RESUMEN
The T helper cell-derived cytokine interleukin-17A (IL-17A) is a variably glycosylated disulfide-linked homodimer of 34-38 kDa. Its polypeptide monomer contains one canonical N-glycosylation site at Asn68, and human recombinant IL-17A was partly N-glycosylated when expressed in human kidney (HEK293) cells as a fusion protein with a melittin signal sequence and an N-terminal hexahistidine tag. Orbitrap mass analyses of the tryptic N-glycopeptide 63-69 indicated that the N-glycosylation was of the GalNAc-terminated type characteristic of cultured kidney cells. The mass spectrum of IL-17A monomer also included peaks shifted by +948 Da from the respective masses of unglycosylated and N-glycosylated polypeptides. These were caused by unpredicted partial O-glycosylation of Thr26 with the mucin-like structure -GalNAc(-NeuNAc)-Gal-NeuNAc. Identical O-glycosylation occurred in commercially sourced recombinant IL-17A also expressed in HEK293 cells but with a different N-terminal sequence. Therefore, the kidney host cell line not only imposed its characteristic pattern of N-glycosylation on recombinant IL-17A but additionally created an O-glycosylation not known to be present in the T cell-derived cytokine. Mammalian host cell lines for recombinant protein expression generally impose their characteristic patterns of N-glycosylation on the product, but this work exemplifies how a host may also unpredictably O-glycosylate a protein that is probably not normally O-glycosylated.
Asunto(s)
Interleucina-17/biosíntesis , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicosilación , Células HEK293 , Humanos , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/química , Interleucina-17/química , Meliteno/biosíntesis , Meliteno/química , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Señales de Clasificación de Proteína , Proteínas Recombinantes de Fusión/química , Espectrometría de Masas en TándemRESUMEN
Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Femenino , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Regiones Constantes de Inmunoglobulina/aislamiento & purificación , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Masculino , Periplasma/genética , Periplasma/inmunología , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/inmunología , Proteínas Periplasmáticas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Interleukin (IL) 22 is a type II cytokine that is produced by immune cells and acts on nonimmune cells to regulate local tissue inflammation. As a product of the recently identified T helper 17 lineage of CD4(+) effector lymphocytes, IL-22 plays a critical role in mucosal immunity as well as in dysregulated inflammation observed in autoimmune diseases. We used comprehensive mutagenesis combined with mammalian cell expression, ELISA cell-based, and structural methods to evaluate how IL-22 interacts with its cell surface receptor, IL-22R/IL-10R2, and with secreted IL-22 binding protein. This study identifies those amino acid side chains of IL-22 that are individually important for optimal binding to IL-22R, considerably expands the definition of IL-22 surface required for binding to IL-10R2, and demonstrates how IL-22 binding protein prevents IL-22R from binding to IL-22. The IL-22R and IL-10R2 binding sites are juxtaposed on adjacent IL-22 surfaces contributed mostly by helices A, D, and F and loop AB. Our results also provide a model for how IL-19, IL-20, IL-24, and IL-26 which are other IL-10-like cytokines, interact with their respective cell surface receptors.
Asunto(s)
Subunidad beta del Receptor de Interleucina-10/química , Subunidad beta del Receptor de Interleucina-10/metabolismo , Interleucinas/química , Interleucinas/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión/genética , Línea Celular , Humanos , Técnicas In Vitro , Subunidad beta del Receptor de Interleucina-10/genética , Interleucinas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Receptores de Interleucina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Termodinámica , Interleucina-22RESUMEN
IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.
Asunto(s)
Interleucina-17/fisiología , Receptores de Interleucina-17/fisiología , Receptores de Interleucina/fisiología , Transducción de Señal/inmunología , Línea Celular , Dimerización , Relación Dosis-Respuesta Inmunológica , Humanos , Interleucina-17/antagonistas & inhibidores , Interleucina-17/química , Interleucina-17/metabolismo , Unión Proteica/inmunología , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17/metabolismoRESUMEN
IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Regulación de la Expresión Génica/inmunología , Interleucina-17/inmunología , Activación de Linfocitos/inmunología , Secuencias de Aminoácidos , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células CHO , Cricetinae , Cricetulus , Cisteína/genética , Cisteína/inmunología , Dimerización , Expresión Génica , Regulación de la Expresión Génica/genética , Humanos , Inmunidad Celular , Interleucina-17/biosíntesis , Interleucina-17/genética , Interleucina-17/farmacología , Activación de Linfocitos/efectos de los fármacosRESUMEN
Th17 cells are a distinct lineage of effector CD4(+) T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-22, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th2 cells. Similar to IL-17A, IL-22 expression was initiated by transforming growth factor beta signaling in the context of IL-6 and other proinflammatory cytokines. The subsequent expansion of IL-22-producing cells was dependent on IL-23. We further demonstrate that IL-22 was coexpressed in vitro and in vivo with both IL-17A and IL-17F. To study a functional relationship among these cytokines, we examined the expression of antimicrobial peptides by primary keratinocytes treated with combinations of IL-22, IL-17A, and IL-17F. IL-22 in conjunction with IL-17A or IL-17F synergistically induced the expression of beta-defensin 2 and S100A9 and additively enhanced the expression of S100A7 and S100A8. Collectively, we have identified IL-22 as a new cytokine expressed by Th17 cells that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity.