Mapping of fruit allergens by 2D electrophoresis and immunodetection

Proteomic analyses of fruits are confronted with a series of specific obstacles: a general low protein content in plant tissues, allergen extraction from highly complex matrices and protein determination in the presence of interfering compounds. Different methods are currently being introduced to achieve higher protein yields and a simultaneous removal of interfering substances, such as polyphenols and polysaccharides. However, no universal protocol suitable for protein purification from any given plant species is available. Protein profiling by 2DE-western blotting offers a powerful tool for the detection and characterization of known and novel plant allergens. Moreover, the detection of IgE-reactive proteins from fruits is improved by combining western blot and alternative visualization techniques. The recent developments in bioinformatics and databases facilitate the interpretation of profiling studies with regard to novel potential fruit allergens.     

Lipid transfer proteins from Rosaceae fruits share consensus epitopes responsible for their IgE-binding cross-reactivity

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.     

How reliable is the structural prediction of IgE-binding epitopes of allergens? The case study of plant lipid transfer proteins

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.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family

Pear is known as an allergenic food involved in the ‘oral allergy syndrome’ which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n=16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.     

Characterisation of Mal d 1-related genes in <Emphasis Type="Italic">Malus</Emphasis>

It has been suggested that there are at least 15 Mal d 1-related (PR10) genes in one genotype of apple (Malus×domestica Borkh.). We sequenced cDNA libraries of cultivar Royal Gala and identified 12 members of the Mal d 1 family, including the previously reported Mal d 1b and Mal d 1d, an allelic variant of the previously reported Mal d 1a. Eight Mal d 1 gene products were expressed in tree-ripened fruit, in either the cortex or the skin, and most of these were also expressed in leaves in response to challenge with Venturia inaequalis—a fungal disease of apple. Mal d 1 gene products were identified from a large number of different tissues. Degree of ripeness as measured by standard parameters was shown not to predict either the amount of protein able to bind to a specific monoclonal antibody 5H8, previously shown to bind to an allergenic epitope in Mal d 1b and a/d, or the amount of Mal d 1 mRNA present. Mal d 1d and Mal d 1b were the most highly expressed isoforms in Royal Gala, particularly in the skin of fruit, and these isoforms were also predominant in other cultivars and species of apple. Genotypes, however, differed in relative predominance of Mal d 1b and Mal d 1d. The predominantly expressed Mal d 1 genes in ripe apple fruit were translated in vivo into proteins and proteins binding to the antibody were found in all cultivars and species examined. New Mal d 1 proteins were identified that bound to the 5H8 antibody. At least two new subfamilies have been identified, and while some structural differences are predicted between groups of isoforms, the P-loop motif is identical in all except two isoforms. A role in intracellular signalling in plants is suggested and in vitro expression of the isoforms should help in assessing their relative roles in disease, allergic responses, senescence and nucleotide-, cytokinin- and brassinosteroid-binding.     

Identification of a villin-related tobacco protein as a novel cross-reactive plant allergen

In a paradigmatic approach we identified cross-reactive plant allergens for allergy diagnosis and treatment by screening of a tobacco leaf complementary DNA (cDNA) library with serum IgE from a polysensitized allergic patient. Two IgE-reactive cDNA clones were isolated which code for proteins with significant sequence similarity to the actin-binding protein, villin. Northern- and Western-blotting demonstrate expression of the villin-related allergens in pollen and somatic plant tissues. In addition, villin-related proteins were detected in several plant allergen sources (tree-, grass-, weed pollen, fruits, vegetables, nuts). A recombinant C-terminal fragment of the villin-related protein was expressed in Escherichia coli, purified and shown to react specifically with allergic patients IgE. After profilin, villin-related proteins represent another family of cytoskeletal proteins, which has been identified as cross-reactive plant allergens. They may be used for the diagnosis and treatment of patients suffering from multivalent plant allergies.     

植物源过敏蛋白

植物过敏原是最普遍的一类过敏原,也是与人类关系最密切的一类过敏原。本文简单介绍了植物中蛋白质类过敏原的种类、特性及低敏植物性食品开发等研究新进展。     

TaCOLD1 defines a new regulator of plant height in bread wheat

Plant height is among the most important agronomic traits that influence crop yield. However, in addition to the Rht‐1 alleles, the molecular basis of plant height in bread wheat remains largely unclear. Based on wheat gene expression profiling analysis, we identify a light‐regulated gene from bread wheat, designated as TaCOLD1, whose encoding protein is homologous to cold sensor COLD1 in rice. We show that TaCOLD1 protein is localized to the endoplasmic reticulum (ER) and plasma membrane. Phenotypic analyses show that overexpression of a mutated form of TaCOLD1 (M187K) in bread wheat cultivar Kenong199 (Rht‐B1b) background resulted in an obvious reduction in plant height. Further, we demonstrate that the hydrophilic loop of TaCOLD1 (residues 178–296) can interact with TaGα‐7A (the α subunit of heterotrimeric G protein) protein but not TaGα‐1B, and the mutation (M187K) in TaCOLD1 remarkably enhances its interaction with TaGα‐7A. Physical interaction analyses show that the C‐terminal region of TaGα‐7A, which is lacking in the TaGα‐1B protein, is necessary for its interaction with TaCOLD1. Intriguingly, the C‐terminal region of TaGα‐7A is also physically associated with the TaDEP1 protein (an atypical Gγ subunit). Significantly, we discover that TaCOLD1 and mTaCOLD1 (M187K) can interfere with the physical association between TaGα‐7A and TaDEP1. Together, this study reveals that TaCOLD1 acts as a novel regulator of plant height through interfering with the formation of heterotrimeric G protein complex in bread wheat and is a valuable target for the engineering of wheat plant architecture.     

Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns

Plants feature a particularly diverse population of short (s)RNAs, the central component of all RNA silencing pathways. Next generation sequencing techniques enable deeper insights into this complex and highly conserved mechanism and allow identification and quantification of sRNAs. We employed deep sequencing to monitor the sRNAome of developing tomato fruits covering the period between closed flowers and ripened fruits by profiling sRNAs at 10 time-points. It is known that microRNAs (miRNAs) play an important role in development but very little information is available about the majority of sRNAs that are not miRNAs. Here we show distinctive patterns of sRNA expression that often coincide with stages of the developmental process such as flowering, early and late fruit maturation. Moreover, thousands of non-miRNA sRNAs are differentially expressed during fruit development and ripening. Some of these differentially expressed sRNAs derived from transposons but many derive from protein coding genes or regions that show homology to protein coding genes, several of which are known to play a role in flower and fruit development. These findings raise the possibility of a regulative role of these sRNAs during fruit onset and maturation in a crop species. We also identified six new miRNAs and experimentally validated two target mRNAs. These two mRNAs are targeted by the same miRNA but do not belong to the same gene family, which is rare for plant miRNAs. Expression pattern and putative function of these targets indicate a possible role in glutamate accumulation, which contributes to establishing the taste of the fruit.     

Kiwellin, a Novel Protein from Kiwi Fruit. Purification, Biochemical Characterization and Identification as an Allergen*

Kiwellin is a novel protein of 28 kDa isolated from kiwi (Actinidia chinensis) fruit. It is one of the three most abundant proteins present in the edible part of this fruit. Kiwellin has been purified by ion exchange chromatography. Its N-terminal amino acid sequence revealed high identity with that previously reported for a 28 kDa protein described as one of the most important kiwi allergens. This observation prompted us to fully characterize this protein. The complete primary structure, elucidated by direct sequencing, indicated that kiwellin is a cysteine-rich protein. Serological tests and Western Blotting analysis showed that kiwellin is specifically recognized by IgE of patients allergic to kiwi fruit. *The protein sequence data reported in this paper will appear in the Swiss-Prot and TrEMBL knowledgebase under the accessionnumber P84527.     

Specific conformational epitope features of pathogenesis-related proteins mediating cross-reactivity between pollen and food allergens

Selected members of plant pathogenesis-related and seed storage proteins represent specific groups of proteins with potential characteristics of allergens. Efforts to understand the mechanism by which pathogenesis-related proteins mediate a broad cross-reactivity in pollen-plant food allergens are still limited. In this study, computational biology approach was used to reveal specific structural implications and conservation of different epitopes from members of Bet v 1 and nsLTP protein families mediating cross-reactivity between pollen and food (fruits, vegetables, legumes, and nut/seeds) allergens. A commonly shared epitope conservation was found among all pollen and food Bet v 1 and nsLTP protein families, respectively. However, other allergenic epitopes were also specifically detected in each family. The implication of these conserved epitopes in a broad cross-reactivity for allergy clinical trials is here discussed.     

Mango profilin: cloning,expression and cross-reactivity with birch pollen profilin Bet v 2

Mango can cause severe anaphylactic reactions. Profilin has been assumed partly responsible for the cross-reactivity between mango fruit and other allergens but has not been finally clarified. In this study, two isoforms of mango fruits profilin were amplified by RT-PCR and 3'RACE from total RNA. Each mango profilin cDNA includes an open reading frame coding for 131 amino acids. The deduced amino acid sequence of the corresponding protein show high identity with other allergenic profilins. Expression of the recombinant mango profilin was carried out in Escherichia coli BL21(DE3) using vector PET28a and the purification of the recombinant protein was performed via affinity chromatography with Ni+ coupled to sepharose. IgE reactivity of recombinant mango profilin was investigated by immunoblot and 8 of 18 mango-allergic patients tested presented specific IgE-antibodies to recombinant mango profilin. IgE-inhibition and ELISA inhibition experiments were performed to analyze mango profilin cross-reactivity with profilins from birch pollen and high cross-reactivities have been found.     

Biochemical aspects of the developing and ripening banana

The peel and pulp of the banana fruit and the pseudostem were examined for glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and aldolase activities and protein, phenolics, chlorophyll and starch. The peel-pulp ratio at various stages of fruit development on the plant and in detached fruits showing incipient ripening were used as an index of the physiological age of the fruit. The enzymes exhibited maximum activity at a stage corresponding to the initiation of the climacteric. GPT level at this stage was higher than that of GOT. An initial increase in the protein content was followed by a decline in both peel and pulp, the level reaching a minimum in climacteric fruits. Astringency, measured in terms of total phenolics, decreased with development; in mature fruits, peel contained 4–5 × as much phenolics as pulp. Chlorophyll in mature fruits was 10 × higher than in young fruits and decreased in ripe fruits. The onset of ripening was attended with a pronounced decrease in the starch. The various analyses were carried out also on the pseudostem removed from the plant soon after flower formation.     

High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2

Allergic reactions to peanuts are a serious health problem because of their high prevalence, associated with potential severity, and chronicity. One of the three major allergens in peanut, Ara h 2, is a member of the conglutin family of seed storage proteins. Ara h 2 shows high sequence homology to proteins of the 2S albumin family. Presently, only very few structural data from allergenic proteins of this family exist. For a detailed understanding of the molecular mechanisms of food-induced allergies and for the development of therapeutic strategies knowledge of the high-resolution three-dimensional structure of allergenic proteins is essential. We report a method for the efficient large-scale preparation of properly folded Ara h 2 for structural studies and report CD-spectroscopic data. In contrast to other allergenic 2S albumins, Ara h 2 exists as a single continuous polypeptide chain in peanut seeds, and thus heterologous expression in Escherichia coli was possible. Ara h 2 was expressed as Trx-His-tag fusion protein in E. coli Origami (DE3), a modified E. coli strain with oxidizing cytoplasm which allows the formation of disulfide bridges. It could be shown that recombinant Ara h 2, thus overexpressed and purified, and the allergen isolated from peanuts are identical as judged from immunoblotting, analytical HPLC, and circular dichroism spectra.     

Aerobiology, biodiversity and chemistry of plant trichomes in the tropics at Bodh Gaya, India – a biopollutant and the suspected human allergen

Aerobiology, diversity and chemistry of some air borne plant trichomes were studied at Magadh University campus, Bodh Gaya, India between September 1995 and August 1996. Thirty-three morphotypes of trichomes were tapped using a Gravity Air sampler. The different types of trichomes showed pronounced seasonality in their distribution pattern. The simple non-septate trichomes of grasses were predominant throughout the year. The other commonly observed trichomes were comparable to that of Chrysanthemum, Tridax, Solanum, Amaranthus and Martynia. Trichomes. The Shannon diversity index of trichomes fluctuated widely (1.063–2.867) during different months of the year. This suggests that seasonality had definite bearing on the composition of aerobiota. Relative humidity was found to play greater role in the case of a number of morphotypes (67%), diversity index (50%) and species richness (61%) but in the case of index of dominance (62%), rainfall played a greater role at P < 0.01 level. The trichomes of a few selected plant species, the pollen grains of which are reported allergens, were chemically analysed by collecting them in bulk. They were found to be fairly rich in their total protein (9.58 to 93.60 mg/g) and carbohydrate (10.0 to 20.20 mg/g) contents.     

Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3)

Non-specific lipid transfer proteins belonging to LTP1 family represent the most important allergens for non pollen-related allergies to Rosaceae fruits in the Mediterranean area. Peach LTP1 (Pru p 3) is a major allergen and is considered the prototypic allergenic LTP. On the contrary, pear allergy without pollinosis seems to be under-reported when compared to other Rosaceae fruits suggesting that the as-yet-uncharacterized pear LTP1 (Pyr c 3) has in vivo a low allergenicity. We report here on the identification of four cDNAs encoding for LTP1 in pear fruits. The two isoforms exhibiting amino acid sequences most similar to those of peach and apple homologues were obtained as recombinant proteins. Such isoforms exhibited CD spectra and lipid binding ability typical of LTP1 family. Moreover, pear LTP1 mRNA was mainly found in the peel, as previously shown for other Rosaceae fruits. By means of IgE ELISA assays a considerable immunoreactivity of these proteins to LTP-sensitive patient sera was detected, even though allergic reactions after ingestion of pear were not reported in the clinical history of the patients. Finally, the abundance of LTP1 in protein extracts from pear peel, in which LTP1 from Rosaceae fruits is mainly confined, was estimated to be much lower as compared to peach peel. Our data suggest that the two isoforms of pear LTP1 characterized in this study possess biochemical features and IgE-binding ability similar to allergenic LTPs. Their low concentrations in pear might be the cause of the low frequency of LTP-mediated pear allergy.     

Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients

Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.     

Plant-based heterologous expression of Mal d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein

Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA's open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein's eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N.benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens.     

Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7-A

The structure of a thaumatin-like protein from banana (Musa acuminata) fruit, an allergen with antifungal properties, was solved at 1.7-A-resolution, by X-ray crystallography. Though the banana protein exhibits a very similar overall fold as thaumatin it markedly differs from the sweet-tasting protein by the presence of a surface exposed electronegative cleft. Due to the presence of this electronegative cleft, the banana thaumatin-like protein (Ban-TLP) acquires a strong (local) electronegative character that eventually explains the observed antifungal activity. Our structural analysis also revealed the presence of conserved residues of exposed epitopic determinants that are presumably responsible for the allergenic properties of banana fruit towards susceptible individuals, and provided evidence that the Ban-TLP shares some structurally highly conserved IgE-binding epitopes with thaumatin-like proteins from fruits or pollen from other plants. In addition, some overlap was detected between the predicted IgE-binding epitopes of the Ban-TLP and IgE-binding epitopes previously identified in the mountain cedar Jun a 3 TLP aeroallergen. The presence of these common epitopes offers a molecular basis for the cross-reactivity between aeroallergens and fruit allergens.