The disulphide mapping,folding and characterisation of recombinant Ber e 1, an allergenic protein,and SFA8, two sulphur-rich 2S plant albumins

We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.     

Solution Structure,Copper Binding and Backbone Dynamics of Recombinant Ber e 1–The Major Allergen from Brazil Nut

Background

The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners.

Methodology/Principal Findings

The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using ¹⁵N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu²⁺ in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale.

Conclusions/Significance

The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu²⁺ ions. This Cu²⁺ binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu²⁺ can in turn affect the interaction that is essential to provoke an allergenic response.     

The major human structural IgE epitope of the Brazil nut allergen Ber e 1: a chimaeric and protein microarray approach

A protein microarray system containing different dilutions of 77 related and non-related proteins was used to show that IgE from subjects allergic to Brazil nut specifically recognise the seed 2S albumin protein (Ber e 1). Further, correctly folded chimaeric 2S albumin proteins containing structural epitope replacement were constructed and directed to the secretion pathway of the methylotropic yeast Pichia pastoris. Through the use of a chimaeric protein microarray system together with sera from a panel of 18 well-characterised Brazil nut allergic subjects, a structural IgE epitope of Ber e 1 was mapped to a helix-loop-helix region. The same structural region has been previously reported as the immunodominant region in related food allergens by different techniques. In conclusion, the combination of chimaeric proteins and protein microarrays will greatly facilitate the screening of a large number of individuals for a particular structural epitope and help to further our understanding of how proteins are recognised by the adaptive immune system.     

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.     

A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies [correction of interspecie] cross-reactivity

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.     

Characterization of potential allergens in fenugreek (Trigonella foenum-graecum) using patient sera and MS-based proteomic analysis

BackgroundFenugreek is a legume plant used as an ingredient of curry spice. Incidents of IgE-mediated food allergy to fenugreek have been reported. Coincidence with allergy to peanut, a major food allergen, seems to be common suggesting a rather high rate of cross-reactivity.ObjectiveCharacterization of fenugreek allergens using patient sera and mass spectrometry-based proteomic analysis.MethodsAllergenic fenugreek proteins were detected by immunoblotting, using sera from 13 patients with specific IgE to peanut and fenugreek. IgE-binding proteins were analyzed by peptide mass fingerprinting and peptide sequencing.ResultsA fenugreek protein quintet in the range from 50 kDa to 66 kDa showed high IgE-affinity, the protein at 50 kDa reaching the strongest signals in all patients. Proteomic analyses allowed the classification of several fenugreek proteins to a number of allergen families. Fenugreek 7S-vicilin and 11S-legumin were partly sequenced and revealed considerable homologies to peanut Ara h 1 and Ara h 3, respectively. The presence of a fenugreek 2S albumin and pathogenesis-related (PR-10) plant pollen protein was assumed by database searching results.ConclusionIn this study, individual fenugreek proteins were characterised for the first time. Observed homologies to major peanut allergens provide a molecular explanation for clinical cross-reactivity.     

Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.     

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.     

Brachypodium distachyon as a model for defining the allergen potential of non-prolamin proteins

Epitope databases and the protein sequences of published plant genomes are suitable to identify some of the proteins causing food allergies and sensitivities. Brachypodium distachyon, a diploid wild grass with a sequenced genome and low prolamin content, is the closest relative of the allergen cereals, such as wheat or barley. Using the Brachypodium genome sequence, a workflow has been developed to identify potentially harmful proteins which may cause either celiac disease or wheat allergy-related symptoms. Seed tissue-specific expression of the potential allergens has been determined, and intact epitopes following an in silico digestion with several endopeptidases have been identified. Molecular function of allergen proteins has been evaluated using Gene Ontology terms. Biologically overrepresented proteins and potentially allergen protein families have been identified.     

Expression of the major olive pollen allergen Ole e 10 in the yeast Pichia pastoris: evidence of post-translational modifications

Olive pollen allergy is a clinical disorder that affects around 20% of the population in Mediterranean areas. The major olive pollen allergen, Ole e 10, is involved in cross-reactivity phenomena and asthma induction in allergic patients, and, besides its clinical interest, Ole e 10 is the first member of a new family of plant proteins. Ole e 10-specific cDNA has been cloned in the plasmid pPICZalphaA and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has been purified in a two chromatographic-step procedure. N-Terminal sequencing, mass spectrometry, IgG, and IgE binding assays were employed to characterize the recombinant allergen. These analyses revealed that the product undergoes a proteolytic cleavage in the N-terminal end with the loss of the first six residues. Different strategies were used to solve this problem, such as changes in the fermentation conditions and the employment of protease-deficient yeast strains. Proteolytic cleavage was minimized and about 51% of rOle e 10 was obtained as a full-length protein. Moreover, a covalent modification was found in the N-terminal end of the full-length rOle e 10. Peptide mapping and mass spectrometry analyses pointed to the existence of a phosphorylation located in a serine residue of the N-terminal segment of rOle e 10 and it was confirmed after treatment of the sample with alkaline phosphatase. Finally, both full-length and truncated rOle e 10 retained most of the IgG- and IgE-binding capabilities of the natural protein isolated from the pollen.     

Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry

Snow crab (Chionoecetes opilio) proteins have been recognized as an important source of both food and occupational allergens. While snow crab causes a significant occupational allergy, only one novel allergen has recently been fully characterized. The muscle proteins from snow crab legs were profiled by SDS-PAGE. Several of these proteins were characterized using tandem mass spectrometry. Five proteins were identified; sarcoplasmic Ca-binding (20kDa), arginine kinase (40), troponin (23kDa) and α-actine (42kDa) and smooth endoplasmic reticulum Ca(2+)ATPase (113kDa). Immunoblotting using serum of sixteen allergic patients resulted in strong reactivity with the 40-kDa protein in seven patients (43%). This protein was purified by chromatography and subsequently de novo sequenced using matrix assisted laser desorption ionization and electrospray tandem mass spectrometry. We identified a second important allergen, arginine kinase, in snow crab, designated Chi o 3. Based on identity and homology analysis, using bioinformatics tools, a signature peptide was identified as a chemical surrogate for arginine kinase. The suitability of this signature peptide was tested for analytically representing the arginine kinase, by performing a multi-reaction monitoring tandem mass spectrometry approach on actual air filter samples collected from a simulated crab processing plant.     

Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea-polyacrylamide gel electrophoresis (AU-PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU-PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3-10. AU-PAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)-PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient's serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were alpha-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS-PAGE or AU-PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis.     

IgG Expression upon Oral Sensitization in Association with Maternal Exposure to Ovalbumin

The role of maternal allergen exposure in the allergenicity of the offspring remains controversial. Some studies have shown that maternal exposure is a risk factor for allergy in the offspring, whereas other studies have shown that maternal exposure induces immune tolerance and protects offspring from allergy disease. Therefore, we utilized maternal rat allergen exposure model to evaluate the offspring immune reactions to ovalbumin protein and to determine whether the Brown Norway (BN) rat model is a suitable animal model for studying the allergenicity of food proteins. For three generations, rats received an allergens or non-allergens by gavage during the pregnancy and lactation periods. After weaning, the offspring rats were used for oral sensitization experiment. In the sensitization experiment, the control rat, which had maternal exposure to phosphate-buffered saline (PBS), exhibited full response of IgG to oral exposure to OVA. The IgG level was significantly lower in F1 rats that were sensitized by maternal exposure to ovalbumin(OVA). Moreover, the lowest IgG level was found for the F3b sensitized by maternal rats exposed to OVA allergen for three continuous generations. Compared with maternal OVA exposure prior to postnatal sensitization, the sensitization via maternal PBS led to a higher serum level of OVA-specific IgG. However, the OVA-specific IgG levels for the two generations of maternal PBS exposure prior to postnatal sensitization was not higher than that for the one generation of maternal rats exposed to PBS prior to postnatal sensitization. Our studies demonstrate that maternal OVA exposure during the pregnancy and lactation can affect the results of oral sensitization studies using ovalbumin protein. BN rats must be bred in non-allergen conditions for at least one generation to avoid problems in rat models for studying the allergenicity of food proteins.     

Plant food allergens--structural and functional aspects of allergenicity

The three dominating plant food allergen groups belong to the prolamin and cupin superfamilies and to the family 10 of pathogenesis-related proteins. The prolamin superfamily comprises allergenic 2S albumins, nonspecific lipid transfer proteins and cereal alpha-amylase/trypsin inhibitors. These allergens have related structures and are stable to thermal processing and proteolysis. The cupin superfamily comprises the allergenic 7S and 11S globulin storage proteins from peanuts, soybean and tree nuts which are heat stable and can form immunogenicity enhancing aggregates. The Bet v 1 family of allergens includes tree pollinosis-associated food allergens with low stability which induce the symptoms of the oral allergy syndrome.     

Epitopic hexapeptide sequences from Baltic cod parvalbumin beta (allergen Gad c 1) are common in the universal proteome

The aim of this study was to analyze the distribution of hexapeptide fragments considered as epitopes of Baltic cod parvalbumin beta (allergen Gad c 1) in the universal proteome. Cod (Gadus morhua subsp. callarias) parvalbumin hexapeptides cataloged in the Immune Epitope Database were used as query sequences. The UniProt database was screened using the WU-BLAST 2 program. The distribution of hexapeptide fragments was investigated in various protein families, classified according to the presence of the appropriate domains, and in proteins of plant, animal and microbial species. Hexapeptides from cod parvalbumin were found in the proteins of plants and animals which are food sources, microorganisms with various applications in food technology and biotechnology, microorganisms which are human symbionts and commensals as well as human pathogens. In the last case possible coverage between epitopes from pathogens and allergens should be avoided during vaccine design.     

Lack of Detectable Allergenicity in Genetically Modified Maize Containing “Cry” Proteins as Compared to Native Maize Based on In Silico & In Vitro Analysis

BackgroundGenetically modified, (GM) crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release.ObjectiveTo compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize.MethodsAn integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE) and Immunoblot using food sensitized patients sera (n = 39) to non GM and GM maize antigens was performed.ResultsIn silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF.ConclusionCry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize.     

A novel dwarfing mutation in a green revolution gene from Brassica rapa

Mutations in the biosynthesis or signaling pathways of gibberellin (GA) can cause dwarfing phenotypes in plants, and the use of such mutations in plant breeding was a major factor in the success of the Green Revolution. DELLA proteins are GA signaling repressors whose functions are conserved in different plant species. Recent studies show that GA promotes stem growth by causing degradation of DELLA proteins via the ubiquitin-proteasome pathway. The most widely utilized dwarfing alleles in wheat (Triticum aestivum; e.g. Rht-B1b and Rht-D1b) encode GA-resistant forms of a DELLA protein that function as dominant and constitutively active repressors of stem growth. All of the previously identified dominant DELLA repressors from several plant species contain N-terminal mutations. Here we report on a novel dwarf mutant from Brassica rapa (Brrga1-d) that is caused by substitution of a conserved amino acid in the C-terminal domain of a DELLA protein. Brrga1-d, like N-terminal DELLA mutants, retains its repressor function and accumulates to high levels, even in the presence of GA. However, unlike wild-type and N-terminal DELLA mutants, Brrga1-d does not interact with a protein component required for degradation, suggesting that the mutated amino acid causes dwarfism by preventing an interaction needed for its degradation. This novel mutation confers nondeleterious dwarf phenotypes when transferred to Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica napus), indicating its potential usefulness in other crop species.