Effect of in vitro gastric and duodenal digestion on the allergenicity of grape lipid transfer protein.
ABSTRACT Severe grape allergy has been linked to lipid transfer protein (LTP) sensitization. LTPs are known to be resistant to pepsin digestion, although the effect of gastroduodenal digestion on its allergenicity has not been reported.
We sought to investigate the effect of gastric and gastroduodenal digestion on the allergenic activity of grape LTP.
The proteolytic stability of grape LTP was investigated by using an in vitro model of gastrointestinal digestion. The allergenicity of LTP and its digesta was assessed in vitro by means of IgE immunoblotting, RASTs, and in vivo skin prick tests in the same patients with grape allergy.
Grape LTP was resistant to gastric digestion, and yielded a 6000-d relative molecular mass C-terminally trimmed fragment after duodenal digestion. This fragment retained the in vitro IgE reactivity of the intact protein. Inclusion of phosphatidylcholine during gastric digestion protected the LTP to a limited extent against digestion. Digestion did not affect the in vivo (skin prick test) biologic activity of LTP.
The allergenic activity of grape LTP was highly resistant to in vitro digestion. This property might facilitate sensitization through the gastrointestinal tract and might also potentiate the ability of LTPs to elicit severe allergic reactions in sensitized individuals.
Purified natural allergens will facilitate the development of component-resolved diagnostic approaches, including allergen chips. This study contributes to our understanding of the role digestion plays in symptom elicitation in true food allergy.
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ABSTRACT: 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.Biochimica et Biophysica Acta 06/2004; 1698(2):175-86. · 4.66 Impact Factor
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ABSTRACT: Allergy to apple and Prunus fruits is frequently associated with birch pollinosis, with the principal cross-reacting allergens involved being members of the Bet v 1 family. However, a major 13-kd component, nonimmunologically related to Bet v 1, has been implicated as allergen in patients allergic to Prunoideae fruit but not to birch pollen. We sought to isolate and characterize the 13-kd allergen present in apple and peach. Sera from patients allergic to both fruits were selected on the basis of clinical symptoms, skin prick tests responses, and specific IgE levels. Allergens were purified by reverse-phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, specific IgE immunodetection, and immunoblot inhibition assays. A 13-kd protein band was recognized in crude apple and peach extracts by 9 of 10 and 10 of 10 sera from patients allergic to fruit, respectively. The isolation and characterization of the corresponding allergens allowed their identification as lipid-transfer proteins, with a molecular mass of 9058 d for the apple protein and 9138 d for the peach protein. Both purified allergens were recognized by sera from patients allergic to fruit and fully inhibited the IgE binding by the 13-kd component present in the 2 crude fruit extracts. Lipid-transfer proteins are relevant apple and peach allergens and, considering their ubiquitous distribution in tissues of many plant species, could be a novel type of panallergen of fruits and vegetables.Journal of Allergy and Clinical Immunology 04/1999; 103(3 Pt 1):514-9. · 12.05 Impact Factor
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ABSTRACT: Allergy to fresh fruits and vegetables is mostly observed in subjects with pollinosis, especially from birch, because of cross-reacting allergens in vegetable foods and pollens. However, allergic reactions to fruits, specifically Rosaceae fruits, have been reported in subjects without pollinosis. This study evaluated the pattern of IgE reactivity, identifying the allergen responsible in 2 groups of patients with oral allergy syndrome to peach with or without birch pollinosis. The allergenic components of peach were detected by SDS-PAGE and immunoblotting. The major peach allergen was purified by HPLC with a cation-exchange column followed by gel filtration chromatography. Its IgE-binding capacity and its homology with the protein of the crude extract were demonstrated by immunoblotting inhibition techniques. To better characterize this allergen, periodic acid-Schiff stain and isoelectrofocusing were used. The amino acid sequencing was done with a gas-phase sequencer. SDS-PAGE and immunoblotting of the 15 patients allergic to peach, 8 without and 7 with birch pollinosis, showed that they all recognized a protein with a molecular weight of 9 kd. This was the only allergen recognized by patients not sensitized to pollen, whereas the birch pollen-sensitive patients had IgE binding to other allergenic proteins at higher molecular weights. The purified 9-kd protein retained its IgE-binding capacity, was negative to periodic acid-Schiff stain, and had an isoelectric point value of greater than 9. A search in the Swiss Prot Bank showed this was a lipid transfer protein, belonging to a group of molecules involved in the defensive system of plants. The major allergen of peach is a 9-kd protein belonging to the group of lipid transfer proteins. This is the only allergen recognized by patients allergic to peach but not sensitized to birch pollen.Journal of Allergy and Clinical Immunology 04/1999; 103(3 Pt 1):520-6. · 12.05 Impact Factor