Characterization of peach thaumatin-like proteins and their identification as major peach allergens.

BACKGROUND: Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. OBJECTIVE: To identify members of the TLP family in peach fruit and to characterize putative allergens. METHODS: Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N-terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (beta-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). RESULTS: Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N-asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed beta-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. CONCLUSION: Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area.

Plant non-specific lipid transfer proteins: an interface between plant defence and human allergy.

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.

Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms.

BACKGROUND: Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients. OBJECTIVE: To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population. METHODS: A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs. RESULTS: The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity. CONCLUSION: Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

New alpha-amylase and trypsin inhibitors among the CM-proteins of barley (Hordeum vulgare).

Barley CM-proteins are a group of at least five salt-soluble components (CMa-e) that can be selectively extracted from endosperm with chloroform/methanol mixtures. N-terminal sequences of proteins CMa, CMb and CMc have been determined and found to be homologous to those previously determined for CMd and CMe, an observation which confirms that their structural genes are members of a dispersed multi-gene family. The purified CM-proteins were tested against trypsin and against alpha-amylases from saliva, pancreas, Aspergillus oryzae, Tenebrio molitor and barley. Besides CMe, which was known to be a trypsin inhibitor, CMc also showed antitrypsin activity, whereas CMa was specifically active against the alpha-amylase from T. molitor and no inhibitory activity was found for proteins CMb and CMd. The evolutionary implications of these findings are discussed.

Latex-vegetable syndrome due to custard apple and aubergine: new variations of the hevein symphony.

An increasing number of vegetables with crossreactions to latex are being described in patients with latex-vegetable syndrome. We present two of these vegetables, custard apple linked in two previous cases with latex sensitisation, and aubergine, that had not been described up to now in patients with latex sensitisation. The diagnosis of both cases was based on the clinical history, positive skin prick test (SPT) and specific IgE to the offending vegetables, as well as to positive SPT and specific IgE levels to latex and the major fruits involved in the latex-fruit syndrome (avocado, banana, and chestnut). Further, crude extracts from latex, custard apple and aubergine, as well as the purified allergens Hev b 6.02 and Prs a 1 were used in in vitro and in vivo assays: IgE immunodetection, histamine release (HRT) and basophil activation (BAT) tests and skin prick tests. In case 1, both purified Hev b 6.02 and Prs a 1 induced positive responses in skin prick tests, high levels of basophil activation and histamine release. Specific IgE immunodetection uncovered a reactive band of 45 kd in the crude custard apple extract, which was also recognized by anti-chitinase monospecific antibodies. The serum from patient 1 also detected Prs a 1 in immunodetection. Hev b 6.02 produced positive skin responses and showed high biological activity in HRT and BAT in the case of patient 2. However, Prs a 1 was reactive neither in SPT nor in IgE immunodetection. In fact, no band was detected using the serum of patient 2 in avocado or aubergine extracts. By contrast, Prs a 1 reached high values of basophil activation and over 10% of histamine release in case 2.

Biotic and abiotic stress can induce cystatin expression in chestnut.

A cysteine proteinase inhibitor (cystatin) from chestnut (Castanea sativa) seeds, designated CsC, has been previously characterized. Its antifungal, acaricide and inhibitory activities have allowed to involve CsC in defence mechanisms. The CsC transcription levels decreased during seed maturation and increased throughout germination, an opposite behavior to that shown by most phytocystatins. No inhibition of endogenous proteinase activity by purified CsC was found during the seed maturation or germination processes. CsC message accumulation was induced in chestnut leaves after fungal infection, as well as by wounding and jasmonic acid treatment. Induction in roots was also observed by the last two treatments. Furthermore, CsC transcript levels strongly raised, both in roots and leaves, when chestnut plantlets were subjected to cold- and saline-shocks, and also in roots by heat stress. All together, these data suggest that chestnut cystatin is not only involved in defence responses to pests and pathogen invasion, but also in those related to abiotic stress.

A major baker's asthma allergen from rye flour is considerably more active than its barley counterpart.

A rye flour protein of about 13.5 kDa, as well as its barley homologue, have been isolated. The rye component was recognized in vitro by IgE of allergic patients and provoked positive responses in 15 out of 21 baker's asthma patients (71%) when skin prick tests were performed. Its barley homologue showed no detectable in vitro reactivity and caused positive responses in only one-third of patients. Although no inhibitory activity against different alpha-amylases or trypsin was found for these two proteins, their N-terminal sequencing revealed considerable similarity to several members of the cereal alpha-amylase/trypsin inhibitor family.

Members of the alpha-amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma.

We have identified the major antigens or IgE binding components from wheat flour. Thirty-five sera from patients with baker's asthma were used to analyze the reaction with wheat salt-soluble proteins. We found a 15 kDa SDS-PAGE band which reacted with all sera tested. Purified members of the alpha-amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two-dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low-molecular-weight allergens.

A barley flour inhibitor of insect alpha-amylase is a major allergen associated with baker's asthma disease.

A barley salt-soluble protein of 14.5 kDa, which inhibits the alpha-amylase from the insect Tenebrio molitor, has been identified as a major IgE-binding component of sera from baker's asthma patients. The N-terminal amino acid sequence of this protein indicates that it is a member of a previously described family of alpha-amylase/trypsin inhibitors.

The role of plant panallergens in sensitization to natural rubber latex.

Latex allergy represents an increasing occupational problem, mainly among healthcare workers. An association between latex allergy and hypersensitivity to some plant foods, particularly fruits (the latex-fruit syndrome), has been established. Class I chitinases with an N-terminal hevein-like domain from avocado, chestnut, banana and other foods, and latex hevein seem to be the allergens responsible for the cross-reactions involved in the latex-fruit syndrome. The potential role of other latex allergens, such as profilin, Hev b 5, Hev b 7 and beta-1,3-glucanases, in the co-sensitization to latex and plant foods is also discussed.

Two different profiles of peach allergy in the north of Spain.

BACKGROUND: Peach allergy has two different patterns: central Europe with oral allergy syndrome (OAS) related to a primary sensitization to birch pollen Bet v 1 and profilins and southern Europe with mostly systemic symptoms, in many cases due to sensitization to lipid-transfer proteins. METHODS: Thirty peach-allergic patients with positive skin and food challenge tests and 29 control subjects were included. Skin prick tests (SPT) with inhalant allergens, commercial peach and apple extracts and native Pru p 3 were performed. In vitro specific immunoglobulin (Ig) E to grass pollen, birch pollen, peach, apple, rBet v 1, rBet v 2 and rPhl p 12 was determined by CAP, and rBet v 1, rMal d 1, rMal d 4, rMal d 3 and rPru p 3 using the ADVIA-Centaur platform. Basophil activation test (BAT) with commercial peach extract, commercial apple extract, nPru p 3, rMal d 3, rMal d 1 and rMal d 4 was also performed. RESULTS: Pru p 3 was the major allergen in the patient group from northern Spain. Sensitization to this allergen was found in 100% of the patients with systemic symptoms or contact urticaria. Only 60% of OAS patients were sensitized to Pru p 3, being all of them sensitized to profilins and 60% of them to allergens of the Bet v 1 family. Specific IgE determination and BAT using recombinant allergens (rPru p 3) show specificity and sensitivity values close to 100%. CONCLUSIONS: Most peach-allergic patients coming from the north of Spain present systemic symptoms after ingestion of peach, Pru p 3 being the main allergen. Patients with OAS present profilin-Bet v 1-related sensitization. Thus, in the north of Spain our patients show a mixed central-south Europe pattern with LTP-profilin-Bet v 1 sensitization depending on the symptoms presented. The use of natural and recombinant plant allergens, allows establishing the sensitization patterns to the different allergens studied.

Allergic hypersensitivity to the lentil pest Bruchus lentis.

BACKGROUND: Lentils are the most common legume involved in allergic reactions in the Mediterranean area and India. Lentil crops could be attacked by a wide range of insect species. Occupational asthma caused by the lentil pest Bruchus lentis has been described in the present study. OBJECTIVE: We studied the possibility of immunoglobulin (Ig)E-mediated hypersensitivity to lentil pests in 16 patients who suffered from allergic symptoms (asthma and anaphylaxis) related to inhalation of lentil particles or ingestion of lentils, in which sensitization to legume proteins was not clear. METHODS: Extracts prepared either from noninfested and infested lentils, and from the lentil parasite B. lentis were used for skin prick testing (SPT), bronchial and oral challenges and in vitro determinations. RESULTS: Skin prick test were positive to infested lentils and B. lentis in all patients and negative to noninfested lentil extracts. Five asthmatic patients reacted in the bronchial challenge test with Bruchus extract. Oral challenges performed with boiled infested lentils were positive in six of seven patients. Immunoglobulin E immunoblotting with Bruchus and infested lentils extracts revealed protein bands that were reactive to serum IgE from six and four, respectively, out of 16 patients. These patients had no IgE to lentil-specific proteins as determined by immunoblotting and Len c 1-specific IgE test. CONCLUSIONS: Lentil pest proteins can be a cause of IgE-mediated rhinoconjunctivitis and asthma in patients eating or inhaling infested lentil particles.

Allergenic reactivity of the melon profilin Cuc m 2 and its identification as major allergen.

BACKGROUND: Melon allergy is commonly associated with oral allergy syndrome (OAS) and with hypersensitivity to pollens and other plant foods. No melon allergen responsible for these clinical characteristics has yet been isolated, although profilin has been proposed as a potential target. OBJECTIVE: To isolate natural and recombinant melon profilin, to evaluate its in vivo and in vitro reactivity, and to analyse its behaviour in simulated gastric fluid (SGF) and heat treatments. METHODS: A pool or individual sera from 23 patients, and an additional group of 10 patients, all of them with melon allergy, were analysed by in vitro and in vivo tests, respectively. Natural melon profilin (nCuc m 2) and its recombinant counterpart (rCuc m 2) were isolated by poly-l-proline affinity chromatography, and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization analysis, DNA sequencing of cDNAs encoding rCuc m 2, and immunodetection with anti-profilin antibodies. In vitro analysis included IgE immunodetection, specific IgE determination, ELISA-inhibition assays, and histamine release (HR) tests. In vivo activity of nCuc m 2 was established by skin prick testing (SPT). The effect of SGF and heat treatment on rCuc m 2 was followed by immunodetection, ELISA inhibition, and HR assays. RESULTS: Both purified forms of melon profilin were recognized by rabbit anti-profilin antibodies and IgE of sera from allergic patients, and showed molecular sizes typical of the profilin family. nCuc m 2 had a blocked N-terminus, whereas rCuc m 2 rendered the expected N-terminal amino acid sequence, its full protein sequence being highly similar (98--71% identity) to those of profilins from plant foods and pollens. The natural allergen displayed a slightly higher IgE-binding capacity than its recombinant counterpart. Specific IgE to nCuc m 2 and rCuc m 2 was found in 100% and 78% of the 23 individual sera analysed, respectively. nCuc m 2 evoked positive SPT responses in all (10/10) patients tested, and rCuc m 2 induced HR in two out of three sera assayed. SGF treatment readily inactivated rCuc m 2, as shown by its loss of recognition by anti-profilin antibodies, lack of IgE binding, and inability to induce HR. In contrast, heat treatment did not affect the IgE-binding capacity of rCuc m 2. CONCLUSIONS: Profilin is highly prevalent in melon-allergic patients, and promptly inactivated by SGF, as expected for an allergen mainly linked to OAS.

Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen.

BACKGROUND: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables. METHODS: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays. RESULTS: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract. CONCLUSION: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.

Aneuploid analysis of low molecular weight gliadins from wheat.

The chromosomal location of genes affecting five components from the low molecular weight gliadin (LMWG) fraction from wheat endosperm has been investigated by aneuploid analysis. Genes controlling these proteins were assigned to chromosomes 4B, 7A and 7D. Chromosomes from homoeologous groups 1, 2 and 6, where genes controlling classical gliadins are located, are not involved in the control of LMWG.

A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests.

Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11,275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.

Subunits of tetrameric α-amylase inhibitors of Hordeum chilense are encoded by genes located in chromosomes 4H(ch) and 7H (ch.).

Three proteins (components 1, 2, and 4) of the non-prolamin, 70% ethanol soluble fraction from the endosperm of Hordeum chilense have been identified as putative subunits of the tetrameric inhibitors active against insect α-amylases. In experiments carried out with the synthetic alloploid Tritordeum (H. chilense x Triticum turgidum conv. durum), previously described proteins from T. turgidum, designated CM2, CM3 and CM 16, have been also identified as subunits of α-amylase inhibitors. Genes for components 1 and 4 of H. chilense have been located in chromosomes 4H(ch) and 7H(ch), based on the analysis of H. chilense-T.turgidum addition lines. Subunits of the inhibitors from wheat and from cultivated barley had been previously assigned to chromosomes of the same homoeology groups.

In vivo allergenic activities of eleven purified members of a major allergen family from wheat and barley flour.

Eleven purified members of the alpha-amylase/trypsin inhibitor family from wheat and barley that showed very different IgE-binding capacities when previously assayed in vitro, were used in double blind in vivo diagnostic tests to further evaluate their allergenic activity. These tests were carried out in 31 patients who showed allergic sensitization to wheat flour as verified by skin test, RAST and challenge test. The three members of the protein family with highest IgE binding in vitro (the glycosylated subunits of tetrameric alpha-amylase inhibitors CM16* from wheat and CMb* from barley, and the barley monomeric inhibitor BMAI-1) were found to be the strongest allergens as indicated by skin sensitivity in prick tests.

Wheat Inhibitors of Heterologous alpha-Amylases : Characterization of Major Components from the Monomeric Class.

The four major components of the wheat monomeric alpha-amylase inhibitors (WMAI) from wheat, Triticum aestivum, endosperm have been isolated and characterized. Two of them, WMAI-1 and WMAI-2, are highly active against the alpha-amylase from the insect Tenebrio molitor and their N-terminal amino acid sequences indicate that they are closely related to each other (86% identical residues) and to the other members of the family (subunits of dimeric and tetrameric alpha-amylase inhibitors and trypsin inhibitors). WMAI-1, which is identical to the previously described 0.28 inhibitor, is encoded by a gene located in the short arm of chromosome 6D and WMAI-2 by a gene in the short arm of chromosome 6B. Components 3 and 4, which have blocked N-terminal residues, have identical internal amino acid sequences and are a separate class of proteins with respect to WMAI-1 and WMAI-2, although their amino acid composition and apparent molecular weights are quite similar. Their inhibitory activity versus alpha-amylases is either unstable during the purification process or due to contamination with other inhibitors.

What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?

BACKGROUND: Class I chitinases are the major panallergens in fruits associated with the latex-fruit syndrome. These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain. The role of these domains in their allergenic capacity is still controversial. OBJECTIVE: We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model. METHODS: Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector. Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography. The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection. Immunoblot, RAST and CAP inhibition assays were also performed. RESULTS: Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies. In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex-fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays. Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5. RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70-90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results. CONCLUSIONS: Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro. The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments. RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens.