RESUMO
N-glycosylation on IgG modulates Fc conformation and effector functions. An IgG-Fc contains a human sialo-complex type (hSCT) glycan of biantennary structure with two α2,6-sialylations and without core-fucosylation is an optimized glycoform developed to enhance the antibody dependent cellular cytotoxicity (ADCC). hSCT modification not only enhances the binding affinity to Fc receptors in the presence of antigen but also in some cases provides gain-of-function effector activity. We used enzymatic glyco-engineering to prepare an IgG-Fc with homogeneous hSCT attached to each CH2 domain and solved its crystal structure. A compact form and an open form were observed in an asymmetric unit in the crystal. In the compact structure, the double glycan latches from the two hSCT chains stabilize the CH2 domains in a closed conformation. In the open structure, the terminal sialic acid (N-acetylneuraminic acid or NeuNAc) residue interacts through water-mediated hydrogen bonds with the D249-L251 helix, to modulate the pivot region of the CH2-CH3 interface. The double glycan latches and the sialic acid modulation may be mutually exclusive. This is the first crystal structure of glyco-engineered Fc with enhanced effector activities. This work provides insights into the relationship between the structural stability and effector functions affected by hSCT modification and the development of better antibodies for therapeutic applications.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Polissacarídeos/química , Engenharia de Proteínas/métodos , Cristalografia por Raios X , Desenho de Fármacos , Glicosilação , Humanos , Estrutura Molecular , Ácido N-Acetilneuramínico/químicaRESUMO
A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 microM) for SARS CoV and 5.2 log (at 1.25 microM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.
Assuntos
Antivirais/síntese química , Carbamatos/síntese química , Cisteína Endopeptidases/química , Dipeptídeos/síntese química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Animais , Antivirais/química , Antivirais/farmacologia , Carbamatos/química , Carbamatos/farmacologia , Linhagem Celular , Chlorocebus aethiops , Coronavirus Humano 229E/efeitos dos fármacos , Proteases 3C de Coronavírus , Cristalografia por Raios X , Dipeptídeos/química , Dipeptídeos/farmacologia , Estabilidade de Medicamentos , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Estrutura Molecular , Ratos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Mice that lack IL-15 or the IL-15R alpha-chain (IL-15Ralpha) are deficient in peripheral CD8(+), but not in CD4(+), T cells. This CD8(+) T cell-specific deficiency has now been investigated further by characterization of a new strain of IL-15Ralpha(-/-) mice. The adult mutant mice exhibited a specific reduction in the percentage of CD8-single positive TCR(high) thymocytes. The expression of Bcl-2 was reduced in both CD8(+) thymocytes and naive T cells of the mutant animals, and the susceptibility of these cells to death was increased. Memory CD8(+) cells were profoundly deficient in IL-15Ralpha(-/-)mice, and the residual memory-like CD8(+) cells contained a high percentage of dead cells and failed to up-regulate Bcl-2 expression compared with naive CD8(+) cells. Moreover, exogenous IL-15 both up-regulated the level of Bcl-2 in and reduced the death rate of wild-type and mutant CD8(+) T cells activated in vitro. These results indicate that IL-15 and IL-15Ralpha regulate the expression of Bcl-2 in CD8(+) T cells at all developmental stages. The reduced Bcl-2 content in CD8(+) cells might result in survival defect and contribute to the reduction of CD8(+) cells in IL-15Ralpha(-/-)mice.