RESUMEN
The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.
Asunto(s)
Antibacterianos/metabolismo , Desarrollo de Medicamentos , Glicósido Hidrolasas/metabolismo , Trastuzumab/metabolismo , alfa-L-Fucosidasa/metabolismo , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Biotinilación , Relación Dosis-Respuesta a Droga , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/metabolismo , Glicósido Hidrolasas/genética , Fenómenos Magnéticos , Estructura Molecular , Mutación , Relación Estructura-Actividad , Trastuzumab/química , Trastuzumab/aislamiento & purificación , alfa-L-Fucosidasa/genéticaRESUMEN
Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Polisacáridos/química , Rituximab/química , Acetilglucosamina/química , Acetilglucosamina/inmunología , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Proteínas Bacterianas/metabolismo , Bacteroides fragilis/enzimología , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/prevención & control , Ingeniería de Proteínas , Receptores de IgG/inmunología , Rituximab/inmunología , Ácidos Siálicos/química , Ácidos Siálicos/inmunología , Streptococcus pyogenes/enzimología , Relación Estructura-Actividad , Trastuzumab/química , Trastuzumab/inmunología , alfa-L-Fucosidasa/metabolismoRESUMEN
The first systematic investigation of the effect of high mannose, hybrid, and bi- and tri-antennary complex type glycans on the effector functions of antibodies was achieved by the discovery of novel Endo-S2 mutants generated by site-directed mutagenesis as glycosynthases with broad substrate specificity.
Asunto(s)
Anticuerpos/química , Glicosiltransferasas/química , Polisacáridos/química , Anticuerpos/metabolismo , Glicósido Hidrolasas/genética , Glicosilación , Glicosiltransferasas/genética , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Receptores de IgG/metabolismo , Streptococcus pyogenes/enzimología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Correction for 'Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies' by Sachin S. Shivatare et al., Chem. Commun., 2018, 54, 6161-6164.
RESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) plays a key role in the regeneration of NADPH and maintenance of cellular redox balance. In the present study, we investigate the effect of G6PD deficiency on H(2)O(2)-elicited signaling in HepG2 cells. H(2)O(2) was found to inhibit cellular protein tyrosine phosphatase (PTP) activity, resulting in activation of MAPKs. MKP-1 expression increased in the late phase of H(2)O(2) signaling. Using RNAi technology, we found that G6PD knockdown enhanced the inhibitory effect of H(2)O(2) on PTPs and led to sustained MAPK activation. This was accompanied by delayed expression and inhibition of MKP-1. Using a pharmacological inhibitor and siRNA, we demonstrate that MKP-1 acts as a regulator of MAPK activation in H(2)O(2) signaling. The prolonged MAPK activation in G6PD-knockdown cells was associated with an increased susceptibility to H(2)O(2)-induced apoptosis and growth retardation. Treatment with p38 and JNK inhibitors or N-acetylcysteine ameliorated such cellular effect, while triptolide and MKP-1-siRNA did the opposite. Glucose oxidase treatment had similar effects as addition of H(2)O(2). Taken together, these findings suggest that G6PD knockdown enhances the magnitude and duration of H(2)O(2)-induced MAPK signaling through inhibition of cellular PTPs, and the resultant anomalous signaling may lead to cell demise.
Asunto(s)
Apoptosis/efectos de los fármacos , Glucosafosfato Deshidrogenasa/genética , Peróxido de Hidrógeno/farmacología , Acetilcisteína/farmacología , Antracenos/farmacología , Fosfatasa 1 de Especificidad Dual/antagonistas & inhibidores , Fosfatasa 1 de Especificidad Dual/metabolismo , Flavonoides/farmacología , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Piridinas/farmacologíaRESUMEN
Mutations in the CLCN1 gene frequently associate with myotonia congenita (MC). We have recently reported several CLCN1 mutants in Taiwanese patients. To further elucidate the correlation between the genotypes and phenotypes, in this study, we used Xenopus oocyte as a system to investigate the functional effects of these mutants. The fs793X and G482R mutants, which were suggested to have a dual inheritance pattern, were found to cause a functional loss of CLCN1 channels. While co-expression of fs793X and wild-type (WT) showed a reduction of chloride conductance by about half of WT channels, the activation curve of voltage-dependence was not shifted. A compound heterozygous mutant, P575S/D644G, was found in a patient. When both mutants were co-expressed in oocytes, they caused a shift of the voltage-dependence of activation curve to more positive values than individual mutant. This indicates that both P575S and D644G mutants may contribute cooperatively to change the gating property of CLCN1 channel. Interestingly, the S471F mutant did not cause significant alternation of functional properties. Consistent with the fact that T631I mutant was found in three asymptomatic individuals, the electrophysiological parameters of T631I were similar to those of WT CLCN1 channels, suggesting that T631I is a neutral mutation. These results further clarify the correlation between the mutations and their functional implications of CLCN1 channels.