Effect of in vitro gastric and duodenal digestion on the allergenicity of grape lipid transfer protein
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.
Available from: sciencedirect.com
- "The authors further reported that the lower transport rate of a hypoallergenic peach nsLTP was associated with a significantly lower expression of TH2-related cytokines compared with Pru p 3 (Table I). The presence of gastric phospatidylcholine in an in vitro digestion assay had a protective effect on the grape nsLTP.67 The breakdown of the protein by duodenal enzymes was slowed down, resulting in a slightly higher ability of the allergen to induce basophil histamine release and to elicit skin reactions in 4 patients with grape allergy. "
[Show abstract] [Hide abstract]
ABSTRACT: Allergic sensitization is a multifactorial process that is not only influenced by the allergen and its biological function per se but also by other small molecular compounds, such as lipids, that are directly bound as ligands by the allergen or are present in the allergen source. Several members of major allergen families bind lipid ligands through hydrophobic cavities or electrostatic or hydrophobic interactions. These allergens include certain seed storage proteins, Bet v 1-like and nonspecific lipid transfer proteins from pollens and fruits, certain inhalant allergens from house dust mites and cockroaches, and lipocalins. Lipids from the pollen coat and furry animals and the so-called pollen-associated lipid mediators are codelivered with the allergens and can modulate the immune responses of predisposed subjects by interacting with the innate immune system and invariant natural killer T cells. In addition, lipids originating from bacterial members of the pollen microbiome contribute to the outcome of the sensitization process. Dietary lipids act as adjuvants and might skew the immune response toward a TH2-dominated phenotype. In addition, the association with lipids protects food allergens from gastrointestinal degradation and facilitates their uptake by intestinal cells. These findings will have a major influence on how allergic sensitization will be viewed and studied in the future.
Journal of Allergy and Clinical Immunology 05/2014; 134(3). DOI:10.1016/j.jaci.2014.04.015 · 11.48 Impact Factor
Available from: Bernadett Berecz
- "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). "
[Show abstract] [Hide abstract]
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 · 3.39 Impact Factor
Available from: Barbara Prandi
- "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. "
[Show abstract] [Hide abstract]
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 · 2.25 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.