RESUMEN
BACKGROUND AND HYPOTHESIS: Volenrelaxin, is a half-life-extended recombinant human relaxin protein developed for improving kidney perfusion and cardiorenal function. This study assessed the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of volenrelaxin following single- and multiple-ascending doses (SAD and MAD) administration. METHODS: In this Phase 1, 4-part, randomized, double-blinded, placebo-controlled SAD and MAD study in healthy participants, SAD participants (n = 56) received an intravenous (IV) or subcutaneous (SC) dose of volenrelaxin or placebo in a dose-ascending manner. MAD participants (n = 77) received volenrelaxin or placebo SC once weekly for 5 weeks. Effective renal plasma flow (ERPF) and measured glomerular filtration rate (mGFR) were determined by para-aminohippurate and iohexol clearance, respectively. RESULTS: Volenrelaxin demonstrated an extended half-life and increased acute and chronic placebo-adjusted ERPF change from baseline by 50% and 44%, respectively (p < 0.0001). Measured GFR was unchanged, while filtration fraction and afferent/efferent renal arteriolar resistances were reduced. Systolic and diastolic blood pressures decreased, and pulse rate increased with increasing volenrelaxin exposures, demonstrating maximal model-derived placebo-adjusted changes (90% confidence interval) of -6.16 (-8.04, -4.28) mmHg, -6.10 (-7.61, -4.58) mmHg, and + 4.39 (3.38, 5.39) bpm, respectively. Adverse events were mild, with no difference in orthostatic hypotension between volenrelaxin and placebo. CONCLUSION: Volenrelaxin was well-tolerated, safe and suitable for weekly SC dosing. Volenrelaxin showed a sustained improvement in kidney perfusion upon repeated dosing, supporting further clinical development in chronic kidney disease and chronic heart failure. Clinical trial registration: NCT04768855.
RESUMEN
γδ T cells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. In vitro blocking of plexin B2 or CD100 inhibited γδ T cell activation. Furthermore, CD100 deficiency in vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T cell response to keratinocyte damage. Ligation of CD100 in γδ T cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support.
Asunto(s)
Antígenos CD/inmunología , Células de Langerhans/inmunología , Proteínas del Tejido Nervioso/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Semaforinas/inmunología , Linfocitos T/inmunología , Factores Despolimerizantes de la Actina/metabolismo , Animales , Antígenos CD/metabolismo , Células CHO , Comunicación Celular/inmunología , Forma de la Célula , Cricetinae , Epidermis/inmunología , Epidermis/lesiones , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Queratinocitos/inmunología , Queratinocitos/metabolismo , Células de Langerhans/metabolismo , Activación de Linfocitos/inmunología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Semaforinas/metabolismo , Análisis de Secuencia de Proteína , Resonancia por Plasmón de Superficie , Linfocitos T/metabolismo , Cicatrización de Heridas/inmunologíaRESUMEN
During somatic hypermutation (SHM), deamination of cytidine by activation-induced cytidine deaminase and subsequent DNA repair generates mutations within immunoglobulin V-regions. Nucleotide insertions and deletions (indels) have recently been shown to be critical for the evolution of antibody binding. Affinity maturation of 53 antibodies using in vitro SHM in a non-B cell context was compared with mutation patterns observed for SHM in vivo. The origin and frequency of indels seen during in vitro maturation were similar to that in vivo. Indels are localized to CDRs, and secondary mutations within insertions further optimize antigen binding. Structural determination of an antibody matured in vitro and comparison with human-derived antibodies containing insertions reveal conserved patterns of antibody maturation. These findings indicate that activation-induced cytidine deaminase acting on V-region sequences is sufficient to initiate authentic formation of indels in vitro and in vivo and that point mutations, indel formation, and clonal selection form a robust tripartite system for antibody evolution.
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Regiones Determinantes de Complementariedad/genética , Mutación INDEL , Hipermutación Somática de Inmunoglobulina , Regiones Determinantes de Complementariedad/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Femenino , Humanos , MasculinoRESUMEN
Recent advances are described for the isolation and affinity maturation of antibodies that couple in vitro somatic hypermutation (SHM) with mammalian cell display, replicating key aspects of the adaptive immune system. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID). AID-directed SHM in vitro in non-B cells, combined with mammalian display of a library of human antibodies, initially naïve to SHM, can be used to isolate and affinity mature antibodies via iterative cycles of fluorescence-activated cell sorting (FACS) under increasingly stringent sort conditions. SHM observed in vitro closely resembles SHM observed in human antibodies in vivo in both mutation type and positioning in the antibody variable region. In addition, existing antibodies originating from mouse immunization, in vivo based libraries, or alternative display technologies such as phage can also be affinity matured in a similar manner. The display system has been developed to enable simultaneous high-level cell surface expression and secretion of the same protein through alternate splicing, where the displayed protein phenotype remains linked to genotype, allowing soluble secreted antibody to be simultaneously characterized in biophysical and cell-based functional assays. This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.
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Anticuerpos Monoclonales/genética , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Antígenos/inmunología , Secuencia de Bases , Separación Celular , Cartilla de ADN/genética , Evolución Molecular Dirigida , Descubrimiento de Drogas , Citometría de Flujo , Biblioteca de Genes , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de ProteínasRESUMEN
A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.
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Empalme Alternativo , Anticuerpos Monoclonales/biosíntesis , Afinidad de Anticuerpos/genética , Evolución Molecular Dirigida , Expresión Génica , Inmunoglobulina G/biosíntesis , Anticuerpos Monoclonales/genética , Células HEK293 , Humanos , Inmunoglobulina G/genética , Precursores del ARN/biosíntesis , Precursores del ARN/genéticaRESUMEN
A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hßNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hßNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.
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Anticuerpos/química , Regiones Determinantes de Complementariedad/metabolismo , Mutación , Animales , Anticuerpos Monoclonales/química , Antígenos/química , Secuencia de Bases , Unión Competitiva , Separación Celular , Codón , Citocinas/metabolismo , Citometría de Flujo , Células HEK293 , Humanos , Técnicas In Vitro , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Ingeniería de Proteínas/métodos , Transducción de SeñalRESUMEN
BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.
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Cardiopatías , Insuficiencia Cardíaca , Relaxina , Animales , Femenino , Humanos , Ratones , Embarazo , Ratas , Cardiomegalia/tratamiento farmacológico , Cardiopatías/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Relaxina/farmacología , Relaxina/uso terapéutico , Relaxina/metabolismo , Volumen SistólicoRESUMEN
Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu(H47)Trp and Arg(H100)Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 Leu(H47)Trp/Arg(H100)Trp double mutant (1E9dm) as an unliganded Fab at 2.05 A resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 A. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.
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Anticuerpos/química , Anticuerpos/inmunología , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Esteroides/metabolismo , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Receptores Inmunológicos/inmunología , Esteroides/químicaRESUMEN
Development of biotherapeutics is hampered by the inherent risk of immunogenicity, which requires extensive clinical assessment and possible re-engineering efforts for mitigation. The focus in the pre-clinical phase is to determine the likelihood of developing treatment-emergent anti-drug antibodies (TE-ADA) and presence of pre-existing ADA in drug-naïve individuals as risk-profiling strategies. Pre-existing ADAs are routinely identified during clinical immunogenicity assessment, but their origin and impact on drug safety and efficacy have not been fully elucidated. One specific class of pre-existing ADAs has been described, which targets neoepitopes of antibody fragments, including Fabs, VH, or VHH domains in isolation from their IgG context. With the increasing number of antibody fragments and other small binding scaffolds entering the clinic, a widely applicable method to mitigate pre-existing reactivity against these molecules is desirable. Here is described a structure-based engineering approach to abrogate pre-existing ADA reactivity to the C-terminal neoepitope of VH(H)s. On the basis of 3D structures, small modifications applicable to any VH(H) are devised that would not impact developability or antigen binding. In-silico B cell epitope mapping algorithms were used to rank the modified VHH variants by antigenicity; however, the limited discriminating capacity of the computational methods prompted an experimental evaluation of the engineered molecules. The results identified numerous modifications capable of reducing pre-existing ADA binding. The most efficient consisted of the addition of two proline residues at the VHH C-terminus, which led to no detectable pre-existing ADA reactivity while maintaining favorable developability characteristics. The method described, and the modifications identified thereby, may provide a broadly applicable solution to mitigate immunogenicity risk of antibody-fragments in the clinic and increase safety and efficacy of this promising new class of biotherapeutics.
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Factores Biológicos/inmunología , Simulación del Acoplamiento Molecular , Anticuerpos de Dominio Único/química , Linfocitos B/inmunología , Factores Biológicos/química , Epítopos/química , Epítopos/inmunología , Humanos , Unión Proteica , Anticuerpos de Dominio Único/inmunologíaRESUMEN
The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.
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Alérgenos/genética , Alérgenos/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Poaceae/genética , Polen/genética , Ingeniería de Proteínas , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Alérgenos/administración & dosificación , Alérgenos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Desensibilización Inmunológica/métodos , Femenino , Humanos , Inmunoglobulina E/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/fisiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Plantas/administración & dosificación , Proteínas de Plantas/metabolismo , Poaceae/inmunología , Polen/inmunología , Conejos , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/prevención & control , Vacunas de Subunidad/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/metabolismoRESUMEN
Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.
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Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Modelos Moleculares , Muramidasa/química , Tiburones/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Epítopos , Células Germinativas/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Isotipos de Inmunoglobulinas/química , Isotipos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Muramidasa/inmunología , Mutación , Estructura Terciaria de ProteínaRESUMEN
The molecular characterization of allergens has accelerated significantly since the widespread implementation of modern analytical methods. The combination of gene cloning and heterologous protein expression has generated an extensive array of allergens that is available for comparative analysis, as well as clinical applications. Several internet-accessible allergen databases integrate the accumulated information from biomedical research and clinical practice. Innovations in classical biophysical methods, such as mass spectrometry, X-ray crystallography, and nuclear magnetic resonance spectroscopy, have rendered complex biological macromolecules amenable to detailed structural analysis. The modern scientific era has realized the synthesis of bioinformatics, molecular biology, biochemistry, biophysics, and immunology, and given us the means needed to decipher the remaining mysteries of allergies. This article addresses how the synergism of modern scientific techniques has hastened our understanding of allergies, how these techniques are applied in the identification and characterization of allergens, and how these methods assist the rational development of clinical tools for allergy diagnosis and treatment.
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Alérgenos/química , Polen/inmunología , Conformación Proteica , Secuencia de Aminoácidos , Dicroismo Circular/métodos , Biología Computacional/métodos , Cristalografía por Rayos X/métodos , Humanos , Resonancia Magnética Nuclear Biomolecular/métodos , Polen/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Most of the 400 million grass pollen-allergic patients worldwide are co-sensitized to several unrelated grass pollen allergens. Based on frequent co-sensitization patterns determined in 200 grass pollen-allergic patients, three recombinant hybrid molecules were developed by polymerase chain reaction-based mending of cDNAs coding for the major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, Phl p 6) for vaccination against grass pollen allergy. The hybrids rP2-P6, rP6-P2, and rP5-P1 contained most of the epitopes of natural grass pollen extract and induced stronger lymphoproliferative responses in cultured mononuclear cells of grass pollen-allergic patients than did equimolar mixtures of the individual allergens. Immunization of mice with the hybrids yielded higher antibody titers than did immunization with the individual allergen components or grass pollen extract, which suggests that the individual components of the hybrids can serve as molecular scaffolds for each other to enhance their immunogenicity. Antibodies induced with the hybrids in mice inhibited the binding of grass pollen-allergic patients' immunoglobulin E to each of the individual allergens and grass pollen extract and may thus represent protective antibodies. The principle of increasing the immunogenicity of antigens by engineering hybrids thereof may be applied not only for the treatment of polysensitized allergic patients but also for general vaccine development.
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Alérgenos/genética , Alérgenos/inmunología , Poaceae/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Anticuerpos Bloqueadores/inmunología , Células Cultivadas , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina G/biosíntesis , Cinética , Activación de Linfocitos , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Rinitis Alérgica Estacional/terapia , Vacunas Combinadas/inmunologíaRESUMEN
TLRs are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system and its biochemical, as well as ligand binding, properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chains containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED.
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Diglicéridos/metabolismo , Glucolípidos/metabolismo , Lipopéptidos/metabolismo , Mycobacterium/metabolismo , Mycoplasma/metabolismo , Staphylococcus aureus/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Proteínas Bacterianas , Baculoviridae/genética , Diglicéridos/síntesis química , Glucolípidos/síntesis química , Interacciones Huésped-Patógeno , Humanos , Insectos , Ligandos , Lipopéptidos/síntesis química , Lipopolisacáridos , Fosfatidilinositoles/química , Unión Proteica , Estructura Terciaria de Proteína/genética , Células Sf9 , Ácidos Teicoicos , Receptor Toll-Like 2/genéticaRESUMEN
The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.
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Autoantígenos/química , Inmunoglobulina E/inmunología , Transactivadores/química , Secuencia de Aminoácidos , Autoantígenos/genética , Autoantígenos/aislamiento & purificación , Secuencia de Bases , Línea Celular , Cromatografía de Afinidad , Dermatitis Atópica/inmunología , Humanos , Activación de Linfocitos , Chaperonas Moleculares , Datos de Secuencia Molecular , Isoformas de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Transactivadores/genética , Transactivadores/aislamiento & purificaciónRESUMEN
Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αß T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML) protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer.
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ADN Complementario/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/química , Cadenas lambda de Inmunoglobulina/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Cricetinae , Humanos , Cadenas lambda de Inmunoglobulina/genética , Ratones , Estructura Secundaria de Proteína , Ratas , Receptores de Antígenos de Linfocitos T gamma-delta/inmunologíaRESUMEN
γδ T cells bridge innate and adaptive immunity and function in immunosurveillance, immunoregulation, tumor cell recognition, and as first line of defense against microbial infection. Costimulation of epithelial γδ T cell activation by the JAML receptor can be induced by interaction with its endogenous ligand CAR or by binding of the stimulatory antibody HL4E10. We, therefore, determined the crystal structure of the JAML-HL4E10 Fab complex at 2.95 Šresolution. HL4E10 binds the membrane-proximal domain of JAML through hydrophobic interactions that account for nanomolar affinity and long half-life, contrasting with the fast kinetics and micromolar affinity of the hydrophilic CAR interaction with the membrane-distal JAML domain. Thus, despite different binding sites and mechanisms, JAML interaction with these two disparate ligands leads to the same functional outcome, namely JAML triggering and induction of cell signaling. Several characteristics of the HL4E10 antibody might then be harnessed in therapeutic applications, such as promoting healing of acute or chronic wounds.
Asunto(s)
Anticuerpos Heterófilos/farmacología , Anticuerpos Monoclonales/farmacología , Moléculas de Adhesión Celular/metabolismo , Células de Langerhans/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/metabolismo , Animales , Anticuerpos Heterófilos/química , Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Línea Celular , Proliferación Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Cricetinae , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fab de Inmunoglobulinas/química , Células de Langerhans/citología , Ratones , Fosfatidilinositol 3-Quinasas/fisiología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Receptores Virales/química , Receptores Virales/metabolismo , Proteínas Recombinantes de Fusión/química , Propiedades de Superficie , Linfocitos T/inmunologíaRESUMEN
Coxsackie and adenovirus receptor (CAR) is the primary cellular receptor for group B coxsackieviruses and most adenovirus serotypes and plays a crucial role in adenoviral gene therapy. Recent discovery of the interaction between junctional adhesion molecule-like protein (JAML) and CAR uncovered important functional roles in immunity, inflammation, and tissue homeostasis. Crystal structures of JAML ectodomain (2.2 angstroms) and its complex with CAR (2.8 angstroms) reveal an unusual immunoglobulin-domain assembly for JAML and a charged interface that confers high specificity. Biochemical and mutagenesis studies illustrate how CAR-mediated clustering of JAML recruits phosphoinositide 3-kinase (P13K) to a JAML intracellular sequence motif as delineated for the alphabeta T cell costimulatory receptor CD28. Thus, CAR and JAML are cell signaling receptors of the immune system with implications for asthma, cancer, and chronic nonhealing wounds.
Asunto(s)
Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Virales/química , Receptores Virales/metabolismo , Transducción de Señal , Animales , Sitios de Unión , Antígenos CD28/metabolismo , Células CHO , Fenómenos Químicos , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Cricetinae , Cricetulus , Cristalización , Cristalografía por Rayos X , Epitelio/inmunología , Glicosilación , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Ratones , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Gammadelta T cells present in epithelial tissues provide a crucial first line of defense against environmental insults, including infection, trauma, and malignancy, yet the molecular events surrounding their activation remain poorly defined. Here we identify an epithelial gammadelta T cell-specific costimulatory molecule, junctional adhesion molecule-like protein (JAML). Binding of JAML to its ligand Coxsackie and adenovirus receptor (CAR) provides costimulation leading to cellular proliferation and cytokine and growth factor production. Inhibition of JAML costimulation leads to diminished gammadelta T cell activation and delayed wound closure akin to that seen in the absence of gammadelta T cells. Our results identify JAML as a crucial component of epithelial gammadelta T cell biology and have broader implications for CAR and JAML in tissue homeostasis and repair.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Epidermis/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores Virales/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Citocinas/metabolismo , Células Epidérmicas , Epidermis/lesiones , Células Epiteliales , Epitelio/inmunología , Epitelio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Queratinocitos/metabolismo , Ligandos , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Cicatrización de HeridasRESUMEN
BACKGROUND: Cluster of differentiation 36 (CD36) is a transmembrane glycoprotein involved in many biological processes, such as platelet biology, angiogenesis and in the aetiopathology of atherosclerosis and cardiovascular diseases. Toll-like receptors (TLRs) are one of the most important receptors of the innate immune system. Their main function is the recognition of conserved structure of microorganisms. This recognition triggers signaling pathways that activate transcription of cytokines and co-stimulatory molecules which participate in the generation of an immune response against microbes. In particular, TLR2 has been shown to recognize a broad range of ligands. Recently, we showed that CD36 serves as a co-receptor for TLR2 and enhances recognition of specific diacylglycerides derived from bacteria. METHODOLOGY/ PRINCIPAL FINDINGS: Here, we investigate the mechanism by which CD36 contributes to ligand recognition and activation of TLR2 signaling pathway. We show that the ectodomain of murine CD36 (mCD36ED) directly interacts with negatively charged diacylglycerol ligands, which explains the specificity and selectivity of CD36 as a TLR2 co-receptor. We also show that mCD36ED amplifies the pro-inflammatory response to lipoteichoic acid in macrophages of wild-type mice and restores the pro-inflammatory response of macrophages from mice deficient in CD36 (oblivious), but not from mice deficient in cluster of differentiation 14 (CD14) (heedless). CONCLUSION/ SIGNIFICANCE: These data indicate that the CD36 ectodomain is the only relevant domain for activation of TLR2 signaling pathway and that CD36 and CD14 have a non-redundant role for loading ligands onto TLR2 in the plasma-membrane. The pro-inflammatory role of soluble CD36 can be relevant in the activation of the immune response against pathogens, as well as in the progression of chronic diseases. Therefore, an increased level of soluble forms of CD36, which has been reported to be increased in type II diabetic patients, could accelerate atherosclerosis by increasing the pro-inflammatory response to diacylglycerol ligands.