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- Materials and methods
Background: Non-specific lipid transfer proteins (LTPs) are involved in allergy to fresh and processed fruits. We have investigated the effect of thermal treatment and glycation on the physico-chemical and IgE-binding properties of the LTP from apple (Mal d 3).
Methods: Mal d 3 was purified from apple peel and the effect of heating in the absence and presence of glucose investigated by CD spectroscopy, electrospray and MALDI-TOF mass spectrometry. IgE reactivity was determined by RAST and immunoblot inhibition, SPT and basophil histamine release test.
Results: The identity and IgE reactivity of purified Mal d 3 was confirmed. Mild heat treatment (90°C, 20 min) in the absence or presence of glucose did not alter its IgE reactivity. More severe heat treatment (100°C, 2 h) induced minor changes in protein structure, but a significant decrease in IgE-binding (30-fold) and biological activity (100- to 1000-fold). Addition of glucose resulted in up to four glucose residues attached to Mal d 3 and only a 2- and 10-fold decrease of IgE-binding and biological activity, respectively.
Conclusions: Only severe heat treatment caused a significant decrease in the allergenicity of Mal d 3 but glycation had a protective effect. The presence of sugars in fruits may contribute to the thermostability of the allergenic activity of LTP in heat-processed foods.
Lipid transfer proteins (LTP) have been identified as allergens in various Rosaceae fruits including apple peach, apricot, plum and cherry (1) and found to be highly homologous (2). In addition, immunological cross-reactivity between LTPs from many botanically unrelated fruits and vegetables has been reported (3, 4). As members of the prolamin superfamily (5), LTPs share a conserved cysteine skeleton with four intra-molecular disulphide bridges stabilizing a bundle of four α-helices and a C-terminal coil (6, 7). This compact structure makes LTP highly resistant to proteolytic attack and to food processing. IgE reactivity of proteins can be unchanged, decreased or increased during food processing (8). LTPs retain their allergenic properties in thermally-treated products such as purees, nectars and juices (9) and polenta (10), indicating the heat stability of purified LTPs extends to the whole food (11). In addition to protein unfolding and aggregation, various chemical modifications can occur during processing, one of the most important being the Maillard reaction (12). Maillard adducts can modify the IgE reactivity of peanut (13) and cherry allergens (14, 15) however the basis of the effects of Maillard reactions on allergenicity of proteins are poorly characterized at a molecular level. This is essential if food manufacturers are to move towards knowledge-based ways of managing allergen risks during processing and to inform the allergenic risk assessment process, which forms part of the regulatory framework pertaining to novel foods and processes. In addition, well-defined and well-characterized allergens are needed for a precise, rapid and reliable detection of hypersensitivity to this allergen in complex food matrix (16) and for future immunotherapy (17, 18). This report describes a molecular study of the effect of heating, (including glycation), on the physico-chemical and IgE-binding properties of the LTP from apple, Mal d 3.
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- Materials and methods
Mild heat treatment (90°C, 20 min) of native Mal d 3 did not significantly alter its IgE binding capacity or its ability to trigger mediator release. This is probably because the unfolding of Mal d 3 is reversible after mild heating, as has been described for the LTP from barley (28). The presence of four intra-molecular disulphide bonds most likely assists in the refolding process. More severe thermal treatment (100°C, 2 h) resulted in the appearance of two additional masses, which could not unequivocally be assigned. Although efforts were made to exclude oxygen during heating, traces are likely to be still present. The additional masses might therefore be explained by oxidation of a disulphide bond by molecular oxygen. Such partial oxidation would account for the appearance of an LTP molecule with a mass increased by 32 Da. In turn, this molecule has the potential, under nucleophilic attack, to eliminate sulphur dioxide giving rise to the other molecular mass observed, with a thioether bond replacing the disulphide bond, and a mass decreased by 32 Da compared to the native LTP. Such modifications would most likely affect refolding of Mal d 3 upon cooling, explaining the reduced IgE binding potency of h2Mal d 3. Molecular dynamics simulations of LTPs have indicated that indeed one disulphide bond is less stable and consequently probably more susceptible to oxidation (29). Loss of a disulphide bond would mean that native folding would not be fully recovered during cooling, affecting conformational but not linear epitopes and hence significantly reducing (30-fold), but not completely abolishing, the IgE binding capacity of the protein. Alternatively, a fraction of the LTP molecules remain unmodified and hence can fully refold, which would account for the reduced but complete inhibitory potency. The observation that the inhibition curves of native Mal d 3 and h2Mal d 3 have very similar shapes might favor the latter explanation.
Apple-based products represent ideal conditions for Maillard reactions due to the presence of sugars and the high temperatures used during processing. In this reaction the anomeric carbon reacts with the free β-amino groups of lysine residues forming a Schiff's base and later the corresponding ketoamine (12). We observed minimal glycation following mild heating, indicating that mild thermal treatments, such as UHT where juices are rapidly heated to 135–140°C for 2–5 s, or the dearomatization procedures (110°C for 1–70 s) used during fruit juice manufacture, might not modify Mal d 3 structure. Severe heat treatment also only induced limited glycation, without aggregation or rearrangement of the sugars to cross-link the Mal d 3. Intriguingly, glycation appeared to protect the IgE binding capacity of Mal d 3 compared to h2Mal d 3. Based on the 3D structure of maize LTP (6) and the peach LTP model (7), all four lysines of Mal d 3 (Q9M5X7) are likely to be surface accessible, representing potential sites of non-enzymatic glycation. The electrospray results are consistent with this, with up to four glucose adducts being found on the Mal d 3. A molecular model of the homologous peach LTP shows that the lysine residues are located in α-helical regions of the protein, which from our CD studies may unfold to a limited extent at high temperature, and suggest that glycation may stabilize the protein conformation at these sites. Since homologous regions of Mal d 3 corresponding to two of the major peach LTP IgE-epitopes identified by Garcia-Casado et al. (7) do not contain lysine residues, glycation would not be expected to dramatically alter IgE reactivity of the apple/peach allergic patients from this study.
The observations that thermally processed foods such as cooked apples (11), commercial peach nectars (9), polenta (10) and cooked cherries (15) are still able to elicit allergic reactions in individuals sensitized towards LTPs is consistent with our observation that glycation has only a moderate effect (2-fold) on IgE-binding although the biological activity showed a 10-fold reduction. Unfortunately, SPT could not be performed with severely heated Mal d 3 due to a lack of material. However, these results maybe clinically relevant since a significant number of patients from this study tolerated processed apple products. Mal d 3 behaved in a contrasting fashion to the recombinant LTP allergen of cherry, Pru av 3, to which IgE reactivity was not diminished (15). However, glycation of the Bet v 1 allergen from cherry, Pru av 1, also reduced IgE binding by 1- to 2-fold although the biological relevance of this was not determined (14). The way in which processing modifies allergenicity is clearly complex. This hampers the development of simple strategies to reduce allergenicity of food products by heat treatment. Greater understanding of its impact at a molecular level will be essential for industry to develop knowledge-based means of reducing allergenicity in the future.