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.
- SourceAvailable from: Bernadett Berecz
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- "This ability to bind lipid affects the stability of some LTPs and their resistance to hydrolysis (Breiteneder & Mills, 2005; Douliez et al., 2000; Vassilopoulou et al., 2006). The 2S albumin and LTP protein families both include a number of major allergens, including SFA8 from sunflower (Yagami, 2010), Ara h 2 from peanut (Burks et al., 1991) and Ber e 1 from Brazil nut (Pastorello et al., 1998); and LTPs from peach (Pru p 1) (Wijesinha- Bettoni et al., 2010), barley (Wijesinha-Bettoni et al., 2010) apple (Sancho et al., 2005) and grape (Vassilopoulou et al., 2006). "
ABSTRACT: In order for a protein to elicit a systemic allergic response it must reach the circulatory system through the intestinal mucosa as a sufficiently large fragment with adequate structural integrity. Sunflower LTP and 2S albumins (SFA8 and three mixed fractions of Alb1 and Alb2) were digested in simulated gastric fluid (SGF) for 2h and the conditions were then changed to mimic the intestinal environment for a further 2h digestion. The effects of phosphatidylcholine (PC) and emulsification on the digestibility of the proteins were investigated. PC protected all of the proteins studied against both gastric and intestinal digestive enzymes but to different extents. Emulsification of SFA8 resulted in strong protection against digestion, which was further enhanced by the presence of PC in the SGF. These results highlight the importance of considering real food structures such as emulsified systems and also the gastrointestinal environment that proteins are exposed to once consumed when assessing allergenicity.Food Chemistry 06/2013; 138(4):2374-81. DOI:10.1016/j.foodchem.2012.12.034 · 3.26 Impact Factor
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- "Mass Spectrom. 2012, 26, 2905–2912 proportion is consistent with that found for grape LTP (9%),  but considerably smaller than that for peach (35%).  The breakdown of Pru ar 3 determined in this experiment is more extensive than that obtained for Pru p 3 in a previous study  and this might partially explain its lower allergenicity; however, other additional factors that play a role in the physiological digestion of a complex food matrix (instead of a purified protein) might also have an influence. "
ABSTRACT: Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4) HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology. Copyright © 2012 John Wiley & Sons, Ltd.Rapid Communications in Mass Spectrometry 12/2012; 26(24):2905-12. DOI:10.1002/rcm.6416 · 2.64 Impact Factor
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ABSTRACT: Grape allergy is considered rare; grape lipid transfer protein (LTP; Vit v 1), an endochitinase and a thaumatin-like protein (TLP) have been reported as grape allergens. A considerable number of patients have referred to our department for severe reactions to grapes, and several IgE binding proteins were detected. The aim of this study was to identify and characterise the allergens involved in severe allergic reactions to grapes and describe the population in which they occur. Patients with reported severe allergic reactions to grapes (n = 37) are described. Grape allergens were purified/fractionated by a combination of chromatographic techniques, identified by proteomic analysis and biochemically characterised. Immunoreactivity was assessed by blot (inhibitions) and RAST (inhibitions), and skin prick tests were performed with the isolated allergens. All subjects were polyallergic, sensitised and reactive to several additional foods and pollen. All patients were sensitised to grape LTP. A 28-kDa expansin, a 37.5-kDa polygalacturonase-inhibiting protein, a 39-kDa beta-1,3-glucanase and a 60-kDa protein were identified as minor grape allergens. Endochitinase and TLP did not play a role. Inhibition experiments revealed the possible cross-reactive role of LTP for clinical sensitivities to other LTP-containing plant foods, but also the involvement of cross-reactive carbohydrate determinants of minor allergens in IgE cross-reactivity. LTP is the major grape allergen, while additional minor allergens may contribute to clinical reactivity. Severe grape allergy presents in atopic patients who frequently react to other LTP-containing, plant-derived foods. The 'LTP syndrome' is the appropriate term to describe this condition.International Archives of Allergy and Immunology 02/2007; 143(2):92-102. DOI:10.1159/000098657 · 2.43 Impact Factor