|High-throughput NMR assessment of the tertiary structure of food allergens.|
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|PMID: 22768312 Owner: NLM Status: MEDLINE|
|BACKGROUND: In vitro component-resolved diagnosis of food allergy requires purified allergens that have to meet high standards of quality. These include the authentication of their conformation, which is relevant for the recognition by specific IgE antibodies from allergic patients. Therefore, highly sensitive and reliable screening methods for the analysis of proteins/allergens are required to assess their structural integrity. In the present study one-dimensional 1H Nuclear Magnetic Resonance (1D 1H-NMR) analysis was adopted for the assessment of overall structural and dynamic properties and authentication of a set of relevant food allergens, including non-specific lipid transfer proteins from apple, peach and hazelnut, 7/8S seed storage globulins from hazelnut and peanut, 11S seed storage globulins from hazelnut and peanut, caseins from cows' and goats' milk and tropomyosin from shrimp.
METHODOLOGY/PRINCIPAL FINDINGS: Two sets of 1D 1H-NMR experiments, using 700 MHz and 600 MHz instruments at 298 K were carried out to determine the presence and the extent of tertiary structure. Structural similarity among members of the individual allergen families was also assessed and changes under thermal stress investigated. The nuclear magnetic resonance (NMR) results were compared with structural information available either from the literature, Protein Data Bank entries, or derived from molecular models.
CONCLUSIONS/SIGNIFICANCE: 1D (1)H-NMR analysis of food allergens allowed their classification into molecules with rigid, extended and ordered tertiary structures, molecules without a rigid tertiary structure and molecules which displayed both features. Differences in thermal stability were also detected. In summary, 1D (1)H-NMR gives insights into molecular fold of proteins and offers an independent method for assessing structural properties of proteins.
|Stefano Alessandri; Ana Sancho; Stefan Vieths; Clare E N Mills; Jean-Michel Wal; Peter R Shewry; Neil Rigby; Karin Hoffmann-Sommergruber|
|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2012-07-02|
|Title: PloS one Volume: 7 ISSN: 1932-6203 ISO Abbreviation: PLoS ONE Publication Date: 2012|
|Created Date: 2012-07-06 Completed Date: 2012-11-13 Revised Date: 2013-07-12|
Medline Journal Info:
|Nlm Unique ID: 101285081 Medline TA: PLoS One Country: United States|
|Languages: eng Pagination: e39785 Citation Subset: IM|
|CERM, Centro di Ricerca di Risonanze Magnetiche and Department of Agricultural Biotechnology, University of Florence, Florence, Italy.|
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Antigens, Plant / chemistry*, classification, immunology
Carrier Proteins / chemistry*, classification, immunology
Corylus / chemistry*, immunology
Food Analysis / methods
Food Hypersensitivity / immunology
Magnetic Resonance Spectroscopy
Malus / chemistry*, immunology
Plant Proteins / chemistry*, classification, immunology
Protein Structure, Tertiary
Prunus / chemistry*, immunology
|//Biotechnology and Biological Sciences Research Council|
|0/Allergens; 0/Antigens, Plant; 0/Carrier Proteins; 0/Plant Proteins; 0/lipid transfer protein|
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Publisher: Public Library of Science, San Francisco, USA
Alessandri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received Day: 27 Month: 2 Year: 2012
Accepted Day: 30 Month: 5 Year: 2012
collection publication date: Year: 2012
Electronic publication date: Day: 2 Month: 7 Year: 2012
Volume: 7 Issue: 7
E-location ID: e39785
PubMed Id: 22768312
Publisher Id: PONE-D-12-05896
|High-Throughput NMR Assessment of the Tertiary Structure of Food Allergens Alternate Title:NMR Assessment of Food Allergens|
|Clare E. N. Mills2¤b|
|Peter R. Shewry5|
|Vladimir N. Uverskyedit1||
1CERM, Centro di Ricerca di Risonanze Magnetiche and Department of Agricultural Biotechnology, University of Florence, Florence, Italy
2Institute of Food Research, Norwich, United Kingdom
3Paul-Ehrlich-Institut, Langen, Germany
4INRA, UR496 Immuno-Allergie Alimentaire, CEA/iBiTeC-S/SPI, CEA de Saclay, Gif sur Yvette, France
5Rothamsted Research, Harpenden, United Kingdom
6Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria
|University of South Florida College of Medicine, United States of America
|Correspondence: * E-mail: Karin.email@example.com
Contributed by footnote: Conceived and designed the experiments: SA KHS. Performed the experiments: SA. Analyzed the data: SA KHS. Contributed reagents/materials/analysis tools: SA AS SV CM JMW PS NR KHS. Wrote the paper: SA KHS.
¤aCurrent address: Department of Molecular Biology, University of Salzburg, Salzburg, Austria
¤bCurrent address: School of Translational Medicine, Manchester Academic Health Science Centre, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom
IgE-mediated food allergies are caused by non-toxic dietary proteins of animal or plant origin. Once sensitized, predisposed individuals develop allergic symptoms upon intake. Clinical manifestations range from mild, local reactions to generalised severe, even life-threatening conditions. However, only a restricted number of proteins are capable of inducing allergic sensitization and elicitation .
Conventional in vitro allergy diagnosis is based on the detection of specific IgE antibodies directed against allergens present in total extracts of food materials. In the recent past the concept of component-resolved diagnosis, in which total extracts are replaced by panels of single allergens for each food, has been introduced aiming at improved sensitivity, reproducibility and specificity of the assay –. However, the quality of the test performance depends on the quality of the analytical tools used. Therefore, the concept of an allergen library was developed within the EuroPrevall project  which included purified members of the allergen families known as non-specific lipid transfer proteins (nsLTPs), 7S seed storage globulins, 11S seed storage globulins, caseins from cows' and goats' milk and tropomyosin from shrimp. State of the art methods for the purification of allergenic food proteins were collated and a catalogue of quality criteria was agreed, including the evaluation of analytical methods for their authentication .
IgE antibodies produced by allergic individuals to a food allergen display different specificities and may be directed against linear and conformational epitopes. Particularly for the conformational, discontinuous lgE epitopes a 3-dimensional structure of the purified allergen closely resembling that of the protein in its natural environment is mandatory for reliable in vitro diagnosis. Therefore, highly efficient methods to assess the structural integrity of these proteins are required. It is particularly important to confirm that individual batches of purified proteins resemble the naturally occurring protein in their structures and properties, displaying either rigid, intermediate or flexible tertiary structures the latter being important for allergens that may exist in partially-unfolded states induced by thermal processing , .
Nuclear Magnetic Resonance (NMR) spectroscopy is one of the main techniques used to study the structural properties of native and processed proteins and is capable of atomic resolution in aqueous solutions. i.e. under conditions similar to the physiological state. It provides information on the positions, bonds and movements of specific atoms with nuclei which have a spins different from zero, primarily 1H, and on their environment. Such information can be used for the characterization of a protein in terms of structural, chemical and dynamic properties , . However, 3D-structure determination may require isotopically-labelled protein to allow such highly resolved structures to be determined. In addition, the instrumental and human resources required for this type of NMR experiment, and the concentrations of proteins that are required, also make it impossible to currently define them as “high-throughput”. An alternative is to use 1D NMR spectra as an initial screening tool, to differentiate between protein chains with rigid and ordered tertiary structures, from those that are randomly coiled and those proteins that are more mobile, disordered or flexible. 1D 1H-NMR spectra already contain enough information to evaluate whether a protein has some tertiary structure, although the signals are not assigned and the determination of 1D 1H-NMR spectra is much less time consuming than other kind of NMR experiment. Close inspection of one-dimensional 1H spectra also provides information about the extent of tertiary structure in partially-structured proteins, about mixtures where only one fraction is structured or about mixture of conformers like molten globules . Aggregation can be also detected by observing the line width of the signals because the width of the signals in a solution NMR spectrum is correlated with the size of the molecule. Such NMR screening is considered a useful tool for identifying targets which have a high probability of producing crystals of sufficient quality for x-ray diffraction studies or are amenable to structural analysis by NMR. 1D 1H-NMR screening allows structural genomics and proteomics studies to be focused on the most promising targets and, therefore, significantly reduces the cost of production of novel 3D protein structures , .
Several examples of the range of structural types that are represented by allergens were chosen, from totally unstructured molecules (caseins), to proteins belonging to families characterized by a rigid and ordered tertiary structure (non specific lipid transfer proteins, nsLTP), representing the most important plant food allergens families , . These included the nsLTPs as representatives of the prolamin superfamily and are identified as allergens in fruits, vegetables and nuts. In Rosaceae fruits they are major allergens accounting for severe allergic reactions in patients. Non specific lipid transfer proteins are stabilised by four disulfide bridges and typically resistant to thermal and enzymatic treatment . The structure of Pru p 3, the major allergen from peach, was determined by X-ray crystallography  (PDB entries 2alg and 2b5s). Another dominant plant food allergen family are the cupins, to which seed storage proteins belong. These can be divided into two groups based on their sedimentation coefficients. These include the 7S vicilin-type globulins which are typically trimeric proteins  and the 11S legumin-type globulins, after post-translational processing, typically consist of six subunit pairs that interact noncovalently. Allergens from this protein family are identified from legumes, nuts and seeds. The 11S or 7S globulins can form both trimeric and hexameric structures: reversible aggregation into hexamers is possible, for 7S globulins, depending on the ionic strength and 11S globulins are assembled via a trimeric intermediate .
Representatives of the animal food allergen families were tropomyosins and caseins. Tropomyosins are known as major allergens in seafood and minor inhalant allergens in cockroaches and mites. Typically non vertebrate tropomyosin has a molecular mass of 33 kDa and is a homodimer of two parallel alpha helical molecules with a flexible coiled-coil structure each  that allows the tropomyosin to assemble around the actin filaments .
Caseins are typically unfolded proteins and account for the majority in the protein fraction of mammalian milk. Cow's milk casein is known as a major food allergen especially in young children. Whole caseins are mixtures of four different proteins: alpha-S1-casein (199 AA, 23.6 kDa), alpha-S2-casein (207 AA 25.2 kDa), beta-casein (209 AA, 24 kDa), k-casein (169 AA, 19 kDa; ). Bovine caseins consist of flexible disordered and mobile loops, with only few residues included in short portions that could be organised at the secondary level.
The aim of the current study was to apply 1D 1H-NMR spectroscopy to structural analysis of a range of allergens from major allergen families and to assess its potential as an effective high throughput method. The structural characteristics of the allergens within a protein family and reversible and irreversible structural transitions due to temperature changes were also investigated.
Non-specific lipid transfer proteins were purified from apple (Malus domestica, Mal d 3), peach (Prunus persica, Pru p 3) and hazelnut (Corylus avellana, Cor a 8) as described previously –. 7S and 11S seed storage globulins were purified from hazelnut (Corylus avellana, Cor a 11, Cor a 9) and peanut (Arachis hypogaea, Ara h 1, Ara h 3) as described previously , . Caseins were purified from cows' (Bos domesticus, Bos d 8) and goats' milk (Whole caprine casein, WCC ). Tropomyosin, the major allergen from brown shrimp (Penaeus aztecus, Pen a 1) was produced as a recombinant protein and purified accordingly . The natural and recombinant allergens were tested for their IgE-binding activity using allergic patients sera as previously described and their primary and secondary structures verified by N-terminal sequencing, MALDI-TOF-MS analysis and far-UV CD-spectroscopy . An overview of the allergens used in this study, their acronyms and physicochemical parameters together with the experimental conditions of the NMR analyses are summarised in Table 1.
Allergen solutions were prepared in H2O/D2O 9∶1, (0.50 ml, with concentration ranging from 0.39 mM to 0.03 mM) in aqueous buffer systems (pH ranging from 7.0 to 7.8) as detailed in the legends for figures. The solutions were transferred into high-quality NMR tube with argon as headspace gas. Before and after the NMR experiments, the allergens were stored at 253 K (−20°C). Two High Resolution NMR experiments were carried out, using an Avance 700 spectrometer by Bruker, operating at a proton resonance frequency of 700 MHz (11.7 Tesla). The two experiments differed in the method of managing the water signal. The pulse program of the first experiment included a 1D sequence with pre-saturation to minimize the water signal . The second experiment is based on gradient-based water suppression by 1D excitation sculpting using 180° water-selective pulses . It increases the overall sensitivity and decreases the distortion of the baseline, but some signal near the (suppressed) water peak can be suppressed. This can be checked by the pre-saturation experiment.
For each sample and for each experiment 256 scans at 298 K (25°C) were routinely programmed, due to the low sample concentration. More scans were required for proteins with higher molecular mass and more diluted protein solutions (Mal d 3, Ara h 3/4, Cor a 9, Ara h 1, Cor a 11) as specified in the legends of the corresponding figures, where variations in the number of scans and of the field strength are reported, with the molecular weights and the molarities of the samples and the buffer conditions. The spectra were initially evaluated considering the dispersion of the signals in the regions of the methyl protons (−0.5–1.5 ppm), alpha-protons (3.5–6.0 ppm), and amide protons (6.0–10.0 ppm).
The consistency of the allergen spectra with information available in the scientific literature and with structures either deposited into the Protein Data Bank (PDB) or supported by a publication, was routinely checked. If no experimental structure was available, the comparison with a modelled structure was performed – and its consistency with the NMR spectra checked.
The stability of the nsLTPs (Mal d 3, Pru p 3 and Cor a 8) to thermal stress was studied using a 600 MHz spectrometer equipped with a probe suited to work at high temperature. A series of experiments was performed consisting of stepwise heating of each sample from 298 K (25°C) to a maximum 358 K (85°C), by 5 K intervals. After each step an NMR water pre-saturation (zgpr) experiment was launched and the spectrum evaluated. If significant structural changes were detectable, the sample was cooled to 298 K and scanned to verify whether the detected structural change was reversible. If the maximum temperature allowed by the probe had not been already reached, the sample was heated to the next threshold.
A minimum of 128 scans was programmed for each NMR experiment, extended to 512 for the most diluted samples (Mal d 3). A minimum of 20 minutes was required to reach the thermal equilibrium after each 5 K heating step. A minimum of 90 minutes was required after each cooling step and after each following heating step. All the experiments were conducted under near-neutral pH condition. The thermal stability of Pru p 3 was also studied in acidic solution (adjusting to pH 3 by addition of HCl).The pH was checked before and after the experiments.
The modelled structures were obtained using the fully automated protein structure homology-modelling server SWISS-MODEL , ,  and were selected based on the protein family of the template, the sequence identity between the target and the template, the modelled residue range of the target, the QMEAN Z-Score and the QMEAN4 global score of the model , –. Models of low quality are expected to have strongly negative QMEAN Z-scores. The QMEAN describes the likelihood that a given model is of comparable quality to experimental structures. The QMEAN4 ranges between 0 and 1 with higher values indicating better models among alternative models of the same target.
Since large chemical shift dispersions, especially in the methyl group region between 1.0 ppm and −1.0 ppm and in the amide region down-field of 8.5 ppm can be used as a reliable indicator for protein folding ,  this approach was taken to assessing the structural attributes of the selected allergens. Few broad peaks around 8.3 ppm are a good indicator of a protein without an ordered tertiary structure, because this is a region characteristic of backbone amides in random-coil configuration. By contrast, several peaks, possibly narrow and resolved, dispersed beyond 8.5 ppm (8.5–11 ppm) are the signature of a rigid and ordered tertiary structure. However, the signals of the amide protons can be broadened due to chemical exchange especially at non-acidic pH. This can be a drawback at protein concentrations close to the detection limit when amide signals are to be observed. Isolated methyl group signals upfield of 0.5 ppm can be more sensitive and reliable probes for folded protein as they are not affected by chemical exchange broadening. In addition, their signal intensities are higher because three equivalent protons contribute rather than one proton of the amide group. Therefore, a wide signal dispersion in the aliphatic region of the spectrum between 1.0 and −1.0 ppm, versus a steep flank of the dominant peak at approximately 1 ppm reliably discriminates rigid and ordered tertiary structures from random-coil or flexible structures. This is because the loss of high field methyl peaks indicates a reduction in tertiary side chain organization, which can be considered complete when the NMR spectrum is characterized by few, rather narrow, but poorly dispersed peaks that are indicative of extremely mobile residues. This condition can be usually achieved as a result of protein unfolding in strongly acidic condition usually in the presence of a high concentration of urea .
A thermodynamically distinct state of proteins showing properties of both native and denatured proteins is “molten globule” . Molten globules are considered as intermediates on the protein folding/unfolding process but in some cases, molten globules exist as stable conformers, with rigid but different tertiary structure . The NMR spectrum of a molten globule protein is characterized by few, extremely broad peaks, especially for most of the backbone amide groups.
Whilst spectral signals from certain allergens (Ara h 1, Cor a 9, Cor a 11, Mal d 3) were weak and spectra were dominated by buffer signals this did not affect the readability of the critical spectral regions.
All the spectra were consistent with the corresponding structures deposited in the PDB, or with structures that could be modelled starting from the sequence and anticipated on the basis of the proteins family membership. Analysis of the 1D 1H-NMR spectra then allowed the classification of the allergens under study as follows.
The NMR spectra of these nsLTPs (Fig. 1 and Fig. 2) show the unmistakable features of rigid and extended tertiary structures which is not affected by low pH environment and is in line with the 2D studies performed with Pru p 3 by Wijeshina-Bettoni et al . There are many narrow and resolved peaks throughout the whole spectral window, notably in the methyl and amide-aromatic regions. The spectra of these allergens are very similar: for example, the extreme methyl region between 1 and 0 ppm and the aromatic region between 6 and 8 ppm. This fact gives experimental support to the similarity of the modelled structures of Mal d 3 and Cor a 8, and the experimental structure of Pru p 3 (Fig. 3), as expected from the high degree of sequence homology and conserved cysteine residues forming disulfide bridges. Thus Mal d 3 (Uni Prot entry: Q9M5X7), and Cor a 8 (Uni Prot entry: Q9ATH2), share 81.3% and 59% sequence identity with Pru p 3 (Uni Prot entry: P81402), respectively. The overall intact 3D structure is relevant for IgE-binding activity of Pru p 3 and is reduced when alkylation and reduction of the protein was performed . Therefore, Pru p 3 was subjected to stepwise heat treatment to investigate whether heating processes have an impact on its overall structure.
During thermal treatment the spectra gradually change towards the denatured state. Under acidic conditions (Fig. 4, pH 3) the increasing loss of tertiary structure of Pru p 3 is apparent from the decreasing dispersion of peaks either in the methyl region (around 0 ppm) or in the amide-aromatic region (6–10 ppm). Irreversible denaturation is not detectable until 358 K (85°C) and the 298 K (25°C) spectra, scanned before and after heating, overlap perfectly. These data are in good agreement with the studies on heat resistance of barley LPT from Perrocheau et al. . Under neutral conditions (Fig. 4, pH 7) two major differences can be observed during thermal denaturation of natural Pru p 3. The former is an irreversible structural transition occurring between 353 K (80°C) and 358 K (85°C): the final room temperature spectrum shows only a few broad and poorly dispersed peaks in the amide-aromatic and in the methyl region and the absence of resonances upfield and downfield. The latter represents the disappearance of amide resonances (7–9 ppm) as the temperature rises. This is due to proton exchange with the solvent, and depends on pH, temperature and accessibility of exchangeable protons, which increases with unfolding, and can be due to the transition to a molten-globule state. Native Mal d 3 shares the same behaviour as Pru p 3 when heated in neutral conditions (data not shown). Heating was stopped at 348 K (75°C) for Cor a 8 due to instrumental problems. The protein refolds to the 25°C tertiary structure after the heat-induced unfolding at the maximum tested temperature (data not shown).
The higher molecular mass of globulins as compared to nsLTPs and their propensity to aggregate into more complex quaternary structures seriously limits the resolution of their NMR spectra (Fig. 5). This could be a particular problem for Cor a 9, due to the possible presence of the hexameric form. A series of signals, though basically unresolved, can be observed in the extreme methyl region of all the spectra (around 0.5 ppm) indicating regions of rigid tertiary structure. On the other hand, the presence of extended mobile regions must be considered, due to the few broad peaks that can be observed in the whole spectral window, notably in the HN region between 6 and 8 ppm. By contrast, the same region of the Cor a 11 spectrum is populated by several better resolved and narrow resonances that give a clear indication of rigid and ordered regions. Both, Cor a 9 and Cor a 11 show a noticeable signal broadening in the backbone NH region between 8.0 and 8.7 ppm, that could indicate the presence of a molten globule state. Narrow, weak signals are observed against a background of broad peaks in the Ara h 3/4 spectrum which could be due to the presence of both lighter monomers and more massive aggregations in the mixture (Fig. 5).
Up to now two sequences of 7/8S globulin Ara h 1, from peanut (63.5 kDa) (UniProt entries P43237 and P43238) were characterised . Very recently, the structure from the Ara h 1 core region without the N-terminal extension and the C-terminal flexible region was determined . Both isoforms could be modelled using the 7S globulin from adzuki bean  as a template (PDB accession 2ea7), with 48% of sequence identity (Fig. 6). The template and the modelled monomer can be divided into two similar modules (N- and C-terminal), each showing an extended loop domain. The models could be verified by the crystal based analysis of the Ara h 1 core fragment , . Already identified IgE epitopes, were mapped on the models and it became evident that some of these epitopes were only surface exposed when the trimer formation was disrupted .
A partially resolved structure (PDB entry 3c3v) has been reported as Ara h 3 (now designated: Ara h 3.0101; Swiss prot Entry: O82580) . It consists of an N-terminal domain and a C-terminal domain. Three regions of the N-terminal domain and the tail of C-terminal domain are disordered (Fig. 7). Using this structure as a template a model was built of the isoform reported as Ara h 4 (now designated: Ara h 3.0201; Swiss Prot entry: Q9SQH7) . The model which shares 75.5% sequence identity with “Ara h 3” (Fig. 7) was calculated including the unresolved regions of the experimental structure, and is consistent with the presence of extended, partially structured, mobile regions.
IgE epitopes were identified on the surface of Ara h 3, some of them specific for the peanut allergen and some of them shared with other legumin type allergens highlighting the molecular basis for cross-reactivity .
No experimental structure of hazelnut globulins is known to date. Again a homology model was built (Fig. 6) using the Cor a 11 sequence  starting from its 48 kDa precursor sequence (Swiss Prot entry: Q8S4P9), with 32.5% of identity with the template PDB 2ea7, chain A, corresponding to the adzuki bean 7S globulin . The model is trimeric, with each monomer formed by two very similar modules and each module containing an extended loop domain. Similarly a model was built using the Cor a 9 sequence (GenBank: AAL73404.1) which has 51.8% sequence identity to the almond globulin ,  (PDB 3fz3) (Fig. 8). The structure of this template, and of the derived model, shows similarities with the structure of the 11S globulin Ara h 3/4 (Ara h 3.0101 and Ara h 3.0201; Fig. 7 and Fig. 8).
Narrow, weak signals in are observed against a background of broad peaks in the Ara 3/4 spectrum which could be due to the presence of both lighter monomers and more massive aggregations in the mixture (Fig. 5).
The spectra of all of the globulins provide experimental support for their modelled structures and are consistent with our knowledge of the structural characteristics of this family of proteins including their tendency towards aggregation, the ubiquity of isoforms, and the presence of extended mobile regions in their tertiary structure.
The NMR spectra of the sample of Pen a 1 under study and the spectra of caseins are very similar. These spectra (Fig. 1) show the typical characteristics of random coil structures with extended regions of the spectral window being devoid of signals and a few broad peaks at characteristic intervals. The dominant methyl peak at approximately 1 ppm is a distinctive signature of a random-coil or extremely flexible structure. Single narrow signals can be observed on the envelope of broad peaks which are not resolved to the baseline because they are confined in narrow spectral ranges, confirming the high mobility of the residues of these samples. A model was constructed of Pen a 1 (Accession No. DQ151457.1)  based on the structure of pig cardiac muscle tropomyosin  (PDB 1c1g, chain A) with which it shares 56.2% sequence identity (Fig. 9). This has a coiled-coil structure which can undergo a transition from a coiled coil to molten-like state before complete dissociation of the two chains, chains that are flexible and can be unfolded at physiological temperature . This could explain the observation that the NMR spectrum is characteristic of a mobile, unfolded structure. Detailed studies on the epitope mapping of this major allergen identified linear IgE epitopes with increased antibody binding activity when grafted onto non allergenic tropomyosin .
The spectra of the caseins were similarly characterised by a lack of signals between 6.2 and 6.6 ppm and 0.0 and 0.7 ppm, respectively (Fig. 1) indicative of their highly mobile structure. The fact that unfractionated caseins are mixtures of four different molecules is not apparent because alpha-s1-casein, beta-casein, alpha-s2-casein and k-casein , all share similar molecular weight and lack secondary and tertiary structure, and therefore their residues share very similar structural environment as confirmed also by the great similarity of all these spectra.
On the basis of the NMR analysis the proteins could be classified into three groups. The first group included those molecules whose spectra showed the unquestionable signature of their tertiary structure: nsLTPs (Fig. 1 and Fig. 2) and the hazelnut 7/8S globulin, Cor a 11. However, the NMR data indicate that the latter may contain disordered stretches (Fig. 5) as has been observed for seed storage globulins from other plant species such as soybean .
The second group included those allergens whose spectra clearly showed no fingerprints for tertiary structure (Fig. 1). These were whole caseins from cows' and goats' milk (Bos d 8, WCC) and tropomyosin from brown shrimp (Pen a 1). The spectra for caseins are consistent with other observations of protein mobility which has led to them being termed rheomorphic .
The third group included allergens whose spectra revealed an intermediate structure: the 11S globulin Cor a 9 and the 7/8S globulin Ara h 1. Their spectra (Fig. 5) indicated that the protein could be structured with well organized rigid regions with an ordered tertiary structure, interspersed with disordered regions (as indicated by temperature factors in the x-ray structures of certain seed storage globulins and the fact certain regions had not been resolved and appear to be highly mobile even in a crystal lattice . Alternatively these data indicate the sample could be a mixture of folded and unfolded or differently folded proteins. The spectrum of the 11 S globulin Ara h 3/4 (Fig. 5) also indicated the presence of both disordered and ordered parts.
It is worth noting that some of the allergens investigated displayed molecular masses, (e.g. 60 kDa without considering possible aggregations) that can be considered beyond the limitations of the method and the instruments adopted.
High quality allergen batches with defined physicochemical features are essential for reliable and sensitive in vitro diagnosis for allergy. The 3-dimensional structures of the most important allergen families have important impacts on their IgE binding activity. It has become evident that food processing can affect the structure and thus allergenicity. Therefore, methods to assess the structural integrity of purified allergens are urgently needed. The present study has shown that 1D NMR can be used to generate a fingerprint containing the overall structural and dynamic information of allergenic proteins in a high throughput manner.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was funded by the EU through the EuroPrevall project (FOOD-CT-2005-514000). Rothamsted Research and the Institute of Food Research are funded by the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. KHS is supported by the Grant SFB-F46-B19 from the Austrian (FWF) Science Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
|1.||Hoffmann-Sommergruber K,Mills EN. Year: 2009Food allergen protein families and their structural characteristics and application in component-resolved diagnosis: new data from the EuroPrevall project.Anal Bioanal Chem395253519639305|
|2.||Nicolaou N,Poorafshar M,Murray C,Simpson A,Winell H,et al. Year: 2010Allergy or tolerance in children sensitized to peanut: prevalence and differentiation using component-resolved diagnostics.J Allergy Clin Immunol12519119720109746|
|3.||Hansen KS,Ballmer-Weber BK,Sastre J,Lidholm J,Andersson K,et al. Year: 2009Component-resolved in vitro diagnosis of hazelnut allergy in Europe.J Allergy Clin Immunol1231134114119344939|
|4.||Bublin M,Pfister M,Radauer C,Oberhuber C,Bulley S,et al. Year: 2011Component-resolved diagnosis of kiwifruit allergy with purified natural and recombinant kiwifruit allergens.J Allergy Clin Immunol12568769420061012|
|5.||Hoffmann-Sommergruber K,Mills EN,Vieths S. Year: 2008Coordinated and standardized production, purification and characterization of natural and recombinant food allergens to establish a food allergen library.Mol Nutr Food Res52Suppl 2S15916519031434|
|6.||Sancho AI,Hoffmann-Sommergruber K,Alessandri S,Conti A,Giuffrida MG,et al. Year: 2010Authentication of food allergen quality by physicochemical and immunological methods.Clin Exp Allergy4097398620642576|
|7.||Johnson P,Philo M,Watson A,Mills EN. Year: 2011Rapid Fingerprinting of Milk Thermal Processing History by Intact Protein Mass Spectrometry with Nondenaturing Chromatography.J Agric Food Chem59(23)12420722007861|
|8.||Vissers YM,Iwan M,Adel-Patient K,Stahl Skov P,Rigby NM,et al. Year: 2011Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays.Clin Exp Allergy411631164221801247|
|9.||Rosner HI,Redfield C. Year: 2009The human alpha-lactalbumin molten globule: comparison of structural preferences at pH 2 and pH 7.J Mol Biol39435136219766656|
|10.||Rehm T,Huber R,Holak TA. Year: 2002Application of NMR in structural proteomics: screening for proteins amenable to structural analysis.Structure101613161812467568|
|11.||Page R,Peti W,Wilson IA,Stevens RC,Wuthrich K. Year: 2005NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline.Proc Natl Acad Sci U S A1021901190515677718|
|12.||Jenkins JA,Griffiths-Jones S,Shewry PR,Breiteneder H,Mills EN. Year: 2005Structural relatedness of plant food allergens with specific reference to cross-reactive allergens: an in silico analysis.J Allergy Clin Immunol11516317015637564|
|13.||Jenkins JA,Breiteneder H,Mills EN. Year: 2007Evolutionary distance from human homologs reflects allergenicity of animal food proteins.J Allergy Clin Immunol1201399140517935767|
|14.||Wijesinha-Bettoni R,Alexeev Y,Johnson P,Marsh J,Sancho AI,et al. Year: 2010The structural characteristics of nonspecific lipid transfer proteins explain their resistance to gastroduodenal proteolysis.Biochemistry492130213920121231|
|15.||Pasquato N,Berni R,Folli C,Folloni S,Cianci M,et al. Year: 2006Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens.J Mol Biol35668469416388823|
|16.||Shewry PR,Napier JA,Tatham AS. Year: 1995Seed storage proteins: structures and biosynthesis.Plant Cell79459567640527|
|17.||Albrecht M,Alessandri S,Conti A,Reuter A,Lauer I,et al. Year: 2008High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp.Mol Nutr Food Res52Suppl 2S18619518727010|
|18.||Frye J,Klenchin VA,Rayment I. Year: 2010Structure of the tropomyosin overlap complex from chicken smooth muscle: insight into the diversity of N-terminal recognition.Biochemistry494908492020465283|
|19.||Farrell HM Jr,Jimenez-Flores R,Bleck GT,Brown EM,Butler JE,et al. Year: 2004Nomenclature of the proteins of cows' milk–sixth revision.J Dairy Sci871641167415453478|
|20.||Oberhuber C,Ma Y,Marsh J,Rigby N,Smole U,et al. Year: 2008Purification and characterisation of relevant natural and recombinant apple allergens.Mol Nutr Food Res52Suppl 2S20821918683825|
|21.||Gaier S,Marsh J,Oberhuber C,Rigby NM,Lovegrove A,et al. Year: 2008Purification and structural stability of the peach allergens Pru p 1 and Pru p 3.Mol Nutr Food Res52Suppl 2S22022918384093|
|22.||Rigby NM,Marsh J,Sancho AI,Wellner K,Akkerdaas J,et al. Year: 2008The purification and characterisation of allergenic hazelnut seed proteins.Mol Nutr Food Res52Suppl 2S25126119006093|
|23.||Marsh J,Rigby N,Wellner K,Reese G,Knulst A,et al. Year: 2008Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis.Mol Nutr Food Res52Suppl 2S27228518727014|
|24.||Blanc F,Bernard H,Alessandri S,Bublin M,Paty E,et al. Year: 2008Update on optimized purification and characterization of natural milk allergens.Mol Nutr Food Res52Suppl 2S16617518504704|
|25.||Brereton IM,Field JM,Irving G,Doddrell DM. Year: 1989Water Suppression with Bo Filed Gradient Homospoil Pulse in High-Resolution NMR Spectroscopy.Magnetic Resonance in Medicine918125|
|26.||Peitsch M. Year: 1995Protein modelling by E-mail/.Nature Biotechnology (N Y)13658660|
|27.||Pieper U,Eswar N,Braberg H,Madhusudhan MS,Davis FP,et al. Year: 2004MODBASE, a database of annotated comparative protein structure models, and associated resources.Nucleic Acids Res32D21722214681398|
|28.||Arnold K,Bordoli L,Kopp J,Schwede T. Year: 2006The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling.Bioinformatics2219520116301204|
|29.||Kiefer F,Arnold K,Kunzli M,Bordoli L,Schwede T. Year: 2009The SWISS-MODEL Repository and associated resources.Nucleic Acids Res37D38739218931379|
|30.||Benkert P,Kuenzli M,Schwede T. Year: 2009QMEAN Server for Protein Model Quality Estimation.Nucleic Acids Research 37:Web server issueW510W514|
|31.||Benkert P,Schwede T,Tosatto SC. Year: 2009QMEANclust: estimation of protein model quality by combining a composite scoring function with structural density information.BMC Struct Biol93519457232|
|32.||Benkert P,Biasini M,Schwede T. Year: 2011Toward the estimation of the absolute quality of individual protein structure models.Bioinformatics2734335021134891|
|33.||Scheich C,Leitner D,Sievert V,Leidert M,Schlegel B,et al. Year: 2004Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis.BMC Struct Biol4415113422|
|34.||Baum J,Dobson CM,Evans PA,Hanley C. Year: 1989Characterization of a partly folded protein by NMR methods: studies in the molten globule state of guinea pig alpha-lactalbumin.Biochemistry287132706269|
|35.||Ohgushi M,Wada A. Year: 1983‘Molten-globule state’: a compact form of globular proteins with mobile side-chains.FEBS Lett16421246317443|
|36.||Toda M,Reese G,Gadermaier G,Schulten V,Lauer I,et al. Year: 2011Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen.J Allergy Clin Immunol12810221030 e102721571358|
|37.||Perrocheau L,Bakan B,Boivin P,Marion D. Year: 2006Stability of barley and malt lipid transfer protein 1 (LTP1) toward heating and reducing agents: relationships with the brewing process.J Agric Food Chem543108311316608238|
|38.||Burks AW,Cockrell G,Stanley JS,Helm RM,Bannon GA. Year: 1995Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity.J Clin Invest96171517217560062|
|39.||Cabanos C,Urabe H,Tandang-Silvas MR,Utsumi S,Mikami B,et al. Year: 2011Crystal structure of the major peanut allergen Ara h 1.Mol Immunol4911512321903274|
|40.||Fukuda T,Maruyama N,Salleh MR,Mikami B,Utsumi S. Year: 2008Characterization and crystallography of recombinant 7S globulins of Adzuki bean and structure-function relationships with 7S globulins of various crops.J Agric Food Chem564145415318461964|
|41.||Cabanos C,Urabe H,Masuda T,Tandang-Silvas MR,Utsumi S,et al. Year: 2010Crystallization and preliminary X-ray analysis of the major peanut allergen Ara h 1 core region.Acta Crystallogr Sect F Struct Biol Cryst Commun6610711073|
|42.||Chruszcz M,Maleki SJ,Majorek KA,Demas M,Bublin M,et al. Year: 2011Structural and Immunologic Characterization of Ara h 1, a Major Peanut Allergen.J Biol Chem286393183932721917921|
|43.||Zhang YZ,Jin T,Guo F,Chen YW,Howard A. Year: 2009Crystal structure of Ara h 3, a major allergen in peanut.Molecular Immunology461796180419251323|
|44.||Kleber-Janke T,Crameri R,Appenzeller U,Schlaak M,Becker WM. Year: 1999Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology.Int Arch Allergy Immunol11926527410474031|
|45.||Rouge P,Culerrier R,Sabatier V,Granier C,Rance F,et al. Year: 2009Mapping and conformational analysis of IgE-binding epitopic regions on the molecular surface of the major Ara h 3 legumin allergen of peanut (Arachis hypogaea).Mol Immunol461067107518995911|
|46.||Lauer I,Foetisch K,Kolarich D,Ballmer-Weber BK,Conti A,et al. Year: 2004Hazelnut (Corylus avellana) vicilin Cor a 11: molecular characterization of a glycoprotein and its allergenic activity.Biochem J38332733415233621|
|47.||Beyer K,Grishina G,Bardina L,Grishin A,Sampson HA. Year: 2002Identification of an 11S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions.J Allergy Clin Immunol11051752312209105|
|48.||Jin T,Albillos SM,Guo F,Howard A,Fu TJ,et al. Year: 2009Crystal structure of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.J Agric Food Chem578643865119694440|
|49.||Reese G,Schicktanz S,Lauer I,Randow S,Luttkopf D,et al. Year: 2006Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus.Clin Exp Allergy3651752416630158|
|50.||Whitby FG,Phillips GN Jr. Year: 2000Crystal structure of tropomyosin at 7 Angstroms resolution.Proteins38495910651038|
|51.||Greenfield NJ,Huang YJ,Palm T,Swapna GV,Monleon D,et al. Year: 2001Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle alpha-tropomyosin in an engineered chimeric protein.J Mol Biol31283384711575936|
|52.||Mills EN,Mackie AR. Year: 2008The impact of processing on allergenicity of food.Curr Opin Allergy Clin Immunol824925318560301|
|Protein family||Function||Allergen* designation||Source||Mol Mass||PDB ID**||1-D-1H-NMR Analyses|
|Experimental conditions (protein conc.; buffer)||Structure|
|Plant Food Allergens|
|Non specific lipid transfer proteins||Plant defence, transfer of lipids||Mal d 3||AppleMalus domestica||9 kDa||2alg (Peach)||0.08 mM; 20 mM phosphate; pH 7.4||folded|
|Pru p 3||PeachPrunus persica||9 kDa||2alg, 2b5s||0.39 mM; 25 mM phosphate; pH 7.0||folded|
|Cor a 8||HazelnutCorylus avellana||9 kDa||2alg (Peach)||0.15 mM; 25 mM phosphate, 150 mM NaCl; pH 7.0|
|Vicillin-type (7/8S globulin)||Seed storage||Cor a 11||HazelnutCorylus avellana||48 kDa||2ea7 Chain A (Adzuki Bean)||0.04 mM; 50 mM phosphate, 150 mM NaCl; pH 7.5||folded|
|Ara h 1||PeanutArachis hypogaea||62 kDa||3fz3 (Almond)||0.03 mM; 50 mM MOPS, 20 mM NaCl pH 7.8||predominantely folded|
|Legumin-type (11S)||Seed storage||Cor a 9||HazelnutCorylus avellana||60 kDa||3fz3 (Almond)||0.03 mM; 50 mM phosphate, 150 mM NaCl; pH 7.5||predominantely folded|
|Ara h 3/4Ara h 3.0101Ara h 3.0201||PeanutArachis hypogaea||60 kDa||3c3v (partial structure)||0.14 mM; 50 mM phosphate, 150 mM NaCl; pH 7.5||predominantely folded|
|Animal Food Allergens|
|Casein||Ca2+ binding||Bos d 8||Cow's milkBos domesticus||19–24 kDa||0.08 mM; 40 mM phosphate; pH 7.4||non folded|
|Goat's milkCapra hircus||19–24 kDa||0.19 mM; 40 mM phosphate; pH 7.4||non folded|
|Tropomyosin||Regulator of muscle contraction||Pen a 1||ShrimpPenaeus aztecus||33 kDa||1c1g (Pig)||0.13 mM; 20 mM MOPS, 0.5 M NaCl; pH 7.6||non folded|
*Allergen designations according to the IUIS Allergen nomenclature committee (www.allergen.org).
**Solved and available structures are listed, with their PDB entries. If models were generated then the respective template structure is given.
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