RESUMEN
Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of the pro-inflammatory interleukin-1-mediated activation of the interleukin-1 receptor (IL-1R). Although wild-type IL-1Ra is used for treatment of inflammatory diseases, its effect is moderate and/or short-lived. The objective of this study was to generate IL-1Ra mutants with enhanced antagonistic activity for potential therapeutic use. Using a directed evolution approach in which libraries of IL-1Ra gene mutants were generated and screened in functional assays, mutants with desired properties were identified. Initially, diversity was introduced into the IL-1Ra using random mutagenesis. Mutations resulting in enhanced antagonistic activity were identified by screening in a reporter cell assay. To further enhance the antagonistic activity, selected mutations were recombined using the DNA recombination technology Fragment-INduced Diversity (FIND). Following three rounds of FIND recombination, several mutants with up to nine times enhanced antagonistic activity (mean IC50 +/- SEM value: 0.78 +/- 0.050 vs. 6.8 +/- 1.1 ng/ml for mutant and wild-type, respectively) were identified. Sequence analysis identified the mutations D47N, E52R and E90Y as being most important for this effect, however, the mutations P38Y, H54R, Q129L and M136N further enhanced the antagonistic function. Analysis of identified mutations in protein models based on the crystal structure of the IL-1Ra/IL-1R complex suggested that mutations found to enhance the antagonistic activity had a stabilizing effect on the IL-1Ra mutants or increased the affinity for the IL-1R. Finally, the therapeutic effect of one mutant was compared to that of wild-type IL-1Ra in collagen-induced arthritis in mice. Indeed, the enhanced antagonistic effect of the mutants observed in vitro was also seen in vivo. In conclusion, these results demonstrate that directed evolution of IL-1Ra is an effective means of generating highly potent therapeutic proteins.
Asunto(s)
Sustitución de Aminoácidos , Artritis Experimental/tratamiento farmacológico , Evolución Molecular Dirigida , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Mutación Missense , Animales , Línea Celular , Evolución Molecular Dirigida/métodos , Humanos , Proteína Antagonista del Receptor de Interleucina 1/genética , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Masculino , Ratones , Ratones Endogámicos DBA , Estructura Cuaternaria de Proteína , Receptores de Interleucina-1RESUMEN
Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2-4 mutations in human CPU can prolong the half-life of CPU's activity at 37 degrees C from 0.2 h of wild-type CPU to 0.5-5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.
Asunto(s)
Carboxipeptidasa B2/metabolismo , Fibrinólisis , Mutagénesis , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Coagulación Sanguínea , Carboxipeptidasa B2/química , Carboxipeptidasa B2/genética , Línea Celular , Regulación hacia Abajo , Activación Enzimática , Estabilidad de Enzimas , Fibrina/genética , Fibrina/metabolismo , Calor , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación Puntual , Desnaturalización Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Alineación de SecuenciaRESUMEN
High-throughput proteomics, based on the microarray platform, requires stable, highly functional components that will yield a highly sensitive read-out of low abundance proteins. Although antibodies are the best characterized binding molecules for this purpose, only a fraction of them appear to behave satisfactorily in the chip format. Therefore, high demands need to be placed on their molecular design. In the present study, we have focused on recombinant antibody design based on a single framework for protein chip applications, aiming at defining crucial molecular probe parameters. Our results show that engineered human recombinant scFv antibody fragments that displayed appropriate biophysical properties (molecular [functional] stability in particular) can be generated, making them prime candidates for high-density antibody arrays. In fact, a superior framework that displays both multifaceted adsorption properties and very high functional stability over several months on chips (stored in a dried-out state) was identified. Taken together, designed scFv fragments based on a single molecular scaffold, readily accessible in large phage display libraries, can undoubtedly meet the requirements of probe content in antibody microarrays, particularly for global proteome analysis.
