RESUMEN
As the link between antigen binding and immune activation, the antibody Fc region has received extensive structural study. In this report, the structure of the Fc fragment of the NIST IgG1 mAb (reference material 8671) is described at 2.1â Å resolution in space group P212121, with approximate unit-cell parameters a = 50, b = 80, c = 138â Å. Prior Fc structures with a wide variety of modifications are also surveyed, focusing on those in the same crystal form. To facilitate the analysis of conformations, a reference frame and a two-parameter metric are proposed, considering the CH2 domains as mobile with respect to a fixed dimeric CH3 core. Over several human Fc structures, a significant variation in Fc elbow conformations is observed, which may serve to facilitate the regulation of Fc effector signaling.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Subunidades de Proteína/química , Receptores Fc/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Receptores Fc/genética , Receptores Fc/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , TermodinámicaRESUMEN
Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Dominios Proteicos , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/aislamiento & purificación , Fenómenos Biofísicos , Cromatografía en Gel , Cristalografía por Rayos X , Electroforesis Capilar , Electroforesis en Gel de Poliacrilamida , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Espectrometría de Masas , Ratones , Modelos Moleculares , Unión Proteica/inmunología , Estándares de ReferenciaRESUMEN
Rats will approach and contact a lever whose insertion into the chamber signals response-independent food delivery. This "autoshaping" or "sign-tracking" phenomenon has recently attracted considerable attention as a platform for studying individual differences in impulsivity, drug sensitization, and other traits associated with vulnerability to drug addiction. Here, we examined two basic stimulus selection phenomena-blocking and overshadowing-in the autoshaped lever pressing of rats. Blocking and overshadowing were decidedly asymmetrical. Previously reinforced lever-extension conditioned stimuli (CSs) completely blocked conditioning to auditory cues (Exps. 1 and 2), and previously nonreinforced lever-extension CSs overshadowed conditioning to auditory cues. By contrast, conditioning to lever-extension CSs was not blocked by either auditory (Exp. 3) or lever-insertion (Exp. 4) cues, and was not overshadowed by auditory cues. Conditioning to a lever-insertion cue was somewhat overshadowed by the presence of another lever, especially in terms of food cup behavior displayed after lever withdrawal. We discuss several frameworks in which the apparent immunity of autoshaped lever pressing to blocking might be understood. Given evidence that different brain systems are engaged when different kinds of cues are paired with food delivery, it is worth considering the possibility that interactions among them in learning and performance may follow different rules. In particular, it is intriguing to speculate that the roles of simple cue-reinforcer contiguity, as well as of individual and aggregate reinforcer prediction errors, may differ across stimulus classes.
