Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion.

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.

Mass spectrometry and structural characterization of 2S albumin isoforms from Brazil nuts (Bertholletia excelsa).

Proteomic approaches have been used to characterise the main 2S albumin isoforms from Brazil nuts (Bertholletia excelsa). Whilst most isoforms ( approximately 10 discrete protein species) exhibited molecular masses of around 12 kDa with a high amino acid sequence homology, important charge heterogeneity was found, with pIs varying between 4.6 and 6.6, with one >or=7.0. Proteomic analysis showed that these corresponded to a total of six National Center for Biotechnology Information (NCBI) accessions and that three isoforms had been purified to homogeneity corresponding to gi/384327, 112754 and 99609. The latter sequence corresponds to an isoform, previously only identified at the nucleotide sequence level, had a slightly higher molecular weight (13.4 kDa), and with noticeable differences in the primary structure. Proteins corresponding to six different NCBI accessions were identified, the heterogeneity of which had been increased by posttranslational processing. Evidence was found of cyclization of the N-terminal glutamine residue in two isoforms, together with ragged C-termini, indicative of carboxypeptidase activity within the vacuole following posttranslational processing. No evidence of glycosylation was found. Circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectroscopy indicated all the studied isoforms were predominantly alpha-helical in nature, but that the Mr 13400 species was structurally distinct, with a higher proportion of alpha-helical structure.