lengths) is greater. By combining 3- and 4-pulse DEER time traces using a method coined DEER-Stitch (DEERS) accurate dipole-dipole coupling measurements can be made which combine the sensitivity of the 3-pulse DEER sequence with the deadtime free advantage of the 4-pulse DEER sequence. To develop the DEER-Stitch method three systems were measured: a semi-rigid bis-nitroxide labeled nanowire, the bis-nitroxide labeled protein CD55 with a distance between labels of almost 8 nm and a dimeric copper amine oxidase from Arthrobacter globiformis (AGAO).
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Algoritmos , Amina Oxidasa (conteniendo Cobre)/química , Arthrobacter/química , Antígenos CD55/química , Antígenos CD55/genética , Cristalización , Cisteína/química , ADN/química , ADN/genética , Humanos , Nanocables , Óxidos de Nitrógeno/química , Distribución Normal , Proteínas/química , Marcadores de SpinRESUMEN
BACKGROUND: We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro. METHODS: Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT-PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays. RESULTS: High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT-PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean ± s.e.m.=6.8 ± 0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6 ± 0.8% and 10.7 ± 0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu. CONCLUSION: Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos CD55/química , Antígenos CD55/genética , Antígenos CD59/química , Antígenos CD59/genética , Activación de Complemento , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/inmunología , Citotoxicidad Inmunológica , Regulación hacia Abajo , Femenino , Citometría de Flujo , Humanos , Hibridación Fluorescente in Situ , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Persona de Mediana Edad , Pronóstico , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor ErbB-2/genética , Trastuzumab , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/inmunologíaRESUMEN
Afa/Dr fimbriae constitute the major virulence factor of diffusely adhering Escherichia coli (Afa/Dr DAEC). After recognizing membrane-bound signaling receptors, they trigger cell responses. One of these receptors is the human decay-accelerating factor (hDAF). It has previously been reported that the binding of Afa/Dr fimbriae to hDAF quickly induces recruitment of hDAF around adhering bacteria. The aim of our study is to analyze the role of Src kinases in the Dr fimbria-induced recruitment of hDAF. Using biochemical methods and confocal microscopy followed by 3-dimensional (3D) analysis, we have shown that the activation and cell membrane targeting of Src kinases are necessary for the recruitment and organization of hDAF around adhering bacteria. We identified c-Src to be the specific kinase involved in this process. Using a set of Src-green fluorescent protein mutants, we showed that the catalytic activity and the Src homology 2 (SH2) and SH3 domains of the Src kinases are necessary for Dr fimbria-induced hDAF mobilization to occur. In addition, using mutated Dr fimbriae and a set of mutated hDAFs in which each of the complement control protein (CCP) domains had successively been deleted, we found that the aspartic acids at position 54 in the Dr fimbriae and in CCP domain 4 of hDAF played pivotal roles in the mobilization of the Src kinases and hDAF, respectively.
Asunto(s)
Antígenos CD55/metabolismo , Escherichia coli/patogenicidad , Proteínas Fimbrias/metabolismo , Familia-src Quinasas/metabolismo , Adhesinas de Escherichia coli/química , Adhesinas de Escherichia coli/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Adhesión Bacteriana , Antígenos CD55/química , Células CHO , Moléculas de Adhesión Celular/metabolismo , Cricetinae , Cricetulus , Escherichia coli/fisiología , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/metabolismo , Técnica del Anticuerpo Fluorescente , Glicosilfosfatidilinositoles/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Microdominios de Membrana , ARN Interferente Pequeño , Transducción de Señal , Dominios Homologos src , Familia-src Quinasas/químicaRESUMEN
Proteomic analysis of samples isolated by laser capture microdissection from clinical specimens requires sample preparation and fractionation methods suitable for small amounts of protein. Here we describe a streamlined filter-aided sample preparation (FASP) workflow that allows efficient analysis of lysates from low numbers of cells. Addition of carrier substances such as polyethylene glycol or dextran to the processed samples improves the peptide yields in the low to submicrogram range. In a single LC-MS/MS run, analyses of 500, 1000, and 3000 cells allowed identification of 905, 1536, and 2055 proteins, respectively. Incorporation of an additional SAX fractionation step at somewhat higher amounts enabled the analysis of formalin fixed and paraffin embedded human tissues prepared by LCM to a depth of 3600-4400 proteins per single experiment. We applied this workflow to compare archival neoplastic and matched normal colonic mucosa cancer specimens for three patients. Label-free quantification of more than 6000 proteins verified this technology through the differential expression of 30 known colon cancer markers. These included Carcino-Embryonic Antigen (CEA), the most widely used colon cancer marker, complement decay accelerating factor (DAF, CD55) and Metastasis-associated in colon cancer protein 1 (MACC1). Concordant with literature knowledge, mucin 1 was overexpressed and mucin 2 underexpressed in all three patients. These results show that FASP is suitable for the low level analysis of microdissected tissue and that it has the potential for exploration of clinical samples for biomarker and drug target discovery.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Proteoma/genética , Proteómica/métodos , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/química , Antígenos CD55/análisis , Antígenos CD55/química , Antígenos CD55/genética , Antígeno Carcinoembrionario/análisis , Antígeno Carcinoembrionario/química , Antígeno Carcinoembrionario/genética , Recuento de Células , Línea Celular Tumoral , Cromatografía Liquida , Neoplasias del Colon/química , Dextranos/química , Fijadores/química , Formaldehído/química , Humanos , Rayos Láser , Microdisección , Mucina-1/análisis , Mucina-1/química , Mucina-1/genética , Mucina 2/análisis , Mucina 2/química , Mucina 2/genética , Adhesión en Parafina , Fragmentos de Péptidos , Polietilenglicoles/química , Proteoma/análisis , Proteoma/química , Espectrometría de Masas en Tándem , Transactivadores , Factores de Transcripción/análisis , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Echovirus 7 (EV7) belongs to the Enterovirus genus within the family Picornaviridae. Many picornaviruses use IgG-like receptors that bind in the viral canyon and are required to initiate viral uncoating during infection. However, in addition, some of the enteroviruses use an alternative or additional receptor that binds outside the canyon. Decay-accelerating factor (DAF) has been identified as a cellular receptor for EV7. The crystal structure of EV7 has been determined to 3.1-Å resolution and used to interpret the 7.2-Å-resolution cryo-electron microscopy reconstruction of EV7 complexed with DAF. Each DAF binding site on EV7 is near a 2-fold icosahedral symmetry axis, which differs from the binding site of DAF on the surface of coxsackievirus B3, indicating that there are independent evolutionary processes by which DAF was selected as a picornavirus accessory receptor. This suggests that there is an advantage for these viruses to recognize DAF during the initial process of infection.
Asunto(s)
Antígenos CD55/química , Antígenos CD55/metabolismo , Infecciones por Echovirus/metabolismo , Enterovirus Humano B/química , Enterovirus Humano B/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Microscopía por Crioelectrón , Cristalización , Cristalografía por Rayos X , Infecciones por Echovirus/virología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The efficacy of cancer-immunotherapy with complement-activating monoclonal antibodies is limited by over-expression of one or more membrane-bound complement regulatory proteins (mCRPs: CD46, CD55, CD59) on the surface of neoplastic cells. In this study we designed small interfering RNAs (siRNAs) for posttranscriptional gene knock down of CD46, CD55 and CD59 aiming at to sensitize tumor cells to complement attack and thereby to better exploit complement for tumor cell destruction. Tumor cell lines of different origin, such as Du145 (prostate), BT474 (breast) and K562 (erythroleukemia) were selected for the study. FACS-analysis demonstrated that siRNA anti-CD46(301) reduced CD46 protein expression up to 80%, siRNA anti-CD55(255) diminished CD55 protein expression up to 49%, and CD59 protein expression was inhibited up to 82% by siRNA anti-CD59(1339). Time course experiments revealed a long-lasting silencing effect with >50% complement regulator inhibition up to day 13. Upon mCRP knock down, complement-dependent cytotoxicity (CDC) was augmented by 20-30% for CD46, by up to 24% for CD55 and by up to 55% for CD59. The combined inhibition of all three inhibitors further enhanced CDC by up to 66%. Dependent on the cell line, CD46 and CD55 downregulation increased significantly C3 ospsonization, which is known to support cell-mediated defense mechanisms. mCRP blocking antibodies were only partly able to further augment the tumor cells' susceptibility to complement lysis. Thus, siRNA-induced inhibition of complement regulator expression clearly sensitizes malignant cells to complement attack and, if specifically targeted to the tumor, appears suited as adjuvant to improve antibody-based cancer immunotherapy.
Asunto(s)
Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Activación de Complemento/genética , Proteína Cofactora de Membrana/metabolismo , Neoplasias/genética , Neoplasias/inmunología , ARN Interferente Pequeño/genética , Antígenos CD55/química , Antígenos CD55/genética , Antígenos CD59/química , Antígenos CD59/genética , Activación de Complemento/inmunología , Citometría de Flujo , Humanos , Proteína Cofactora de Membrana/antagonistas & inhibidores , Proteína Cofactora de Membrana/genética , Neoplasias/patología , ARN Mensajero/genética , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Virus Vaccinia/química , Virus Vaccinia/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Antígenos CD55/química , Antígenos CD55/inmunología , Antígenos CD55/metabolismo , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Virus Vaccinia/inmunología , Proteínas Virales/inmunologíaRESUMEN
Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains < 120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (< 150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.
Asunto(s)
Membrana Celular/química , Toxina del Cólera/química , Gangliósido G(M1)/química , Microdominios de Membrana/química , Antígenos CD/química , Antígenos CD55/química , Línea Celular , Colesterol/química , Simulación por Computador , Glicosilfosfatidilinositoles/química , Humanos , Microscopía Confocal/métodos , Método de Montecarlo , Nanotecnología/métodos , Receptores de Transferrina/químicaRESUMEN
Decay accelerating factor (DAF) is a very potent complement regulatory protein which holds promise for clinical usage. Here we report on an improved procedure for refolding both rat and human DAF over-expressed in Escherichia coli. It was shown that 50-70% of the inclusion body could be refolded to soluble active protein. This method excludes the use of L-arginine, which is expensive, and can be used to prepare a large quantity of recombinant DAF for therapeutic studies.