RESUMEN
The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.
Asunto(s)
Toxinas Bacterianas/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Enterotoxinas/uso terapéutico , Proteínas de Escherichia coli/uso terapéutico , Sinapsinas/uso terapéutico , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Células CHO/efectos de los fármacos , Células CHO/metabolismo , Bovinos , Cricetinae , Evaluación Preclínica de Medicamentos , Endocitosis , Enterotoxinas/química , Enterotoxinas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Femenino , Gangliósido G(M1)/metabolismo , Activación de Linfocitos/efectos de los fármacos , Linfocinas/metabolismo , Masculino , Proteína Básica de Mielina/inmunología , Proteína Básica de Mielina/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Distribución Aleatoria , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/uso terapéutico , Método Simple Ciego , Relación Estructura-Actividad , Sinapsinas/química , Sinapsinas/genética , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.