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Les L T P (Lipid transfer proteins)

lundi 23 août 2010, par Allerdata

Les LTP, ou Lipid Transfer Proteins, sont dorénavant un grand classique en allergologie moléculaire. Mais leur identification en tant qu’allergènes est pourtant beaucoup plus récente que celle des profilines ou des Bet v 1-like . Cet intérêt pour les LTP réside dans certaines de leurs caractéristiques :

  • on trouve des LTP dans la plupart des végétaux et ces LTP croisent aisément entre elles, ce qui en fait des panallergènes
  • la grande stabilité des LTP à la chaleur et à la digestion les distingue d’autres protéines comme les profilines et les Bet v 1-like et favorise la survenue fréquente de symptômes sévères chez les patients qui se sont sensibilisés aux LTP
  • enfin une inconnue subsiste sur les raisons qui concentrent les observations d’allergie aux LTP dans la bordure du bassin méditerranéen, notamment l’Italie et l’Espagne . Des hypothèses ont été récemment émises quant au rôle favorisant possible d’un contact avec la LTP de pêche par voie cutanée et/ou respiratoire , mais elles attendent confirmation. Cet aspect est détaillé dans le chapitre dédié aux Rosacées.

De telles particularités ont suscité de fréquentes revues , d’autant que le nombre de LTP IgE-réactives ne cesse de croître : en août 2010, la base Allerdata recensait pas moins de 61 LTP :

  • 44 dans des aliments d’origine végétale
  • 11 dans des pollens
  • 5 dans des produits divers : feuille de cannabis, latex d’hévéa, bouton de rose, bière, Arabidopsis (une plante de laboratoire).

Ces protéines non glycosylées (sauf celles de pariétaire) n’ont pas d’équivalents IgE-réactifs connus en dehors des plantes et des dérivés des plantes.

Fait (trop) peu courant et méritant d’être signalé, les connaissances sur cette famille de protéines ont bénéficié des travaux d’au moins 2 équipes françaises, à Nantes et à Toulouse .

Les termes de « protéines de transfert lipidique » ou de « lipido-transférases » sont parfois rencontrés. Mais l’acronyme anglo-saxon LTP est préférable car universellement utilisé dans la littérature scientifique. De plus, les LTP ne désignent qu’une sorte particulière de protéines parmi toutes celles qui transportent des lipides.

On rencontre aussi l’abréviation « nsLTP », celle-ci signifiant « non specific » pour souligner la multiplicité des ligands hydrophobes pouvant se coupler aux LTP .

Enfin, la classification générale des protéines range les LTP :

  • en 2 familles, les LTP-1 et les LTP-2 : seules les LTP-1 ayant été montrées IgE-réactives, elles sont plus simplement dénommées « LTP » dans la littérature allergologique. Les LTP-2 (env. 7 kDa) n’ont que peu d’homologie séquentielle avec les LTP-1 (9 kDa) : environ 30%
  • par contre, ces 2 sortes de LTP ont en commun un « motif » de 8 cystéines (formant 4 ponts disulfures entre elles), motif qui est retrouvé dans les 2S-albumines, les inhibiteurs amylase/trypsine des céréales et certaines gliadines. Ces différentes familles de protéines, bien que peu homologues sur le plan séquentiel (pas de réactivité croisée d’une famille à une autre), font partie de la super-famille des « prolamines ».

Où trouve-t-on des LTP ? :

  • dans les plantes supérieures (pas dans les mousses ou les lycophytes)
  • dans de nombreux organes, surtout les parties aériennes
  • et particulièrement dans la peau de certains fruits , ce qui est habituellement mis en relation avec l’action antimicrobienne des LTP : ces dernières représentent le groupe PR-14 parmi les protéines de défense végétale .

La réactivité croisée des LTP

Comme pour d’autres familles de protéines, la proximité taxonomique favorise l’homologie et donc la réactivité croisée. L’identité séquentielle entre les LTP s’étend sur un continuum assez vaste, plus vaste qu’entre profilines par exemple. On aura donc une assez bonne réactivité croisée in vitro mais dont la pertinence clinique sera de plus en plus faible à mesure que l’identité séquentielle s’abaisse (ex. < 60%). D’autant qu’au final les épitopes sont conformationnels .

Le tableau ci-dessous résume les comparaisons entre la LTP de pêche, Pru p 3, et les LTP d’autres plantes. Il présente des % d’identité maximaux, sachant que les LTP sont multi-géniques, c’est-à-dire se présentent dans une même plante sous des variantes différant de quelques acides aminés .

On peut remarquer que les % d’identité des LTP polliniques avec Pru p 3 sont relativement faibles. Et, bien qu’une pollinose soit rencontrée chez la plupart des patients présentant un syndrome LTP, le rôle déclenchant d’un pollen dans la sensibilisation aux LTP n’a pas reçu jusqu’à présent de confirmation formelle. Ce point distingue une fois encore les LTP des profilines ou des PR-10.

On trouvera une argumentation plus détaillée des rapports entre LTP de pollens et divers aliments dans les textes concernant :

De même, l’impact d’une sensibilisation vis-à-vis des LTP dans une allergie alimentaire est abordé dans :

A l’heure actuelle il est possible de préciser un tableau clinique ou diagnostique en testant in vitro :

  • rPru p 3 (pêche)
  • rPar j 2 (pariétaire)
  • rCor a 8 (noisette)
  • rAra h 9 (arachide)
  • nArt v 3 (armoise)

Le test Pru p 3 peut servir de témoin d’une sensibilisation LTP, en sachant qu’il est possible, mais peu fréquent, que ce test soit négatif alors qu’une autre LTP serait positive (ex. rCor a 8). Le recombinant rPar j 2 est utile : cette LTP est spécifique d’une sensibilisation aux pariétaires car elle ne croise pas avec les autres LTP. L’intérêt de rAra h 9, bien qu’avancé , est douteux en dehors des zones méditerranéennes. Il en est de même pour nArt v 3.

Un diagnostic différentiel inclut, en plus d’une LTP, la recherche d’une réactivité pour une PR-10 (Bet v 1-like) et pour une profiline, ainsi que l’exclusion d’une participation du latex. Des extraits pour tests cutanés et spécifiques en LTP, profiline ou PR-10 sont à présent disponibles dans certains pays … mais hélas pas en France.

[1] - Pastorello EA, Farioli L, Pravettoni V, Ortolani C, Ispano M, Monza M, et al. The major allergen of peach (Prunus persica) is a lipid transfer protein. J Allergy Clin Immunol 1999;103:520-526
BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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[2] - Sanchez-Monge R, Lombardero M, Garcia Selles FJ, Barber D, Salcedo G. Lipid-transfer proteins are relevant allergens in fruit allergy. J Allergy Clin Immunol 1999;103:514-519
BACKGROUND: 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. OBJECTIVE: We sought to isolate and characterize the 13-kd allergen present in apple and peach. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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[3] - Asero R, Mistrello G, Roncarolo D, de Vries SC, Gautier MF, Ciurana CL, et al. Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion. Int Arch Allergy Immunol 2000;122:20-32
Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. OBJECTIVE: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. RESULTS: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. CONCLUSION: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.
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[4] - Pastorello EA, Pompei C, Pravettoni V, Farioli L, Calamari AM, Scibilia J, et al. Lipid-transfer protein is the major maize allergen maintaining IgE-binding activity after cooking at 100°C, as demonstrated in anaphylactic patients and patients with positive double-blind, placebocontrolled food challenge results. J Allergy Clin Immunol 2003;112:775-783
BACKGROUND: In a previous study a 9-kd lipid-transfer protein (LTP) was identified as the major allergen of raw maize in a population of 22 anaphylactic patients. However, the stability of this protein in cooked maize is unknown . OBJECTIVE: We investigated the allergenicity of 5 maize hybrids and its modification after different thermal treatments by using sera from anaphylactic patients and patients with positive double-blind, placebo-controlled food challenges . METHODS: Five maize hybrids were extracted by using different methods, obtaining the water-soluble, zein, total zein, glutelin, and total protein fractions. The IgE-binding capacity of the different extracts, both raw and after thermal treatment, was investigated by means of SDS-PAGE immunoblotting. A 9-kd heat-stable allergen was purified by means of HPLC and sequenced. Changes in its secondary structure during and after heating from 25 degrees C to 100 degrees C were monitored by means of circular dichroism . RESULTS: All raw maize hybrids showed similar protein and IgE-binding profiles. The SDS-PAGE of all the heat-treated hybrids demonstrated a decreased number of stained bands in respect to the raw samples. The IgE immunoblotting demonstrated that the major allergen of the water-soluble, total zein, total protein, and glutelin fractions was a 9-kd protein identified by means of amino acid sequence as an LTP and a sub-tilisin-chymotrypsin inhibitor (in total zein fraction). The IgE-binding capacity of this 9-kd protein remained unchanged after thermal treatments, even though circular dichroism demonstrated an altered secondary structure . CONCLUSIONS: Maize LTP maintains its IgE-binding capacity after heat treatment, thus being the most eligible candidate for a causative role in severe anaphylactic reactions to both raw and cooked maize.
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[5] - Asero R, Mistrello G, Roncarolo D, Amato S, Falagiani P. Analysis of the heat stability of lipid transfer protein from apple. J Allergy Clin Immunol 2003;112:1009-1011
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[6] - Cavatorta V, Sforza S, Aquino G, Galaverna G, Dossena A, Pastorello EA et al. In vitro gastrointestinal digestion of the major peach allergen Pru p 3, a lipid transfer protein: Molecular characterization of the products and assessment of their IgE binding abilities. Mol Nutr Food Res 2010;54:1452-1457
A simulated gastrointestinal digestion has been carried out on purified peach lipid transfer protein, one of the main allergens among the population of the Mediterranean area and the major allergen of peach allergic patients. The percentage of intact protein, after extensive digestion, measured by comparison with a non-digestible peptide analogue used as internal standard, was found to be about one-third of the original protein content. The peptides formed in digested fraction were characterized by means of LC/MS. The products of the digestion essentially derived from trypsin action, whereas the protein appeared to be resistant to pepsin and chymotrypsin. The identified peptides could be classified as low molecular weight and high molecular weight peptides. The latter consisted of the full protein, with the disulfide bridges still intact, deprived of the smaller peptides. The different digestion products, including the high and low molecular weight peptides, were purified by LC and assessed, together with the intact protein, by dot-blot analysis with sera of allergic patients, allowing to estimate their potential allergenicity. The intact protein and the high molecular weight peptides were found to be recognized by patients' sera, whereas the small peptides were found to be not reactive.
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[7] - Pastorello EA, Ortolani C, Farioli L, Pravettoni V, Ispano M, Borga A, et al. Allergenic cross-reactivity among peach, apricot, plum, and cherry in patients with oral allergy syndrome: an in vivo and in vitro study. J Allergy Clin Immunol 1994;94:699-707
Oral allergy syndrome in response to fruits and vegetables frequently occurs as clusters of hypersensitivity to members of the same botanical family, for which the immunologic basis lies in a number of common allergens, most of them still unidentified. OBJECTIVE: This study was designed to assess the in vivo and in vitro cross-reactivity between fruits of the Prunoideae subfamily (i.e., peach, cherry, apricot, and plum) and to identify their major allergens and the cross-reactivity of the peach extract with grass and birch pollen. METHODS: The in vivo study was conducted by skin prick tests and open food challenges with fresh fruits in 23 patients with oral allergy syndrome for peach and positive skin prick test and RAST results for the other Prunoideae. In vitro sodium dodecylsulfate-polyacrylamide gel electrophoresis was followed by immunoblotting and immunoblotting-inhibition. RESULTS: A 13 kd component was identified as the only major allergen common to all the Prunoideae, the other major allergens were found at 14 kd in peach and at 30 kd in cherry. Immunoblotting inhibition showed wide cross-reactivity within the Prunoideae, whereas grass and birch pollen partially inhibited the peach blotting. CONCLUSIONS: Clinical cross-reactivity to Prunoideae is essentially due to a common 13 kd IgE-binding component, which seems to be the most important major allergen of this subfamily, not shared with grass and birch pollen.
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[8] - Fernández-Rivas M, González-Mancebo E, Rodríguez-Pérez R, Benito C, Sánchez-Monge R, Salcedo G, et al. Clinically relevant peach allergy is related to peach lipid transfer protein, Pru p 3, in the Spanish population. J Allergy Clin Immunol 2003;112:789-795
BACKGROUND: Sensitization to peach and related Rosaceae fruits without clinical expression is commonly observed as the result of the extensive cross-reactivity of IgE antibodies directed toward lipid transfer proteins (LTPs), Bet v 1 homologues, profilins, and carbohydrate determinants . OBJECTIVE: We aimed to study whether there are any clinical or immunologic differences between patients allergic to peach and those who have a current clinically irrelevant sensitization to this fruit . METHODS: One hundred subjects with adverse reactions to peach were evaluated by medical history, skin prick tests with fresh peach and purified peach LTP (Pru p 3), and specific IgE determinations to peach, rBet v 1, and rBet v 2 (birch profilin). Clinical reactivity to peach was established by double-blind, placebo-controlled food challenges. The clinical characteristics and the in vivo and in vitro tests were compared between allergic and nonallergic patients . RESULTS: Peach allergy was confirmed in 76 patients and ruled out in 16; 2 patients dropped out, and the study was not conclusive in 6 individuals (placebo reactors). Pollen allergy was found in 76% of the allergic patients and in 100% of the nonallergic patients. Positive responses to Pru p 3, rBet v 1, and rBet v 2 were observed in 62%, 7%, and 34% of patients allergic to peach, respectively. The sensitization rate to Pru p 3 was significantly higher among subjects allergic than nonallergic to peach (62% vs 31%, P =.02). IgE responses to rBet v 2 were more frequent among subjects allergic to pollen, but no difference was observed in the presence or absence of peach allergy . CONCLUSIONS: Pru p 3 is the major allergen of peach in our population, and the IgE response to this allergen is related to the clinical expression of peach allergy. Sensitization to profilin is observed in those patients with an associated pollen allergy but does not appear to be related to the clinical reactivity to peach.
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[9] - Borghesan F, Mistrello G, Roncarolo D, Amato S, Plebani M, Asero R. Respiratory Allergy to Lipid Transfer Protein. Int Arch Allergy Immunol 2008;147:161-165
BACKGROUND: Due to unclear reasons, allergy to lipid transfer protein (LTP) is frequent in Mediterranean countries but rare in Northern Europe . OBJECTIVE: We report a paradigmatic case of primarily airborne sensitization to LTP that might explain the geographical distribution of this type of food allergy . METHODS: A 21-year-old woman began having severe perennial rhinitis 6 months after she started working in a wholesale fruit storehouse in Southern Italy where large amounts of fruits, including peaches, were handled; symptoms subsided when she left the workplace for >5 days and relapsed as soon as she was back at work. Later on, she developed severe food allergies to peach, hazelnut, peanut, apricot, plum and tomato. The patient underwent a nasal challenge with peach peel extract, and IgE reactivity was assessed by immunoblot analysis . RESULTS: In vivo and in vitro analyses showed sensitivity to LTP. The nasal challenge with peach peel extract (6 microg protein) induced acute, severe respiratory symptoms. On immunoblot with peach peel extract patient's serum reacted uniquely against LTP, as demonstrated by inhibition assays with the recombinant peach protein . CONCLUSION: LTP may induce sensitization via the respiratory tract due to inhalation of air-dispersed food particles, and this may precede the onset of food allergy. If this way of sensitization were effective in the majority of LTP allergic patients (e.g. by exposure to peaches showing intact fuzz in areas where peaches are grown and directly sold on the market) our findings could explain the strange geographical distribution of this type of food allergy.
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[10] - Barber D, de la Torre F, Lombardero M, Antépara I, Colas C, Dávila I et al. Component-resolved diagnosis of pollen allergy based on skin testing with profilin, polcalcin and lipid transfer protein pan-allergens. Clin Exp Allergy 2009;39:1764-1771
Background Allergy diagnosis needs to be improved in patients suffering from pollen polysensitization due to the existence of possible confounding factors in this type of patients. Objective To evaluate new diagnostic strategies by comparing skin responses to pan-allergens and conventional allergenic extracts with specific IgE (sIgE) to purified allergen molecules. Methods One thousand three hundred and twenty-nine pollen-allergic patients were diagnosed by a combination of an in vitro method with a panel of 13 purified allergens, including major allergens and pan-allergens, using a high-capacity screening technology (ADVIA-Centaurs) and skin prick test (SPT) to pan-allergens and conventional extracts. Results There was a high concordance (k index) between in vitro (sIgE to major allergens) and in vivo (SPT to conventional extracts) methods in patients who were not sensitized to panallergens, but SPT with conventional extracts failed to diagnose patients with sensitization to pan-allergens. In patients who were simultaneously sensitized to polcalcins and profilins, there was a duplication both in the number of sensitizations to major allergens and in the years of disease evolution. There was a statistical association between sensitization to profilins and/or lipid transfer proteins and food allergy (Po0.0001). Conclusion The novel diagnostic strategy has proven to be a valuable tool in daily clinical practice. Introduction of routine SPT to pan-allergens is a simple and feasible way of improving diagnostic efficacy. Patients sensitized to pan-allergens should be tested by an adequate panel of allergenic molecules in order to identify the allergens that are responsible for the allergic disease.
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[11] - Asero R. Peach-induced contact urticaria is associated with LTP sensitisation and may precede food allergy by years. Allergy 2010;65(suppl. 92):51
Background: Peach-induced contact urticaria is frequently observed in subjects allergic to this fruit. Objective: To detect whether contact urticaria is associated with a specific allergen protein of the peach. Methods: Ninety two subjects with peach allergy were studied. Patients were diagnosed as being sensitized to lipid transfer protein (Pru p 3) or as having a pollenfood allergy syndrome induced by Pru p 1 and/or profilin, Pru p 4, on the basis of the results of SPT containing such allergen proteins in an isolated form. Specific IgE to peach extract were measured as well. Contact urticaria was confirmed by a contact test with intact peach. A contact test with nectarine was carried out as control. Results: Overall, contact urticaria was present 21% of patients; the peach contact test scored positive in all cases. Contact urticaria was significantly more frequent in patients hypersensitive to lipid transfer protein (63%) than in subjects with pollenfood allergy syndrome (6%; P < 0.001). Contact urticaria was not associated with a higher level of peach-specific IgE. In several cases contact urticaria clearly preceded by years the onset of food allergy. Contact test with nectarine scored negative in 5/5 cases. Conclusions: Peach-induced contact urticaria is associated with sensitization to peach lipid transfer protein. The negative clinical history and contact test with nectarine along with the well-known high concentration of LTP in peach fuzz suggest that peach fuzz plays a role in the pathogenesis of contact urticaria.
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[12] - Salcedo G, Sanchez-Monge R, Diaz-Perales A, Garcia-Casado G, Barber D. Plant non-specific lipid transfer proteins as food and pollen allergens. Clin Exp Allergy 2004;34:1336-1341
Several members of the plant non-specific lipid transfer protein (LTP) family have been identified as relevant allergens in foods and pollens. These allergens are highly resistant to both heat treatment and proteolytic digestion. These characteristics have been related with the induction of severe systemic reactions in many patients, and with the possibility of being primary sensitizers by the oral route. A specific geographical distribution pattern of sensitization to LTP allergens has been uncovered. This allergen family is particularly important in the Mediterranean area, but shows a very limited incidence in Central and Northern Europe. The potential role in the plant, as well as the biochemical and allergenic properties of the LTP family, are reviewed here.
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[13] - Breiteneder H, Mills C. Nonspecific lipid-transfer proteins in plant foods and pollens: an important allergen class. Curr Opin Allergy Clin Immunol 2005;5:275-279
PURPOSE OF REVIEW: Here we focus our attention on the structural stability and physicochemical properties of plant nonspecific lipid-transfer proteins (nsLTPs) as keys to their allergenicity. We further present the current opinions on the route of sensitization and include the latest additions to the nsLTP allergen family. RECENT FINDINGS: Plant nsLTPs are small cysteine-rich lipid-binding proteins that play a key role in plant resistance to biotic and abiotic stress. Besides their relevance for plant-pathogen interactions, nsLTPs have attracted interest as true food allergens which are of high importance to atopics in Mediterranean countries. It is now becoming clear that their molecular properties such as the remarkable stability to proteolysis and thermal denaturation are intrinsically linked to their allergenicity. These properties also facilitate sensitization via the gastrointestinal tract which allows these molecules to act as allergens independently of prior exposure to pollen. In addition, a group of allergenic pollen nsLTPs exists which seem to be only partially linked to the food nsLTPs by cross-reactivity. SUMMARY: Research into the family of nsLTPs will continue to provide insights about the particular molecular properties that make an nsLTP an allergen and how primary sensitization occurs.
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[14] - Salcedo G, Sánchez-Monge R, Barber D, Díaz-Perales A. Plant non-specific lipid transfer proteins: an interface between plant defence and human allergy. Biochim Biophys Acta 2007;1771:781-791
Plant non-specific LTPs (lipid transfer proteins) form a protein family of basic polypeptides of 9 kDa ubiquitously distributed throughout the plant kingdom. The members of this family are located extracellularly, usually associated with plant cell walls, and possess a broad lipid-binding specificity closely related to their three-dimensional structure. The nsLTP fold is characterized by a compact domain composed of 4 alpha-helices, firmly held by a network of 4 conserved disulphide bridges. This fold presents a large internal tunnel-like cavity, which can accommodate different types of lipids. nsLTPs are involved in plant defence mechanisms against phytopathogenic bacteria and fungi, and, possibly, in the assembly of hydrophobic protective layers of surface polymers, such as cutin. In addition, several members of the nsLTP family have been identified as relevant allergens in plant foods and pollens. Their high resistance to both heat treatment and digestive proteolytic attack has been related with the induction by these allergens of severe symptoms in many patients. Therefore, they are probably primary sensitizers by the oral route. nsLTP sensitization shows an unexpected pattern throughout Europe, with a high prevalence in the Mediterranean area, but a low incidence in Northern and Central European countries.
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[15] - Lavaud F, Fontaine JF, Perotin JM, Angelier AS, Meirhaeghe D, Lebargy F. Manifestations cliniques de l’allergie aux protéines de transfert lipidique. Rev Fr Allergol 2009;49:427-432
Les protéines de transfert lipidique (LTP) sont produites par de nombreux végétaux, dans les pollens, fruits ou autres parties de la plante. Ce sont par excellence des panallergènes dont le chef de file est représenté par LTP de la pêche (Pru p 3). La liste des sources d‚allergènes se complète de jour en jour avec de très nombreuses réactions croisées même si l‚origine de la sensibilisation (voie respiratoire, digestive ou cutanée) reste méconnue. Les LTP sont impliquées dans des allergies respiratoires, notamment professionnelles. Ce sont surtout des allergènes alimentaires puissants car thermostables et résistants à la protéolyse, responsables de réactions systémiques sévères. Dans les régions méditerranéennes, ce sont les allergènes majeurs des allergies alimentaires aux rosacées.
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[16] - Egger M, Hauser M, Mari A, Ferreira F, Gadermaier G. The Role of Lipid Transfer Proteins in Allergic Diseases. Curr Allergy Asthma Rep 2010;10:326-335
Nonspecific lipid transfer proteins (LTPs) are important allergens in fruits, vegetables, nuts, pollen, and latex. Despite their wide distribution throughout the plant kingdom, their clinical relevance is largely confined to the Mediterranean area. As they can sensitize via the gastrointestinal tract, LPTs are considered true food allergens, and IgE reactivity to LTPs is often associated with severe systemic symptoms. Although Pru p 3 represents the predominant LTP in terms of patients' IgE recognition, the contribution of pollen LTPs in primary sensitization cannot be ruled out. Due to structural homology, LTPs from different allergen sources are generally IgE cross-reactive. However, sensitization profiles among allergic patients are extremely heterogeneous, and individual cross-reactivity patterns can be restricted to a single LTP or encompass many different LTPs. Molecule-based approaches in allergy research and diagnosis are important for better understanding of LTP allergy and could assist clinicians with providing adequate patient-tailored advice.
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[17] - Douliez JP, Jegou S, Pato C, Larre C, Molle D, Marion D. Identification of a new form of lipid transfer protein (LTP1) in wheat seeds. J Agric Food Chem 2001;49:1805-1808
Recently, this laboratory has isolated from barley and beer extract an isoform of lipid transfer protein (LTP1), which was not fully sequenced (Jegou, S.; Douliez, J. P.; Molle, D.; Boivin, P.; Marion, D. J. Agric. Food Chem. 2000, 48, 5023--5029). It was named LTP1b and exhibited a molecular weight 294 Da higher than that of the known LTP1. This paper reports the finding of an LTP1 isoform in wheat that also exhibits an excess of 294 Da compared to the native protein. Amino acid sequencing, reduction and alkylation, and mass spectrometry showed that this new LTP1b possesses the same N-terminal sequence as the native LTP1, suggesting that the difference resides in the binding of an adduct which has a molecular weight of 294 Da. The aim of the present paper is to highlight various biophysical techniques that afford the identification of such an isoform-like LTP1 and to correlate this finding with other isoforms of LTP1 that were isolated from other plants but not fully sequenced
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[18] - Jégou S, Douliez JP, Mollé D, Boivin P, Marion D. Purification and structural characterization of LTP1 polypeptides from beer. J Agric Food Chem 2000;48:5023-5029
We report on the purification of lipid transfer proteins (LTP) from barley seeds and beer with the aim of investigating the chemical modifications that occur during the brewing process. In seeds, the well-known LTP of 9 kDa (LTP1) has been found together with a second form named LTPb that displays comparable amino acid composition but was not fully sequenced. These two forms have been recovered in beer with marked chemical modifications including disulfide bond reduction and rearrangement and especially glycation by Maillard reaction. The glycation is heterogeneous with variable amounts of hexose units bound to LTPs. Circular dichroism shows that glycated LTP1 having all their disulfide bridges reduced are totally unfolded. These results provide a first basis for understanding how barley LTPs become foam-promoting agents during the malting and brewing process.
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[19] - Blein JP, Coutos-Thevenot P, Marion D, Ponchet M. From elicitins to lipid-transfer proteins: a new insight in cell signalling involved in plant defence mechanisms. Trends Plant Sci 2002;7:293-296
Elicitins and lipid-transfer proteins are small cysteine-rich lipid-binding proteins secreted by oomycetes and plant cells, respectively, that share some structural and functional properties. In spite of intensive work on their structure and diversity at the protein and genetic levels, the precise biological roles of lipid-transfer proteins remains unclear, although the most recent data suggest a role in somatic embryogenesis, in the formation of protective surface layers and in defence against pathogens. By contrast, elicitins are known elicitors of plant defence, and recent work demonstrating that elicitins and lipid-transfer proteins share the same biological receptors gives a new perspective to understand the role played by lipid binding proteins, mainly the early recognition of intruders in plants.
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[20] - Perrocheau L, Bakan B, Boivin P, Marion D. Stability of Barley and Malt Lipid Transfer Protein 1 (LTP1) toward Heating and Reducing Agents: Relationships with the Brewing Process. J Agric Food Chem 2006;54:3108-3113
Barley lipid transfer protein (LTP1) is a heat-stable and protease-resistant albumin that concentrates in beer, where it participates in the formation and stability of beer foam. Whereas the barley LTP1 does not display any foaming properties, the corresponding beer protein is surface-active. Such an improvement is related to glycation by Maillard reactions on malting, acylation on mashing, and structural unfolding on brewing. The structural stability of purified barley and glycated malt LTP1 toward heating has been analyzed. Whatever the modification, lipid adduction or glycation, barley LTP1s are highly stable proteins that resisted temperatures up to 100 degrees C. Unfolding of LTP1 occurred only when heating was conducted in the presence of a reducing agent. In the presence of sodium sulfite, the lipid-adducted barley and malt LTP1 displayed higher heat stability than the nonadducted protein. Glycation had no or weak effect on heat-induced unfolding. Finally, it was shown that unfolding occurred on wort boiling before fermentation and that the reducing conditions are provided by malt extract.
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[21] - Bodinier M, Triballeau S, Pineau F, Legoux MA, Leroy M, Bakan B et al. Allergénicité des LTP de blé. Alim'Inter 2009;14:221-224
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[22] - Borges JP, Culerrier R, Archimbaud N, Barre A, Didier A, Rougé P. Caractérisation d'un épitope consensus des protéines de transfert de lipides de fruits. Rev Fr Allergol Immunol Clin 2006;46:341-342
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[23] - Borges JP, Jauneau A, Brule C, Culerrier R, Barre A, Didier A, et al. The lipid transfer proteins (LTP) essentially concentrate in the skin of Rosaceae fruits as cell surface exposed allergens. Plant Physiol Biochem 2006;44:535-542
The localization and distribution of non-specific lipid transfer proteins (nsLTP) allergens in the skin and pulp of Rosaceae fruits (apple, peach, apricot, plum) has been investigated. nsLTP essentially concentrate in the pericarp of the fruits whereas the pulp contains lower amounts of allergens. Immunolocalization showed they are primarily located in the cytosol but are subsequently excreted and finally accumulate at the plasmalemma-cell wall interface and in the cell wall. However, high discrepancies were observed in the content of allergens among, e.g. different cultivars of apple. As a consequence, the consumption of peeled-off fruits is recommended to reduce the risk of severe allergic reactions (anaphylactic shock) in individuals sensitized to Rosaceae fruits.
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[24] - Borges JP, Barre A, Culerrier R, Granier C, Didier A, Rougé P. Lipid transfer proteins from Rosaceae fruits share consensus epitopes responsible for their IgE-binding cross-reactivity. Biochem Biophys Res Commun 2008;365:685-690
Four IgE-binding epitopes have been characterized that cover a large area (40%) of the molecular surface of lipid transfer protein allergens of Rosaceae (apple, peach, apricot, and plum). They mainly correspond to electropositively charged regions protruding on the molecular surface of the modeled apple (Mal d 3), apricot (Pru ar 3), and plum (Pru d 3) allergens. Two of these epitopes consist of consensus epitopes structurally conserved among the lipid transfer protein allergens from the Rosaceae. Their occurrence in different lipid transfer protein allergens presumably accounts for the IgE-binding cross-reactivity often observed among different Rosaceae fruits. In this respect, LTP consist of phylogenetically- and structurally-related pan allergens. However, the IgE-binding cross-reactivity due to fruit lipid transfer protein has varying degrees of clinical relevance and this cross-reactivity is not necessarily accompanied by a cross-allergenicity to the corresponding fruits.
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[25] - Borges JP, Barre A, Culerrier R, Archimbaud N, Didier A, Rougé P. How reliable is the structural prediction of IgE-binding epitopes of allergens? The case study of plant lipid transfer proteins. Biochimie 2007;89:83-91
The linear IgE-binding epitopes of non-specific lipid transfer proteins (nsLTP) from plants were predicted using a combination of predictive tools including (1) the hydropathic profiles based on different scales of hydrophilicity, flexibility and exposure to the solvent, (2) the hydrophobic cluster analysis plots, (3) the occurrence of charged residues in the predicted amino acid sequence stretches and, (4) the exposition of the predicted linear IgE-binding epitopes checked on the three-dimensional models built for the nsLTP. A reliable prediction was obtained for nsLTP as compared with the previously characterized IgE-binding epitopes of various proteins. A consensual IgE-binding epitope occurring in other plant nsLTP and responsible for some IgE-binding cross-reactivity among fruit nsLTP has been identified and characterized. Despite some discrepancies, a fairly good prediction resulted in applying our combination of predictive methods to longer nsLTP or plant profilins.
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[26] - Barre A, Brulé C, Borges JP, Culerrier R, Jauneau A, Didier A et al. Concentration des LTP dans la peau et la pulpe des fruits. Rev Fr Allergol 2009;49:166-169
Les principaux allergènes des fruits de Rosacées (Prunoidées) correspondent à des protéines de défense (protéines pathogenesis-related [PR]) qui sont essentiellement exprimées en réponse à l‚attaque de la plante par un champignon phytopathogène. Il s‚agit des protéines de transfert des lipides (LTP), des 1,3ß-glucanases et des protéines thaumatin-like (TLP), qui appartiennent respectivement aux familles des protéines PR-14, PR-2 et PR-5. La LTP de pomme (Mal d 3) est principalement localisée dans la peau du fruit et la peau de certaines variétés de pomme (Granny Smith, Golden delicious, Fuji) est particulièrement riche en LTP. La chair du fruit en renferme beaucoup moins. La LTP de pêche (Pru p 3) s‚accumule essentiellement dans les poils formant le revêtement duveteux du fruit. En pratique, l‚épluchage de ces fruits réduit considérablement l‚apport de LTP aux personnes sensibilisées. En revanche, la cuisson des fruits n‚offre aucun intérêt en raison de l‚extrême résistance des LTP à la dénaturation thermique. Dans d‚autres fruits, prune (Pru d 3) et abricot (Pru ar 3), les LTP sont aussi abondantes dans la peau que dans la chair et l‚épluchage de ces fruits ne réduit en rien leur allergénicité. Des résultats préliminaires semblent indiquer que les fruits issus de l‚agriculture biologique possèdent des teneurs en LTP supérieures à celles observées dans les fruits issus de l‚agriculture traditionnelle. Dans les pays du pourtour Méditerranéen, l‚Espagne et l‚Italie en particulier, l‚allergie à la pêche (Pru p 3) s‚accompagne souvent de réactions anaphylactiques sévères.
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[27] - Yeats TH, Rose JKC. The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs). Protein Sci 2008;17:191-198
Plant lipid-transfer proteins (LTPs) are abundant, small, lipid binding proteins that are capable of exchanging lipids between membranes in vitro. Despite their name, a role in intracellular lipid transport is considered unlikely, based on their extracellular localization. A number of other biological roles, including antimicrobial defense, signaling, and cell wall loosening, have been proposed, but conclusive evidence is generally lacking, and these functions are not well correlated with in vitro activity or structure. A survey of sequenced plant genomes suggests that the two biochemically characterized families of LTPs are phylogenetically restricted to seed plants and are present as substantial gene families. This review aims to summarize the current understanding of LTP biochemistry, as well as the evidence supporting the proposed in vivo roles of these proteins within the emerging post-genomic framework.
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[28] - Borges JP, Jauneau A, Brule C, Culerrier R, Barre A, Didier A, et al. The lipid transfer proteins (LTP) essentially concentrate in the skin of Rosaceae fruits as cell surface exposed allergens. Plant Physiol Biochem 2006;44:535-542
The localization and distribution of non-specific lipid transfer proteins (nsLTP) allergens in the skin and pulp of Rosaceae fruits (apple, peach, apricot, plum) has been investigated. nsLTP essentially concentrate in the pericarp of the fruits whereas the pulp contains lower amounts of allergens. Immunolocalization showed they are primarily located in the cytosol but are subsequently excreted and finally accumulate at the plasmalemma-cell wall interface and in the cell wall. However, high discrepancies were observed in the content of allergens among, e.g. different cultivars of apple. As a consequence, the consumption of peeled-off fruits is recommended to reduce the risk of severe allergic reactions (anaphylactic shock) in individuals sensitized to Rosaceae fruits.
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[29] - Malandain H, Lavaud F. Allergénicité des protéines de défense végétale. Rev Fr Allergol Immunol Clin 2004;44:469-475
La synthèse de protéines de défense fait partie des réponses biochimiques que les plantes ont développées pour lutter contre leurs ennemis naturels et contre les stress environnementaux. De nombreuses protéines de défense végétale sont allergisantes : chitinases, protéines de transfert lipidique LTP, protéines Bet v 1-like. Cet article rappelle les principales familles de protéines de défense végétale, leur impact allergique et l'effet de certains procédés agricoles ou technoalimentaires sur l'allergénicité des aliments végétaux.
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[30] - Borges JP, Barre A, Culerrier R, Granier C, Didier A, Rougé P. Lipid transfer proteins from Rosaceae fruits share consensus epitopes responsible for their IgE-binding cross-reactivity. Biochem Biophys Res Commun 2008;365:685-690
Four IgE-binding epitopes have been characterized that cover a large area (40%) of the molecular surface of lipid transfer protein allergens of Rosaceae (apple, peach, apricot, and plum). They mainly correspond to electropositively charged regions protruding on the molecular surface of the modeled apple (Mal d 3), apricot (Pru ar 3), and plum (Pru d 3) allergens. Two of these epitopes consist of consensus epitopes structurally conserved among the lipid transfer protein allergens from the Rosaceae. Their occurrence in different lipid transfer protein allergens presumably accounts for the IgE-binding cross-reactivity often observed among different Rosaceae fruits. In this respect, LTP consist of phylogenetically- and structurally-related pan allergens. However, the IgE-binding cross-reactivity due to fruit lipid transfer protein has varying degrees of clinical relevance and this cross-reactivity is not necessarily accompanied by a cross-allergenicity to the corresponding fruits.
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[31] - Tordesillas L, Pacios LF, Palacin A, Quirce S, Armentia A, Barber D et al. Molecular basis of allergen cross-reactivity: Non-specific lipid transfer proteins from wheat flour and peach fruit as models. Mol Immunol 2009;47:534-540
Peach non-specific lipid transfer protein (Pru p 3; nsLTP) has been characterized as the major food allergen in the adult Mediterranean population. Its wheat homologous protein, Tri a 14 has a relevant inhalant allergen in occupational baker's asthma. Different sensitization patterns to these allergens have been found in patients with this latter disorder. The objective of the present study was to characterize IgE epitopes of Tri a 14 and to compare them with those of Pru p 3 using three complementary strategies: the analysis of IgE-binding capacity of decapeptides bound to membrane, the identification of mimotopes using a phage display random peptide library, and the analysis of the surface electrostatic potential of both allergens. Thus, synthetic overlapping decapeptides, covering the Pru p 3 and Tri a 14 amino acid sequences, were used to identify sequential regions involved in recognition of IgE from baker's asthma patients sensitized to both nsLTPs. A phage display library was screened with total IgE from the same patients, and positive clones sequentially selected using the purified allergens, allowed to identify mimotopes (conformational epitopes) of Tri a 14 and Pru p 3. Both sequential regions and mimotopes were localized in the corresponding 3D molecular surface and their electrostatic properties were analyzed. Common sequential regions with strong IgE-binding capacity (residues 31-40 and 71-80) were identified in Tri a 14 and Pru p 3, whereas regions Tri a 14(51-60) and Pru p 3(11-20) were found specific of each allergen. A major conformational epitope (mimotope), L34H35N36R39S40S42D43G74V75L77P78Y79T80, which comprised the two common sequential epitopes, was located in Tri a 14, and a very similar one in Pru p 3. However, differences were detected on the surface electrostatic potential of both mimotopes: a first part (around residues 31-45) showed similar positive features in both allergens, whereas a second part (around residues 74-80) was markedly negative in Tri a 14 but neutral-positive in Pru p 3. Tri a 14 and Pru p 3 have a similar conformational region involved in IgE-binding, although their electrostatic features are different. Additionally, common and specific sequential IgE-binding regions were mapped in both allergens. These findings could be instrumental in understanding the cross-reactivity and specificity of sensitization to both homologous allergens.
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[32] - Yeats TH, Rose JKC. The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs). Protein Sci 2008;17:191-198
Plant lipid-transfer proteins (LTPs) are abundant, small, lipid binding proteins that are capable of exchanging lipids between membranes in vitro. Despite their name, a role in intracellular lipid transport is considered unlikely, based on their extracellular localization. A number of other biological roles, including antimicrobial defense, signaling, and cell wall loosening, have been proposed, but conclusive evidence is generally lacking, and these functions are not well correlated with in vitro activity or structure. A survey of sequenced plant genomes suggests that the two biochemically characterized families of LTPs are phylogenetically restricted to seed plants and are present as substantial gene families. This review aims to summarize the current understanding of LTP biochemistry, as well as the evidence supporting the proposed in vivo roles of these proteins within the emerging post-genomic framework.
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[33] - Krause S, Reese G, Randow S, Zennaro D, Quaratino D, Palazzo P et al. Lipid transfer protein (Ara h 9) as a new peanut allergen relevant for a Mediterranean allergic population. J Allergy Clin Immunol 2009;124:771-778
BACKGROUND: Nonspecific lipid transfer proteins (LTPs) represent potent pollen and food allergens. However, the allergenic properties of peanut LTP have not been studied. OBJECTIVE: To identify LTP in peanut extract using sera from subjects with peanut allergy and Pru p 3-sensitized subjects from Southern Europe, clone and express this protein, and obtain information on the importance as allergen for these selected patients. METHODS: Peanut LTP (Ara h 9) was cloned and sequenced by using a combination of bioinformatic and molecular biology tools (PCR, immunoblotting, Basic Local Alignment Search Tool [BLAST] searches). The immunologic properties of Ara h 9, Ara h 1, Ara h 2, and Ara h 3 were studied by using sera from subjects with peanut and peach allergy from Italy by immunoblotting and allergen microarray technology. RESULTS: Two Ara h 9 isoforms-Ara h 9.01 and Ara h 9.02-were cloned and expressed. Ara h 9 represented a minor allergen for subjects with peanut allergy. However, including Ara h 9 as single component for serologic detection of sensitization to peanut by component-resolved diagnosis seems crucial, because the frequency of sensitization to the classic major peanut allergens Ara h 1, Ara h 2, and Ara h 3 was low in these patients from Southern Europe. CONCLUSION: Ara h 9 is a new member of the LTP allergen family that seems to play an important role in peanut allergy for patients from the Mediterranean area.
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[34] - Asero R, Jimeno L, Barber D. Component-resolved diagnosis of plant food allergy by SPT. Eur Ann Allergy Clin Immunol 2008;40:115-121
BACKGROUND: Fruits and vegetables may contain both labile and stable allergens. The former induce only OAS, whereas stable allergens may induce systemic reactions. Component-resolved diagnosis (CRD) of allergy to plant foods is therefore essential for the clinical management of allergic patients. METHODS: 80 adults allergic to plant foods underwent SPT with purified natural date palm profilin (Pho d 2), purified Mal d 1, a peach extract containing uniquely LTP, and with a kiwi extract containing uniquely stable allergens. RESULTS: 58 (72%) patients were monosensitized: 24 to Mal d 1, 24 to profilin, 7 to LTP, and 3 to kiwi. 22 patients were multi-sensitised: 14 to Mal d 1 and profilin, 2 to Mal d 1 and kiwi, 1 to LTP and profilin, 3 to LTP and Mal d 1, and 2 to LTP, Mal d 1 and profilin. Mal d 1 and LTP sensitisation were associated with apple and peach allergy, respectively, whereas profilin sensitisation was associated with allergy to melon, watermelon, banana, tomato and citrus fruits. 18/21 kiwi-allergic patients were sensitised to one of the cross-reacting allergens, but 2/18 reacted to kiwi-specific allergens as well. CONCLUSIONS: In patients with allergy to plant-derived foods CRD can be performed by SPT with purified allergen proteins. In the future, the availability of a larger number of purified natural or recombinant allergens for SPT will represent a simple means to classify food-allergic patients properly on the first visit.
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