RESUMEN
BACKGROUND: The ability of an intact protein to reach the circulatory system may be a prerequisite to allergenicity and many allergens, particularly those from plant foods, have been found to be consistently more resistant to digestion by pepsin than other proteins. OBJECTIVE: This study assessed the pepsinolytic stability of native 2S albumins from Brazil nut and sunflower seed and their recombinant versions produced in Pichia pastoris. The physicochemical stability of native and recombinant Brazil nut 2S albumins and recombinant sunflower seed 2S albumin was also assessed. The immunoreactivity of native Brazil nut 2S albumin and recombinant 2S albumins was compared using serum from patients allergic to Brazil nuts and animals immunized with native 2S albumins. METHODS: Digestibility was measured in simulated gastric fluid followed by SDS-PAGE. Circular dichroism spectra were used to analyse unfolding, as proteins were denatured by temperature, pH and guanidinium chloride. Immunoreactivity was assessed by immunoblot, RAST and ELISA. RESULTS: Brazil nut 2S albumin was significantly more resistant to proteolytic digestion than other Brazil nut proteins. It was also resistant to thermally and chemically induced denaturation. Equally high resistance to proteolytic digestion was observed with sunflower seed 2S albumin. The recombinant albumins mirrored their native counterparts in stability and immunoreactivity. CONCLUSION: The important food allergen Brazil nut 2S albumin is as stable to digestion as is sunflower seed 2S albumin, whose allergenicity has yet to be determined. The 2S albumins and their recombinant counterparts could not be easily denatured by physicochemical treatments. The results suggest that 2S albumin is the only Brazil nut protein to reach the gut immune system intact. The production of properly folded recombinant proteins will facilitate mechanistic studies as well as diagnostic testing and antigen-based therapies.
Asunto(s)
Albúminas/química , Nueces/química , Proteínas de Plantas/química , Precursores de Proteínas/química , Semillas/química , Albuminas 2S de Plantas , Albúminas/inmunología , Animales , Antígenos de Plantas , Fenómenos Químicos , Química Física , Digestión , Estabilidad de Medicamentos , Jugo Gástrico/química , Guanidina/farmacología , Helianthus/inmunología , Calor , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Hipersensibilidad a la Nuez/sangre , Nueces/inmunología , Proteínas de Plantas/inmunología , Plantas Comestibles/inmunología , Desnaturalización Proteica , Precursores de Proteínas/inmunología , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Semillas/inmunologíaRESUMEN
Two well known 2 S albumins, Ber e 1 from brazil nut and sunflower 2 S albumin 8 (SFA-8), have been expressed in a eukaryotic system and purified. Analysis of recombinant versions of Ber e 1 and SFA-8 revealed them to be significantly more resistant to digestion by pepsin than BSA, and to be stable for up to 30 min in simulated gastric fluid. Unfolding monitored by CD indicated that both proteins were also very resistant to denaturation induced by heat and low pH. These results suggest that, although the ability of 2 S albumins to reach the circulatory system may be a prerequisite for the allergenicity of this group of proteins, stability is just one of a number of characteristics that provoke a selective immune response.
