RESUMEN
Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.
Asunto(s)
Albuminas 2S de Plantas/inmunología , Alérgenos/inmunología , Antígenos de Plantas/inmunología , Células Dendríticas/metabolismo , Albuminas 2S de Plantas/genética , Albuminas 2S de Plantas/metabolismo , Alérgenos/genética , Alérgenos/metabolismo , Animales , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Bertholletia/metabolismo , Células de la Médula Ósea/citología , Células Dendríticas/inmunología , Endocitosis , Femenino , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/terapia , Glicosilación , Humanos , Inmunoterapia , Interleucina-12/metabolismo , Lectinas Tipo C/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos BALB C , Pichia/metabolismo , Unión Proteica , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Protein microarray is a miniaturized multi-analyte, solid-phased immunoassay where thousands of immobilized individual protein spots on a microscopic slide bind are bound to specific antibodies (immunoglobulins) from serum samples, which are then detected by fluorescent labeling. The image processing and pattern recognition are then quantitatively analyzed using advanced algorithms. Here, we describe the use of an in-house-produced complex protein microarray containing extracts and pure proteins that has been probed with antibodies present in the horse sera and detection by fluorophore-conjugated antibody and data analysis. The flexibility of the number and types of proteins that can be printed on the microarray allows different set of specific IgE immunoassay analysis to be carried out.