RESUMO
WaaL is a membrane enzyme implicated in ligating undecaprenyl-diphosphate (Und-PP)-linked O antigen to lipid A-core oligosaccharide. We determined the periplasmic location of a large (EL5) and small (EL4) adjacent loops in the Escherichia coli K-12 WaaL. Structural models of the EL5 from the K-12, R1 and R4 E. coli ligases were generated by molecular dynamics. Despite the poor amino acid sequence conservation among these proteins, the models afforded similar folds consisting of two pairs of almost perpendicular alpha-helices. One alpha-helix in each pair contributes a histidine and an arginine facing each other, which are highly conserved in WaaL homologues. Mutations in either residue rendered WaaL non-functional, since mutant proteins were unable to restore O antigen surface expression. Replacements of residues located away from the putative catalytic centre and non-conserved residues within the centre itself did not affect ligation. Furthermore, replacing a highly conserved arginine in EL4 with various amino acids inactivates WaaL function, but functionality reappears when the positive charge is restored by a replacement with lysine. These results lead us to propose that the conserved amino acids in the two adjacent periplasmic loops could interact with Und-PP, which is the common component in all WaaL substrates.
Assuntos
Carbono-Oxigênio Ligases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Membrana/química , Periplasma/enzimologia , Sequência de Aminoácidos , Arginina/química , Arginina/genética , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/metabolismo , Sequência Conservada , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligases , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
In nonobese diabetic (NOD) mice, a deficiency in the number and function of invariant natural killer T-cells (iNKT cells) contributes to the onset of type 1 diabetes. The activation of CD1d-restricted iNKT cells by alpha-galactosylceramide (alpha-GalCer) corrects these deficiencies and protects against spontaneous and recurrent type 1 diabetes. Although interleukin (IL)-4 and IL-10 have been implicated in alpha-GalCer-induced protection from type 1 diabetes, a precise role for these cytokines in iNKT cell regulation of susceptibility to type 1 diabetes has not been identified. Here we use NOD.IL-4(-/-) and NOD.IL-10(-/-) knockout mice to further evaluate the roles of IL-4 and IL-10 in alpha-GalCer-induced protection from type 1 diabetes. We found that IL-4 but not IL-10 expression mediates protection against spontaneous type 1 diabetes, recurrent type 1 diabetes, and prolonged syngeneic islet graft function. Increased transforming growth factor-beta gene expression in pancreatic lymph nodes may be involved in alpha-GalCer-mediated protection in NOD.IL-10(-/-) knockout mice. Unlike the requirement of IL-7 and IL-15 to maintain iNKT cell homeostasis, IL-4 and IL-10 are not required for alpha-GalCer-induced iNKT cell expansion and/or survival. Our data identify an important role for IL-4 in the protection against type 1 diabetes by activated iNKT cells, and these findings have important implications for cytokine-based therapy of type 1 diabetes and islet transplantation.