RESUMEN
Neurturin (NRTN) provides trophic support to neurons and is considered a therapeutic agent for neurodegenerative diseases, such as Parkinson's disease. It binds to its co-receptor GFRa2, and the resulting NRTN-GFRa2 complex activates the transmembrane receptors rearranged during transfection (RET) or the neural cell adhesion molecule (NCAM). We report the crystal structure of NRTN, alone and in complex with GFRa2. This is the first crystal structure of a GFRa with all three domains and shows that domain 1 does not interact directly with NRTN, but it may support an interaction with RET and/or NCAM, via a highly conserved surface. In addition, biophysical results show that the relative concentration of GFRa2 on cell surfaces can affect the functional affinity of NRTN through avidity effects. We have identified a heparan sulfate-binding site on NRTN and a putative binding site in GFRa2, suggesting that heparan sulfate has a role in the assembly of the signaling complex. We further show that mutant NRTN with reduced affinity for heparan sulfate may provide a route forward for delivery of NRTN with increased exposure in preclinical in vivo models and ultimately to Parkinson's patients.
Asunto(s)
Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/química , Heparitina Sulfato/química , Complejos Multiproteicos/química , Neurturina/química , Transducción de Señal , Cristalografía por Rayos X , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Neurturina/genética , Neurturina/metabolismo , Dominios Proteicos , Estructura Cuaternaria de ProteínaRESUMEN
Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
Asunto(s)
Anticuerpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Enterotoxinas/metabolismo , Anticuerpos Monoclonales/metabolismo , Toxinas Bacterianas/química , Células CACO-2 , Clostridioides difficile/enzimología , Cristalografía por Rayos X , Citosol/metabolismo , Enterotoxinas/química , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Rubidio/química , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.
Asunto(s)
Antibacterianos/metabolismo , Anticuerpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/enzimología , Enterocitos/metabolismo , Enterotoxinas/metabolismo , Glucosiltransferasas/metabolismo , Modelos Moleculares , Secuencia de Aminoácidos , Antibacterianos/química , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Neutralizantes/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Sitios de Unión de Anticuerpos , Células CACO-2 , Secuencia Conservada , Cristalografía por Rayos X , Enterocitos/efectos de los fármacos , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/toxicidad , Mapeo Epitopo , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/toxicidad , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Secuencias Repetitivas de AminoácidoRESUMEN
Antibody therapeutics offer effective treatment options for a broad range of diseases. One of the greatest benefits of antibody therapeutics is their extraordinarily long serum half-life, allowing infrequent dosing with long-lasting effects. A characteristic of antibodies that drives long half-life is the ability to interact with the recycling receptor, FcRn, in a pH-dependent manner. The benefit of long half-life, however, carries with it liabilities. Although the positive effects of antibody therapeutics are long-lasting, any acute adverse events or chronic negative impacts, such as immunosuppression in the face of an infection, are also long-lasting. Therefore, we sought to develop antibodies with a chemical handle that alone would enjoy the long half-life of normal antibodies but, upon addition of a small-molecule antidote, would interact with the chemical handle and inhibit the antibody recycling mechanism, thus leading to rapid degradation and shortened half-life in vivo Here we present a proof of concept study where we identify sites to incorporate a non-natural amino acid that can be chemically modified to modulate FcRn interaction in vitro and antibody half-life in vivo This is an important first step in developing safer therapeutics, and the next step will be development of technology that can perform the modifying chemistry in vivo.
Asunto(s)
Anticuerpos/química , Antídotos/química , Antígenos de Histocompatibilidad Clase I/química , Receptores Fc/química , Anticuerpos/uso terapéutico , Antídotos/uso terapéutico , Antígenos de Histocompatibilidad Clase I/uso terapéutico , Humanos , Receptores Fc/uso terapéuticoRESUMEN
Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/inmunología , Interleucina-6/antagonistas & inhibidores , Receptores de IgG/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Células HEK293 , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Interleucina-6/inmunología , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Hígado/citología , Hígado/metabolismo , Ratones , Unión Proteica , Receptores de IgG/inmunologíaRESUMEN
UNLABELLED: Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and continue to be a pandemic threat. While vaccines are available, other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. To produce a therapeutic agent that is highly efficacious at low doses and is broadly specific against antigenically drifted H5N1 influenza viruses, we developed two neutralizing monoclonal antibodies and combined them into a single bispecific Fc fusion protein (the Fc dual-affinity retargeting [FcDART] molecule). In mice, a single therapeutic or prophylactic dose of either monoclonal antibody at 2.5 mg/kg of body weight provided 100% protection against challenge with A/Vietnam/1203/04 (H5N1) or the antigenically drifted strain A/Whooper swan/Mongolia/244/05 (H5N1). In ferrets, a single 1-mg/kg prophylactic dose provided 100% protection against A/Vietnam/1203/04 challenge. FcDART was also effective, as a single 2.5-mg/kg therapeutic or prophylactic dose in mice provided 100% protection against A/Vietnam/1203/04 challenge. Antibodies bound to conformational epitopes in antigenic sites on the globular head of the hemagglutinin protein, on the basis of analysis of mutants with antibody escape mutations. While it was possible to generate escape mutants in vitro, they were neutralized by the antibodies in vivo, as mice infected with escape mutants were 100% protected after only a single therapeutic dose of the antibody used to generate the escape mutant in vitro. In summary, we have combined the antigen specificities of two highly efficacious anti-H5N1 influenza virus antibodies into a bispecific FcDART molecule, which represents a strategy to produce broadly neutralizing antibodies that are effective against antigenically diverse influenza viruses. IMPORTANCE: Highly pathogenic H5N1 avian influenza viruses are associated with severe disease in humans and are a pandemic threat. A vaccine is available, but other approaches are required for patients that typically respond poorly to vaccination, such as the elderly and the immunocompromised. The variability of the virus means that such an approach must be broad spectrum. To achieve this, we developed two antibodies that neutralize H5N1 influenza viruses. In mice, these antibodies provided complete protection against a spectrum of H5N1 influenza viruses at a single low dose. We then combined the two antibodies into a single molecule, FcDART, which combined the broad-spectrum activity and protective efficacy of both antibodies. This treatment provides a novel and effective therapeutic agent or prophylactic with activity against highly pathogenic H5N1 avian influenza viruses.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Subtipo H5N1 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Células CHO , Cricetinae , Cricetulus , Perros , Hurones , Técnica del Anticuerpo Fluorescente , Células HEK293 , Pruebas de Inhibición de Hemaglutinación , Humanos , Células de Riñón Canino Madin Darby , Ratones , Pruebas de Neutralización , Infecciones por Orthomyxoviridae/inmunologíaRESUMEN
We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100µg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.
Asunto(s)
Anticuerpos/genética , Anticuerpos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Biblioteca de Péptidos , Adenovirus Humanos/genética , Línea Celular , Antígenos Nucleares del Virus de Epstein-Barr/genética , Vectores Genéticos , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismoRESUMEN
We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell-killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19(-) cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Linfocitos B/inmunología , Linfoma de Células B , Linfocitos T/inmunología , Animales , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Linfocitos B/citología , Complejo CD3/inmunología , Complejo CD3/metabolismo , Comunicación Celular/inmunología , Línea Celular Tumoral , Femenino , Humanos , Linfocinas/inmunología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Sialoglicoproteínas/inmunología , Linfocitos T/citología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. METHODS: DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. RESULTS: DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. CONCLUSION: This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Linfocitos B/efectos de los fármacos , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Receptores de IgG , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Antígenos CD79/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dimerización , Femenino , Humanos , Inmunoglobulinas/metabolismo , Inmunosupresores/inmunología , Inmunosupresores/farmacocinética , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Noqueados , Receptores de IgG/inmunología , Transducción de Señal , Bazo/citología , Andamios del TejidoRESUMEN
The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.
Asunto(s)
Vacunas contra el Carbunco , Carbunco/prevención & control , Antitoxinas/química , Antitoxinas/metabolismo , Toxinas Bacterianas/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Animales , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Vectores Genéticos , Iridoviridae/química , Iridoviridae/inmunología , Masculino , Proteínas de la Membrana/inmunología , Microscopía Electrónica , Nanopartículas , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptores de PéptidosRESUMEN
The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH approximately 6.0 or below when it is bound to ANTXR1, but only at pH approximately 5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation.
Asunto(s)
Carbunco/genética , Bacillus anthracis/genética , Receptores de Péptidos/genética , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Secuencia Conservada , Cartilla de ADN , ADN Bacteriano/genética , Endocitosis/genética , Genes Reporteros , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Mutagénesis , Plásmidos/genéticaRESUMEN
Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis.
Asunto(s)
Carbunco/microbiología , Antígenos Bacterianos/toxicidad , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/toxicidad , Receptores de Péptidos/metabolismo , Animales , Carbunco/inmunología , Carbunco/metabolismo , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Bacillus anthracis/metabolismo , Toxinas Bacterianas/inmunología , Secuencia de Bases , Humanos , Longevidad/efectos de los fármacos , Masculino , Proteínas de la Membrana/inmunología , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Proteínas de Neoplasias/inmunología , Ratas , Ratas Endogámicas F344 , Receptores de Superficie Celular/inmunología , Receptores de Péptidos/inmunologíaRESUMEN
The ability of many retroviruses to cause disease can be correlated to their cytopathic effect (CPE) in tissue culture characterized by an acute period of cell death and viral DNA accumulation. Here, we show that mutants of a subgroup B avian retrovirus (Alpharetrovirus) cause a very dramatic CPE in certain susceptible avian cells that is coincident with elevated levels of apoptosis, as measured by nuclear morphology, and persistent viral DNA accumulation. These mutants also have a broadly extended host range that includes rodent, cat, dog, monkey, and human cells (31). Previously, we have shown that the mutants exhibit diminished resistance to superinfection. The results presented here have important implications for the process of evolution of retroviruses to use distinct cellular receptors.
Asunto(s)
Alpharetrovirus/fisiología , Alpharetrovirus/patogenicidad , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/virología , Alpharetrovirus/genética , Animales , Apoptosis , Evolución Biológica , Línea Celular , Núcleo Celular/metabolismo , Efecto Citopatogénico Viral , ADN Viral/metabolismo , Mutación , Virulencia , Replicación ViralRESUMEN
The three proteins that constitute anthrax toxin self-assemble into toxic complexes after one of these proteins, protective antigen (PA), binds to tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2) cellular receptors. The toxin receptor complexes are internalized, and acidic endosomal pH triggers pore formation by PA and translocation of the catalytic subunits into the cytosol. In this study we show that the pH threshold for conversion of the PA prepore to the pore and for translocation differs by approximately a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry.
Asunto(s)
Antígenos Bacterianos/metabolismo , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/metabolismo , Receptores de Péptidos/metabolismo , Antígenos Bacterianos/fisiología , Endocitosis , Concentración de Iones de Hidrógeno , Membranas Intracelulares , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos , Proteínas de Neoplasias/metabolismo , Porosidad , Receptores de Superficie Celular/metabolismo , Receptores de Péptidos/fisiología , Vesículas TransportadorasRESUMEN
Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60 mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.
Asunto(s)
Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Animales , Fenómenos Biofísicos , Biofisica , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Técnicas de Placa-Clamp , Compuestos de Amonio Cuaternario/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Péptidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Never before has there been such a strong possibility that biological agents might be used indiscriminately on civilian populations. This review focuses on the use of antitoxins - antibodies, receptor decoys, dominant-negative inhibitors of translocation, small-molecule inhibitors and substrate analogues - to counteract those biological weapons for which toxins are an important mechanism of disease pathogenesis.
Asunto(s)
Antitoxinas/farmacología , Bioterrorismo , Toxinas Biológicas/toxicidad , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/toxicidad , Antitoxinas/uso terapéutico , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Toxinas Botulínicas/antagonistas & inhibidores , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/toxicidad , Humanos , Ricina/antagonistas & inhibidores , Ricina/toxicidad , Toxinas Biológicas/metabolismoRESUMEN
Alpharetroviruses provide a useful system for the study of the molecular mechanisms of host range and receptor interaction. These viruses can be divided into subgroups based on diverse receptor usage due to variability within the two host range determining regions, hr1 and hr2, in their envelope glycoprotein SU (gp85). In previous work, our laboratory described selection from a subgroup B avian sarcoma-leukosis virus of an extended-host-range variant (LT/SI) with two adjacent amino acid substitutions in hr1. This virus retains its ability to use the subgroup BD receptor but can also infect QT6/BD cells, which bear a related subgroup E receptor (R. A. Taplitz and J. M. Coffin, J. Virol 71:7814-7819, 1997). Here, we report further analysis of this unusual variant. First, one (L154S) of the two substitutions is sufficient for host range extension, while the other (T155I) does not alter host range. Second, these mutations extend host range to non-avian cell types, including human, dog, cat, mouse, rat, and hamster. Third, interference experiments imply that the mutants interact efficiently with the subgroup BD receptor and possibly the related subgroup E receptor, but they have another means of entry that is not dependent on these interactions. Fourth, binding studies indicate that the mutant SU proteins retain the ability to interact as monomers with subgroup BD and BDE receptors but only bind the subgroup E receptor in the context of an Env trimer. Further, the mutant SU proteins bind well to chicken cells but do not bind any better than wild-type subgroup B to QT6 or human cells, even though the corresponding viruses are capable of infecting these cells.
Asunto(s)
Virus de la Leucosis Aviar/patogenicidad , Virus del Sarcoma Aviar/patogenicidad , Inmunoglobulina G/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Virus de la Leucosis Aviar/genética , Virus del Sarcoma Aviar/genética , Gatos , Línea Celular , Células Cultivadas , Embrión de Pollo , Pollos , Cricetinae , Perros , Humanos , Inmunoglobulina G/genética , Ratones , Datos de Secuencia Molecular , Mutación , Ratas , Recombinación Genética , Especificidad de la Especie , Transfección , Proteínas del Envoltorio Viral/genéticaRESUMEN
Bacillus anthracis secretes two bipartite toxins thought to be involved in anthrax pathogenesis and resulting death of the host. The current model for intoxication is that protective antigen (PA) toxin subunits bind a single group of cell-surface anthrax toxin receptors (ATRs), encoded by the tumor endothelial marker 8 (TEM8) gene. The ATR/TEM8-PA interaction is mediated by the receptor's extracellular domain related to von Willebrand factor type A or integrin inserted domains (VWA/I domains). A metal ion-dependent adhesion site (MIDAS) located within this domain of the ATR/TEM8 protein chelates a divalent cation critical for PA binding. In this report, we identify a second PA receptor encoded by capillary morphogenesis gene 2 (CMG2), which has 60% amino acid identity to ATR/TEM8 within the VWA/I domain, as well as a conserved MIDAS motif. A recombinant CMG2 protein bound PA and mediated toxin internalization when expressed on receptor-deficient cells. Binding between the CMG2 VWA/I domain and PA was shown to be direct and metal-dependent, although the cation specificity of this interaction is different than that observed with ATR/TEM8. Northern blot analysis revealed that CMG2 is widely expressed in human tissues, indicating that this receptor is likely to be relevant for disease pathogenesis. Finally, a soluble version of the CMG2 VWA/I domain inhibited intoxication of cells expressing endogenous toxin receptors when it was added to PA at a 3:1 ratio. These studies distinguish CMG2 as a second anthrax toxin receptor and identify a potent antitoxin that may prove useful for the treatment of anthrax.