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Solution and high-pressure NMR studies of the structure, dynamics and stability of the cross-reactive allergenic cod parvalbumin Gad m 1

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Beta-parvalbumins from different fish species have been identified as the main elicitors of IgE-mediated reactions in fish-allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross-reactivity among fish parvalbumins. In addition, the high-pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca2+-free forms for potential use in immunotherapy. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
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... The Gad m 1 chain contains three tandemly arrayed EF-hands, of which only the two C-terminal motifs named CD and EF bind Ca 2+ . In the cation-bound form, Gad m 1 displays a highly stable monomeric helical globular fold [24]. In contrast, removal of bound Ca 2+ at high protein concentrations triggers Gad m 1 amyloid aggregation through the regions forming helix B and helix D in the globular fold [25][26][27]. ...
... To investigate the influence of amyloids on calcium carbonate precipitation, aggregates were placed under the previous conditions, and the reaction was enabled by controlled diffusion of CO2 from the decomposition of ammonium carbonate. As a control for the non-polymerized and Ca 2+ precomplexed protein form, we used Ca 2+ -bound Gad m 1 wt monomers at similar concentrations [24,25]. The representative crystal structures that formed were visualized by scanning electron microscopy (SEM) (Figure 6). ...
... To investigate the influence of amyloids on calcium carbonate precipitation, aggregates were placed under the previous conditions, and the reaction was enabled by controlled diffusion of CO 2 from the decomposition of ammonium carbonate. As a control for the non-polymerized and Ca 2+ pre-complexed protein form, we used Ca 2+ -bound Gad m 1 wt monomers at similar concentrations [24,25]. The representative crystal structures that formed were visualized by scanning electron microscopy (SEM) (Figure 6). ...
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Acid proteins capable of nucleating Ca2+ and displaying aggregation capacity play key roles in the formation of calcium carbonate biominerals. The helix-loop helix EF-hands are the most common Ca2+-binding motifs in proteins. Calcium is bound by the loop region. These motifs are found in many proteins that are regulated by calcium. Gad m 1, an Atlantic cod β-parvalbumin isoform, is a monomeric EF-hand protein that acts as a Ca2+ buffer in fish muscle; the neutral and acid apo-forms of this protein can form amyloids. Since Ca2+-nucleating proteins have a propensity to form extended β-strand structures, we wondered whether amyloid assemblies of an EF-hand protein were able to influence calcium carbonate crystallization in vitro. Here, we used the Gad m 1 chain as a model to generate monomeric and amyloid assemblies and to analyze their effect on calcite formation in vitro. We found that only amyloid assemblies alter calcite morphology.
... Of these, the segment containing residues 33-44 has been described as highly immunoreactive in β-PV from several fish species 3,19,20,24,25 . Ca 2+ binding stabilizes the proteins in a helical globular fold, as shown by the 3D structures of cod (Gad m 1) and mackerel (Sco j 1) major β-PVs, among others 3,26,27 . Impairment of this fold in the carp β-PV Cyp c 1 by mutation of both acid loops reduces the IgE binding by 95%, supporting the existence of conformational epitopes 28 . ...
... (d) Segments involved in the globular and amyloid folds of β-PV. The globular fold contains three helix-loop-helix (AB, CD and EF) EF-hands, of which only CD and EF bind cations 3,4,26,43 . Regions assembling into amyloids were predicted by AmylPred2, and they are depicted by arrows. ...
... Figure 4a,b show that freshly prepared Ca 2+ -bound proteins cooperatively folded with predominant α-helical secondary structures and hydrodynamic radii close to the R H T of spherical monomers (1.6 ± 0.1 nm). These observations agree with the available 3D structures of gmPV1 and sjPV1 26,43 . However, despite these similarities, Ca 2+ -bound sjPV1 and gmPV2 unfolded at a significantly lower temperature than the other chains (Fig. 4c). ...
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Most fish-allergic patients have anti-β-parvalbumin (β-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the β-PV load is considered a determinant for allergenicity, fish species express distinct β-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken Gadus morhua and Scomber japonicus models, determined their β-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus. The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differences.
... Of these, the segment containing residues 33-44 has been described as highly immunoreactive in β-PV from several fish species 3,19,20,24,25 . Ca 2+ binding stabilizes the proteins in a helical globular fold, as shown by the 3D structures of cod (Gad m 1) and mackerel (Sco j 1) major β-PVs, among others 3,26,27 . Impairment of this fold in the carp β-PV Cyp c 1 by mutation of both acid loops reduces the IgE binding by 95%, supporting the existence of conformational epitopes 28 . ...
... Fig. 4a and 4b show that freshly prepared Ca 2+ -bound proteins cooperatively folded with predominant αhelical secondary structures and hydrodynamic radii close to the R H T of spherical monomers (1.6 ± 0.1 nm). These observations agree with the available 3D structures of gmPV1 and sjPV1 26,43 . However, despite these similarities, Ca 2+ -bound sjPV1 and gmPV2 unfolded at a significantly lower temperature than the other chains (Fig. 4c). ...
... (d) Segments involved in the globular and amyloid folds of β-PV. The globular fold contains three helix-loop-helix (AB, CD and EF) EF-hands, of which only CD and EF bind cations3,4,26,43 ...
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Most fish-allergic patients have anti-β-parvalbumin (β-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the β-PV load is considered a determinant for allergenicity, fish species express distinct β-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken Gadus morhua and Scomber japonicus models, determined their β-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus . The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differences.
... To address the question of whether degradation properties and preservation of monomers involves amyloid-like aggregates we chose β-parvalbumins, the main elicitors of IgE-mediated reactions in fish-allergic individuals [26]. The β-parvalbumins are Ca 2+ binding proteins of about 12 kDa and contribute >2.5 mg per gram to raw fish muscle [26][27][28]. They fold into a structure consisting of three EFhand motifs (AB, CD and EF) ( fig. ...
... They fold into a structure consisting of three EFhand motifs (AB, CD and EF) ( fig. 1A), of which only CD and EF can bind divalent cations (Ca 2+ and/or Mg 2+ ) [26,27]. The major immunologically reactive sites have been found on the junction between the motifs (regions 33-44, 65-74) and on the EF region (segments 88-96 and 95-109) [26,[29][30][31]. ...
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Principles: Amyloids are highly cross-β-sheet-rich aggregated states that confer protease resistance, membrane activity and multivalence properties to proteins, all essential features for the undesired preservation of food proteins transiting the gastrointestinal tract and causing type I allergy. Methods: Amyloid propensity of β-parvalbumin, the major fish allergen, was theoretically analysed and assayed under gastrointestinal-relevant conditions using the binding of thioflavin T, the formation of sodium dodecyl sulphate- (SDS-) resistant aggregates, circular dichroism spectroscopy and atomic force microscopy fibril imaging. Impact of amyloid aggregates on allergenicity was assessed with dot blot. Results: Sequences of β-parvalbumin from species with commercial value contain several adhesive hexapeptides capable of driving amyloid formation. Using Atlantic cod β-parvalbumin (rGad m 1) displaying high IgE cross-reactivity, we found that formation of amyloid fibres under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species under gastrointestinal relevant conditions and for the IgE-recognition in the sera of allergic patients. Incorporation of the anti-amyloid compound epigallocatechin gallate prevents rGad m 1 fibrillation, facilitates its protease digestion and impairs its recognition by IgE. Conclusions: the formation of amyloid by rGad m 1 explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and its epitope architecture as allergen.
... Analysis of the Gad m 1:scFv-gco9 interaction by NMR spectroscopy Interaction of [ 15 N-13 C]-labeled rGad m1 and scFv-gco9 was monitored by analysis of 1 H-15 N HSQC experiments. The production and the solution structure of the [ 15 N-13 C]-labeled rGad m 1 was previously published by our groups [23]. ...
... The CSP was calculated using the formula CSP = | ΔδH| + 0.1 Ã | ΔδN|, where |ΔδH| and Δδ N| are the CSP of 1 H and 15 N nuclei. The molecular dynamics of free Gad m 1 and Gad m 1:scFv-gco9 were monitored by 15 N backbone relaxation experiments measuring the transversal (R 2 ) and longitudinal (R 1 ) relaxation rates measured as described in [23]. ...
Article
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Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.
... Therefore, β -parvalbumin chains that are unable to form amyloids share a diminished IgE interaction. Third, the overlap of epitopes and amyloid folds in the AB motif, which is a hot spot for conformational exchange and lacks Ca 2+ -binding properties, suggests that amyloids could also form in the presence of Ca 2+ as a function of the chain sequence 53,54 . Therefore, unless specifically removed by centrifugation, β -parvalbumin solutions may contain traces of amyloid species that, as function of their analytical use, could mislead reactivity assignments. ...
Article
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Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy.
... Characterization. PV has been extensively studied in bony fish including in Atlantic cod (Gadus morhua) [28,29,[33][34][35][36][37], Alaska Pollack (Theragra chalcogramma) [38,39], common carp (Cyprinus carpio) [24,30,31,37,40], silver carp (Hypophthalmichthy molitrix) [41], Atlantic salmon (Salmo salar) [39,[42][43][44][45][46] and recently Asian seabass (Lates calcarifer) [47,48]. In comparison, studies on PV from cartilaginous fish are limited to leopard shark [25], red stingray [26], Atlantic stingray [49] and thornback ray [50]. ...
Article
Allergy to bony fish is common and probably increasing worldwide. The major heat stable pan-fish allergen, parvalbumin (PV), has been identified and characterized for numerous fish species. In contrast, there are very few reports of allergic reactions to cartilaginous fish despite widespread consumption. The molecular basis for this seemingly low clinical cross-reactivity between these two fish groups has not been elucidated. PV consists of two distinct protein lineages, α and β. The α-lineage of this protein is predominant in muscle tissue of cartilaginous fish (Chondrichthyes), while β-PV is abundant in muscle tissue of bony fish (Osteichthyes). The low incidence of allergic reactions to ingested rays and sharks is likely due to the lack of molecular similarity, resulting in reduced immunological cross-reactivity between the two PV lineages. Structurally and physiologically both protein lineages are very similar, however the amino acid homology is very low with 47% to 54%. Furthermore, PV from ancient fish species such as the coelacanth demonstrates 62% sequence homology to leopard shark α-PV and 70% to carp β-PV. This indicates the extent of conservation of the PV isoforms lineages across millennia. This review highlights prevalence data on fish allergy and sensitization to fish, and details the molecular diversity of the two protein lineages of the major fish allergen PV among different fish groups, emphasizing the immunological and clinical differences in allergenicity. This article is protected by copyright. All rights reserved.
... Figure 2b shows a ribbon representation of the lowest energy structure of Sco j 1. Sco j 1 formed a single, compact domain consisting of six α-helices with residues 9-19 (helix-A), 27-34 (helix-B), 41-51 (helix-C), 61-66 (helix-D), 80-90 (helix-E), and 100-106 (helix-F). These secondary structural elements are in good agreement with those of the other parvalbumin structures 14,19 . Helix pairs AB, CD, and EF form three EF-hand motifs. ...
Article
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Although fish is an important part of the human diet, it is also a common source of food allergy. The major allergen in fish is parvalbumin, a well-conserved Ca²⁺-binding protein found in the white muscle of many fish species. Here, we studied the solution structure of the parvalbumin Sco j 1, derived from the Pacific mackerel, using nuclear magnetic resonance spectroscopy. We mapped the IgE-binding epitope proposed in a recent study onto the present structure. Interestingly, three of four residues, which were elucidated as key residues of the IgE-binding epitope, were exposed to solvent, whereas one residue faced the inside of the molecule. We expect that this solution structure can be used in future studies attempting to analyze the various IgE-binding modes of these allergens.
... Several studies, on different fish species, have demonstrated that calcium-chelating is crucial to the structural preservation of parvalbumin and its IgE-binding capacity (Swoboda et al., 2007;Permyakov et al., 2008;Swoboda et al., 2013;Moraes et al., 2014;Kumeta et al., 2017). Thus, EDTA has been used in several fish allergens-related studies, to induce, in vitro, the apoparvalbumin, i.e., the Ca 2+ -free form of parvalbumin, and explore its reduced IgE-affinity and potential decreased allergenicity. ...
Article
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Food allergy is an abnormal immune response to specific proteins in a certain food. The chronicity, prevalence, and the potential fatality of food allergy, make it a serious socio-economic problem. Fish is considered the third most allergenic food in the world, affecting part of the world population with a higher incidence in children and adolescents. The main allergen in fish, responsible for the large majority of fish-allergic reactions in sensitized patients, is a small and stable calcium-binding muscle protein named beta-parvalbumin. Targeting the expression or/and the 3D conformation of this protein by adding specific molecules to fish diets has been the innovative strategy of some researchers in the fields of fish allergies and nutrition. This has shown promising results, namely when the apo-form of β-parvalbumin is induced, leading in the case of gilthead seabream to a 50% reduction of IgE-reactivity in fish allergic patients.
... This behaviour was described for the segments 25 FDHKAFFTKVGLAAKSSA 42 and 67 FLQNFSAGARAL 78 of Gad m 1 using anti-fibril OC-antibody and sera IgE interactions [15,16]. Interestingly, the N-terminal region of each of the segments (DHKAFFTKV and FLQNFS) form part of helical structures in the native state of Gad m 1 [26]. This conformational multiplicity of the regions forming the IgE-binding epitopes questions the validity of mapping the epitopes singly on the native 3D structures of food allergens. ...
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Several animal food allergens assembly into amyloids under gastric-like environments. These aggregated structures provide Gad m 1 with an enhanced IgE interaction due to the amyloid assembly of the epitope regions. However, whether these properties are unique of Gad m 1 or common to other food allergens has not yet been addressed. Using Bos d 5, Bos d 12 and Gal d 2 as food allergen models and Gad m 1 as control, aggregation reactions and the sera of milk, egg and fish allergic patients we have analyzed the IgE interaction of the distinct amyloids. We found that amyloids formed by Bos d 12 and Gal d 2 full-length and truncated chains are recognized by the IgEs of milk and egg allergic patient sera. As with Gad m 1, in most cases amyloid recognition is higher than that of the precursor structure. Bos d 5 was not recognized under any fold by the IgE of the sera studied. These results support that formation of IgE-binding amyloids might be a common feature to animal food allergen.
... The potential conformational multiplicity of the regions forming the IgE-binding epitopes questions the validity of mapping the epitopes using the native 3D structures of food allergens as unique template. In fact, the IgE binding sequences DHKAFFTKV and FLQNFS form parts of helical structures in the native state of Gad m 1 whereas the amyloid fold adopts a cross β-sheet structure [16,37]. In fact, consideration of the IgE-epitope amyloid folds will simultaneously explain their protease resistance and higher avidity and affinity for any ligand binding process [12,14,15,32]. ...
Article
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Several animal food allergens assemble into amyloids under gastric-like environments. These aggregated structures provide Gad m 1 with an enhanced immunoglobulin E (IgE) interaction due to the fibrillation of the epitope regions. However, whether these properties are unique to Gad m 1 or shared by other food allergens has not yet been addressed. Using Bos d 5, Bos d 12 and Gal d 2 as allergen models and Gad m 1 as the control, aggregation reactions and the sera of milk, egg and fish allergic patients have been analyzed, assessing the IgE interactions of their amyloids. We found that amyloids formed by Bos d 12 and Gal d 2 full-length and truncated chains are recognized by the IgEs of milk and egg allergic patient sera. As with Gad m 1, in most cases amyloid recognition is higher than that of the native structure. Bos d 5 was not recognized under any fold by the IgE of the sera studied. These results suggest that the formation of IgE-binding amyloids could be a common feature to animal food allergens.
... It was reported that Ca 2+ binding was essential for the formation of the conformation structure of calcium-binding proteins, and the consumption of Ca 2+ may lead to the decrement of IgEbinding capacity. 40,41 The influences of Ca 2+ and Nglycosylation sites on the structure of PM and their allergenicity need further research. Sequence alignment indicated that the R. venosa PM was more closely related to P. canaliculata, which belonged to the Gastropoda branch. ...
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Paramyosin (PM) is an important structural protein in molluscan muscles. However, as an important allergen, there is a little information on PM in the molluscs. In this study, a 99 kDa molecular weight allergen protein was purified from Rapana venosa and confirmed as PM by mass spectrometry. The results of immunoglobulin E (IgE)-binding activity and physicochemical characterization showed that R. venosa PM could react with a specific IgE of the sera from sea snail-allergic patients, and the IgE-binding activity could be reduced by thermal treatment. The full-length cDNA of R. venosa PM was cloned, which encodes 859 amino acid residues, and it has a higher homology among molluscan species. According to the circular dichroism results, Fourier transform infrared, and 2D and 3D structure analysis, both PM and tropomyosin are conserved proteins, which are mainly composed of the α-helix structure. These results are significant for better understanding the anaphylactic reactions in sea snail-allergic patients and allergy diagnosis.
... These two proteins exhibit two distinct protein folding patterns (http:// scop.mrc-lmb.cam.ac.uk/); PMI is a typical member of the cupin superfamily dominated by two neighboring b-barrels (Dunwell et al. 2001) while the frog parvalbumin and its close homolog, the fish allergen Gad m 1, consist of three tandem calcium-binding EFhand motifs with a helix-loop-helix topology (Moraes et al. 2014) (Fig. 2). The matched eight contiguous amino acid peptides are located in different structural environments within their respective proteins and assume distinct conformations in the two proteins; In PMI, the sequence DLSDKETT adopts a loop and bstrand in the second double-stranded b-helix barrel while the matched frog peptide is part of the aE helix. ...
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Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.
Preprint
Acid proteins capable of nucleating Ca2+ and displaying aggregation capacity play key roles in the formation of calcium carbonate biominerals. EF-hands are among the largest Ca2+-binding motif in proteins. Gad m 1, an Atlantic cod β-parvalbumin isoform, is a monomeric EF-hand protein that acts as a Ca2+ buffer in fish muscle and is able to form amyloids under acidic conditions. Since nucleating Ca2+ protein have a propensity to form extended β-strand structures, we wondered whether amyloid assemblies of a protein containing refolded EF-hand motifs were able to influence the in vitro calcium carbonate crystallization. Here we have used the Gad m 1 chain as model to generate monomeric and amyloid assemblies and analyze their effect on in vitro calcite formation. We found that only amyloid assemblies alter calcite morphology.
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Amyloid formation is basically featured by a protein-protein interaction in which the reacting regions are the segments assembling into cross β-sheets. To identify these segments both theoretical and experimental tools have been developed. Here, we focus on the use of peptide arrays to probe the binding of several amyloid-specific probes such as the OC and A11 anti-amyloid conformation-selective antibodies and of monomers and preformed fibrils. These arrays use libraries containing partly overlapping peptides derived from the sequence of Gad m 1, the major allergen from Atlantic cod, which forms amyloids under gastrointestinal relevant conditions.
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Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with the molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.
Chapter
Proteins containing EF-hand helix-loop-helix-binding motifs play essential roles in calcium homeostasis and signaling pathways. These proteins have considerable structural and functional diversity by virtue of their cation-binding properties, and occur as either Ca²⁺-bound or Ca²⁺-free states with distinct aggregation propensities. That is the case among β-parvalbumins and S100 proteins, which under certain conditions undergo Ca²⁺-dependent self-assembly reactions with the formation of oligomers, amyloid-type aggregates and fibrils. These phenomena may be particularly relevant in human S100A6 protein and in fish Gad m 1 allergenic protein, which are implicated in human disease processes. Here, we describe detailed methods to generate and monitor the formation of amyloidogenic assemblies and aggregates of these two EF-hand proteins in vitro.
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Gad m 1 is the major allergen from Atlantic cod. It belongs to β-parvalbumin protein family and is characterized by the presence of two calcium-binding sites so called EF-hand motifs. β-Parvalbumins such as Gad m 1 are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization to various fish species in allergic patients. Despite extensive efforts, the complete elucidation of β-parvalbumin-IgE complexes has not been achieved yet. Allergen structural studies are essential for the development of novel immunotherapy strategies, including vaccination with hypoallergenic derivatives and chimeric molecules. Here, we report for the first time the NMR study of a β-parvalbumin: Gad m 1. This report includes: 1H, 13C and 15N resonance assignments of Gad m 1 as well as the second structure information based on the 13C chemical shifts.
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Sarcoplasmic calcium-binding protein (SCP) is an EF-hand Ca2+-binding protein recently identified as a new crustacean allergen. Some EF-hand Ca2+-binding allergens, such as parvalbumin (fish allergen) and Bet v 4 (birch pollen allergen), have been shown to contain conformational-type IgE epitopes associated with the Ca2+-chelating. This study was performed to clarify the relationships between IgE reactivity and structure of the black tiger shrimp SCP. When analyzed by ELISA in the absence and presence of EGTA, the IgE reactivity of the black tiger shrimp SCP was found to be considerably reduced by Ca2+-depletion. Furthermore, circular dichroism spectral data showed a conformational difference between Ca2+-bound and Ca2+-depleted forms of the black tiger shrimp SCP. On the other hand, the synthetic 18 peptides spanning the entire amino acid sequence of the black tiger shrimp SCP were all assessed to have little IgE-binding ability by ELISA. Our results demonstrate that the IgE reactivity of the black tiger shrimp SCP is attributable mostly to conformational-type IgE epitopes, at least part of which is maintained by Ca2+-chelating.
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The impact of pressure on the backbone (15) N, (1) H and (13) C chemical shifts in N-terminally acetylated α-synuclein has been evaluated over a pressure range 1-2500 bar. Even while the chemical shifts fall very close to random coil values, as expected for an intrinsically disordered protein, substantial deviations in the pressure dependence of the chemical shifts are seen relative to those in short model peptides. In particular, the nonlinear pressure response of the (1) H(N) chemical shifts, which commonly is associated with the presence of low-lying "excited states", is much larger in α-synuclein than in model peptides. The linear pressure response of (1) H(N) chemical shift, commonly linked to H-bond length change, correlates well with those in short model peptides, and is found to be anticorrelated with its temperature dependence. The pressure dependence of (13) C chemical shifts shows remarkably large variations, even when accounting for residue type, and do not point to a clear shift in population between different regions of the Ramachandran map. However, a nearly universal decrease in (3) JHN-Hα by 0.22±0.05 Hz suggests a slight increase in population of the polyproline II region at 2500 bar. The first six residues of N-terminally acetylated synuclein show a transient of approximately 15 % population of α-helix, which slightly diminishes at 2500 bar. The backbone dynamics of the protein is not visibly affected beyond the effect of slight increase in water viscosity at 2500 bar.
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A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, ≥90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed (ϕ, ψ) torsion angles of ca 12º. TALOS-N also reports sidechain χ(1) rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.
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Conformational changes are essential for protein-protein and protein-ligand recognition. Here we probed changes in the structure of the protein ubiquitin at low temperatures in supercooled water using NMR spectroscopy. We demonstrate that ubiquitin is well folded down to 263 K, although slight rearrangements in the hydrophobic core occur. However, amide proton chemical shifts show non-linear temperature dependence in supercooled solution and backbone hydrogen bonds become weaker in the region that is most prone to cold-denaturation. Our data suggest that the weakening of the hydrogen bonds in the β-sheet of ubiquitin might be one of the first events that occur during cold-denaturation of ubiquitin. Interestingly, the same region is strongly involved in ubiquitin-protein complexes suggesting that this part of ubiquitin more easily adjusts to conformational changes required for complex formation.
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The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
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The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.
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Because of shifts in the gender ratio and incidence and remission rates of asthma during the teen ages, the methodology of incidence studies among teenagers is important, i.e. if the time intervals between surveys are too long, the incident cases might not be properly identified. The aim was to study the impact of study design on the incidence rates of asthma and wheeze during the teen ages. In a study about asthma and allergic diseases within the OLIN studies (Obstructive Lung Disease in northern Sweden), a cohort of school children (n = 3,430) was followed annually by questionnaire from age 8 yrs. In the endpoint survey (age 18 yrs) 2,582 (75% of original responders) participated. Incident cases from age 12-18 yrs were identified by two methods: annual questionnaire reports (AR) and baseline-endpoint surveys only (BE). The cumulative incidence of asthma and wheeze was significantly higher based on AR compared to BE. Compared to the incidence rates based on all the annual surveys, the calculated average annual rates based on BE were in general lower both among the boys and among the girls. There were no differences between boys and girls in incidence rates of asthma or wheeze during the early teen years. However, from the age of 15 years, the annual incidence rates were significantly or borderline significantly higher among girls than boys. At onset, the additional cases of current asthma identified by AR had significantly less severe asthma than those identified in BE (p < 0.02). the size of the incidence of asthma and wheeze during the teen ages was influenced by study design. By using the conventional prospective study design with longer follow-up time, the incidence was underestimated.
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NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between 13C, 15N and 1H chemical shifts and backbone torsion angles phi and psi (Cornilescu et al. J Biomol NMR 13 289-302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted phi and psi angles, equals +/-13 degrees . Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.
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The Red Queen said, ‘It takes all the running you can do, to keep in the same place.’ Lewis Carrol Motivation: Newly solved protein structures are routinely scanned against structures already in the Protein Data Bank (PDB) using Internet servers. In favourable cases, comparing 3D structures may reveal biologically interesting similarities that are not detectable by comparing sequences. The number of known structures continues to grow exponentially. Sensitive—thorough but slow—search algorithms are challenged to deliver results in a reasonable time, as there are now more structures in the PDB than seconds in a day. The brute-force solution would be to distribute the individual comparisons on a massively parallel computer. A frugal solution, as implemented in the Dali server, is to reduce the total computational cost by pruning search space using prior knowledge about the distribution of structures in fold space. This note reports paradigm revisions that enable maintaining such a knowledge base up-to-date on a PC. Availability: The Dali server for protein structure database searching at http://ekhidna.biocenter.helsinki.fi/dali_server is running DaliLite v.3. The software can be downloaded for academic use from http://ekhidna.biocenter.helsinki.fi/dali_lite/downloads/v3. Contact: liisa.holm@helsinki.fi
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A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.
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The EF-hand motif is the most common calcium-binding motif found in proteins. Several high-resolution structures containing different metal ions bound to EF-hand sites have given new insight into the modulation of their binding affinities. Recently determined structures of members of several newly identified protein families that contain the EF-hand motif in some of their domains, as well as of their complexes with target molecules, are throwing light on the surprising variety of functions that can be served by this simple and ingenious structural motif.
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SWISS-MODEL (http://swissmodel.expasy.org) is a server for automated comparative modeling of three-dimensional (3D) protein structures. It pioneered the field of automated modeling starting in 1993 and is the most widely-used free web-based automated modeling facility today. In 2002 the server computed 120 000 user requests for 3D protein models. SWISS-MODEL provides several levels of user interaction through its World Wide Web interface: in the ‘first approach mode’ only an amino acid sequence of a protein is submitted to build a 3D model. Template selection, alignment and model building are done completely automated by the server. In the ‘alignment mode’, the modeling process is based on a user-defined target-template alignment. Complex modeling tasks can be handled with the ‘project mode’ using DeepView (Swiss-PdbViewer), an integrated sequence-to-structure workbench. All models are sent back via email with a detailed modeling report. WhatCheck analyses and ANOLEA evaluations are provided optionally. The reliability of SWISS-MODEL is continuously evaluated in the EVA-CM project. The SWISS-MODEL server is under constant development to improve the successful implementation of expert knowledge into an easy-to-use server.
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Homology models of proteins are of great interest for planning and analysing biological experiments when no experimental three-dimensional structures are available. Building homology models requires specialized programs and up-to-date sequence and structural databases. Integrating all required tools, programs and databases into a single web-based workspace facilitates access to homology modelling from a computer with web connection without the need of downloading and installing large program packages and databases. SWISS-MODEL workspace is a web-based integrated service dedicated to protein structure homology modelling. It assists and guides the user in building protein homology models at different levels of complexity. A personal working environment is provided for each user where several modelling projects can be carried out in parallel. Protein sequence and structure databases necessary for modelling are accessible from the workspace and are updated in regular intervals. Tools for template selection, model building and structure quality evaluation can be invoked from within the workspace. Workflow and usage of the workspace are illustrated by modelling human Cyclin A1 and human Transmembrane Protease 3. The SWISS-MODEL workspace can be accessed freely at http://swissmodel.expasy.org/workspace/
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IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.
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This review focuses on advances and updates in the epidemiology, pathogenesis, diagnosis, and treatment of food allergy over the past 3 years since our last comprehensive review. On the basis of numerous studies, food allergy likely affects nearly 5% of adults and 8% of children, with growing evidence of an increase in prevalence. Potentially rectifiable risk factors include vitamin D insufficiency, unhealthful dietary fat, obesity, increased hygiene, and the timing of exposure to foods, but genetics and other lifestyle issues play a role as well. Interesting clinical insights into pathogenesis include discoveries regarding gene-environment interactions and an increasing understanding of the role of nonoral sensitizing exposures causing food allergy, such as delayed allergic reactions to carbohydrate moieties in mammalian meats caused by sensitization from homologous substances transferred during tick bites. Component-resolved diagnosis is being rapidly incorporated into clinical use, and sophisticated diagnostic tests that indicate severity and prognosis are on the horizon. Current management relies heavily on avoidance and emergency preparedness, and recent studies, guidelines, and resources provide insight into improving the safety and well-being of patients and their families. Incorporation of extensively heated (heat-denatured) forms of milk and egg into the diets of children who tolerate these foods, rather than strict avoidance, represents a significant shift in clinical approach. Recommendations about the prevention of food allergy and atopic disease through diet have changed radically, with rescinding of many recommendations about extensive and prolonged allergen avoidance. Numerous therapies have reached clinical trials, with some showing promise to dramatically alter treatment. Ongoing studies will elucidate improved prevention, diagnosis, and treatment.
Article
Parvalbumin is a sarcoplasmic Ca2+-binding protein of 12 kDa and represents the major fish allergen. Several peptide segments are identified as immunoglobulin E (lgE)-binding epitopes of cod parvalbumin. However, carp parvalbumin (Cyp c 1) shows a markedly reduced IgE-binding ability upon depletion of Ca2+, suggesting the importance of conformational epitopes associated with Ca2+-chelating. In this study, the IgE reactivity of Pacific mackerel Scomber japonicus parvalbumin (Sco j 1) was demonstrated to be markedly reduced (60–100% reduction) by Ca2+-depletion, similar to Cyp c 1. Three Sco j 1 mutants (D51A, D90A, D51/90A), with modifications in either one or both of the two Ca2+-binding sites, were then constructed by site-directed mutagenesis, followed by expression in Escherichia coli, and evaluated for their IgE reactivity. Interestingly, the double mutant (D51/90A), probably devoid of Ca2+-binding capacity, exhibited a significantly reduced IgE reactivity (equivalent to 0.0–7.5% of the IgE reactivity of natural Sco j 1). The results suggest that the IgE-binding ability of Sco j 1 largely depends on the solid conformation mediated by Ca2+-chelating, and that the hypoallergenic D51/90A will be a useful tool for the specific immunotherapy of fish allergy.
Article
Scope: IgE-epitope mapping of allergens reveal important information about antigen components involved in allergic reactions. The peptide-based microarray immunoassay has been used to map epitopes of some food allergens. We developed a peptide microarray immunoassay to map allergenic epitopes in parvalbumin from Atlantic cod (Gad m 1), the most consumed cod species in Spain. Methods and results: Sera from 13 fish-allergic patients with specific IgE to cod parvalbumin were used. A library of overlapping peptides was synthesized, representing the primary sequence of Gad m 1. Peptides were used to analyze allergen-specific IgE antibodies in patient sera. 100% of the patients recognized one antigenic region of 15 amino acids in length in Gad m 1. This region only partially correlated with one of the three antigenic determinants of Gad c 1 (Allergen M), parvalbumin from Baltic cod (Gadus callarias). In the 3D model of the protein, this region was located on the surface of the protein. Conclusion: We have identified a relevant antigenic region in Gad m 1. This epitope could be considered as a severity marker and provides additional information to improve fish allergy diagnosis and the design of safe immunotherapeutic tools.
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The purpose of the review is to summarize and comment on recent developments regarding the safety of engineered immunotherapy vaccines. In the last 2 years, several studies were published in which allergy vaccines were developed on the basis of chemical modification of natural allergen extracts, the engineering of allergen molecules by recombinant DNA technology and synthetic peptide chemistry, allergen genes, new application routes and conjugation with immune modulatory molecules. Several studies exemplified the general applicability of hypoallergenic vaccines on the basis of recombinant fusion proteins consisting of nonallergenic allergen-derived peptides fused to allergen-unrelated carrier molecules. These vaccines are engineered to reduce both, immunoglobulin E (IgE) as well as allergen-specific T cell epitopes in the vaccines, and thus should provoke less IgE and T-cell-mediated side-effects. They are made to induce allergen-specific IgG antibodies against the IgE-binding sites of allergens with the T-cell help of the carrier molecule. Several interesting examples of allergy vaccines with potentially increased safety profiles have been published. The concept of fusion proteins consisting of allergen-derived hypoallergenic peptides fused to allergen-unrelated proteins that seems to be broadly applicable for a variety of allergens appears to be of particular interest because it promises not only to reduce side-effects but also to increase efficacy and convenience of allergy vaccines.
Article
Fish allergy is associated with IgE-mediated hypersensitivity reactions to parvalbumins, which are small calcium-binding muscle proteins and represent the major and sole allergens for 95% of fish-allergic patients. We performed Fourier transform infrared and tryptophan fluorescence spectroscopy to explore the pressure-temperature (p-T) phase diagram of cod parvalbumin (Gad m 1) and to elucidate possible new ways of pressure-temperature inactivation of this food allergen. Besides the secondary structure of the protein, the Ca(2+) binding to aspartic and glutamic acid residues was detected. The phase diagram was found to be quite complex, containing partially unfolded and molten globule states. The Ca(2+) ions were essential for the formation of the native structure. A molten globule conformation appears at 50 °C and atmospheric pressure, which converts into an unordered aggregated state at 75 °C. At >200 MPa, only heat unfolding, but no aggregation, was observed. A pressure of 500 MPa leads to a partially unfolded state at 27 °C. The complete pressure unfolding could only be reached at an elevated temperature (40 °C) and pressure (1.14 GPa). A strong correlation was found between Ca(2+) binding and the protein conformation. The partially unfolded state was reversibly refolded. The completely unfolded molecule, however, from which Ca(2+) was released, could not refold. The heat-unfolded protein was trapped either in the aggregated state or in the molten globule state without aggregation at elevated pressures. The heat-treated and the combined heat- and pressure-treated protein samples were tested with sera of allergic patients, but no change in allergenicity was found.
Article
In the preceding paper it has been shown that the unique dynamic information on fast internal motions in an NMR relaxation experiment on macromolecules in solution is specified by a generalized order parameter, 8, and an effective correlation time, 7,. This paper deals with the extraction and interpretation of this information. The procedure used to obtain S2 and T, from experimental data by using a least-squares method and, in certain favorable circumstances, by using an analytical formula is described. A variety of experiments are then analyzed to yield information on the time scale and spatial restriction of internal motions of isoleucines in myoglobin, methionines in dihydrofolate reductase and myoglobin, a number of aliphatic residues in basic pancreatic trypsin inhibitor, and ethyl isocyanide bound to myoglobin, hemoglobin, and aliphatic side chains in three random-coil polymers. The numerical values of S2 and 7, can be readily interpreted within the framework of a variety of models. In this way, one can obtain the same physical picture of internal motions as that obtained by using complicated spectral densities to fit the data. The numerical value of the order parameter, unlike the effective correlation time T,, plays a crucial role in determining what models can be used to describe the experiment; models in which the order parameter cannot be reproduced are eliminated. Conversely, any model that can yield the correct value of S works.
Article
A new approach to the interpretation of nuclear magnetic resonance relaxation experiments on macromolecules in solution is presented. This paper deals with the theoretical foundations and establishes the range of validity of this approach, and the accompanying paper demonstrates how a wide variety of experimental relaxation data can be successfully analyzed by using this approach. For both isotropic and anisotropic overall motion, it is shown that the unique imformation on fast internal motions contained in relaxation experiments can be completely specified by two model-independent quantities; (1) a generalized order parameter, S, which is a measure of the spatial restriction of the motion, and (2) an effective correlation time, T/sub e/, which is a measure of the rate of motion. A simple expression for the spectral density involving these two parameters is derived and is shown to be exact when the internal (but not overall) motions are in the extreme narrowing limit. The model-free approach (so called because S² and T/sub e/ have model-independent significance) consists of using the above spectral density to least-squares fit relaxation data by treating S² and T/sub e/ as adjustable parameters. The range of validity of this approach is illustrated by analyzing error-free relaxation data generated by using sophisticated dynamical models. Empirical rules are presented that allow one to estimate the of S² and T/sub e/ extracted by using the model-free approach by considering their numerical values, the resonance frequencies, and the parameters for the overall motion. For fast internal motions, it is unnecessary to use approaches based on complicated spectral densities derived within the framework of a model because all models that can give the correct value of S² work equally well.
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Food allergy (FA) is perceived as a common problem, especially during childhood. Accurate assessment of incidence and prevalence of FA has been difficult to establish, however, due to lack of universally accepted diagnostic criteria. Although many foods are reported to cause IgE-mediated FA, most studies focus on 4 common food groups: cow's milk, hen's egg, peanut/tree nuts, and fish/shellfish. There may be variation in the prevalence of FA in regions of the world and a likely increase in prevalence has been observed in recent decades. This cannot be stated with confidence, however, without the use of consistent methodology and diagnostic criteria.
Article
The interaction of specific IgE antibodies with allergens is a key event in the induction of allergic symptoms, thus representing an important target for therapeutic interventions in Type I allergies. We report here the solution NMR structure of Art v 1, the major mugwort pollen allergen. Art v 1 is the first protein structure with an allergenic defensin fold linked to a polyproline domain, which has not been identified in any reported allergen structure in the PDB. Moreover, the direct interaction of polyclonal IgE antibodies from an allergic patient has been mapped on the surface of an allergen for the first time. The data presented herein provide the basis for the design of tools for safe and effective vaccination against mugwort pollen allergy.
Article
Parvalbumins are the most important fish allergens. Polysensitization to various fish species is frequently reported and linked to the cross-reactivity of their parvalbumins. Studies on cross-reactivity and its association to the allergenicity of purified natural parvalbumins from different fish species are still lacking. In addition, some studies indicate that dark muscled fish such as tuna are less allergenic. Total protein extracts and purified parvalbumins from cod, whiff, and swordfish, all eaten frequently in Spain, were tested for their IgE-binding properties with 16 fish allergic patients' sera from Madrid. The extent of cross-reactivity of these parvalbumins was investigated by IgE ELISA inhibition assays. Additionally, the cDNA sequences of whiff and swordfish parvalbumins were determined. Extractable amounts of parvalbumins from cod were 20 times and from whiff 30 times higher than from swordfish. Parvalbumins were recognized by 94% of the patients in extracts of cod and whiff, but only by 60% in swordfish extracts. Nevertheless, a high cross-reactivity was determined for all purified parvalbumins by IgE inhibition. The amino acid sequence identities of the three parvalbumins were in a range of 62-74%. The parvalbumins of cod, whiff and swordfish are highly cross-reactive. The high amino acid sequence identity among cod, whiff and swordfish parvalbumins results in the observed IgE cross-reactivity. The low allergenicity of swordfish is due to the low expression levels of its parvalbumin.
Article
In double-blind, placebo-controlled, oral food challenges with fish, a 12-fold higher false-negative rate was found compared with other food antigens. In an effort to elucidate this discrepancy, cooked lyophilized fish extracts (used in double-blind, placebo-controlled, oral food challenges) were compared with cooked, nonlyophilized fish extracts (used in open challenges) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ELISA-inhibition assays. Altered fish allergenicity as a result of food processing was examined with canned tuna and salmon. Forty-five children and young adults with food allergies, including 18 patients with IgE-mediated hypersensitivity to fish, were challenged with canned tuna. All 45 challenges with canned tuna were negative. Two of these patients are allergic to salmon and also have negative reactions to challenges with canned salmon. In vitro investigation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of tuna and salmon extracts revealed a striking loss of definable protein fractions in the canned fish extract when compared with raw and cooked fish extracts, and immunoblot analyses demonstrated minimal IgE-specific binding to the canned fish extracts. In addition, decreased allergenicity of the canned tuna and salmon was demonstrated by ELISA-inhibition assay and by negative oral challenges with canned salmon in two patients allergic to salmon. Collectively, these findings suggest that some of the major allergens responsible for IgE-mediated food allergy to fish are more labile than previously recognized.
Article
A FORTRAN 77 computer program has been written to aid with macromolecular modeling and drug design. Called WHAT IF, it provides an intelligent and flexible environment for displaying, manipulating, and analyzing small molecules, proteins, nucleic acids, and their interactions. A relational protein structure database is incorporated to be queried. The program is suitable for most common crystallographic work. The menu-driven operation of WHAT IF, combined with the use of default values wherever user input is required, makes it very easy to use for a novice user while keeping full flexibility for more sophisticated studies. Although there are not too many unique features in WHAT IF, the fact that everything is integrated in one program makes it a unique tool for many purposes.
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Article
A novel program has been developed for the interpretation of 15N relaxation rates in terms of macromolecular anisotropic rotational diffusion. The program is based on a highly efficient simulated annealing/minimization algorithm, designed specifically to search the parametric space described by the isotropic, axially symmetric and fully anisotropic rotational diffusion tensor models. The high efficiency of this algorithm allows extensive noise-based Monte Carlo error analysis. Relevant statistical tests are systematically applied to provide confidence limits for the proposed tensorial models. The program is illustrated here using the example of the cytochrome c' from Rhodobacter capsulatus, a four-helix bundle heme protein, for which data at three different field strengths were independently analysed and compared.
Article
Several Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. To study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. Two olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunoblotting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. Thirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. EF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross-reactivity among members of the same family.
Article
Association of the parvalbumin AB and CD-EF domains was examined in Hepes-buffered saline, pH 7.4, employing fragments from rat alpha and beta. All of the interactions require Ca(2+). In saturating Ca(2+), the alpha AB/alpha CD-EF (alpha/alpha) complex displays an association constant of (7.6 +/- 0.4) x 10(7) M(-1). Ca(2+)-binding data for a mixture of the alpha fragments are compatible with an identical two-site model, yielding an average binding constant of (8.5 +/- 0.2) x 10(5) M(-1). The beta/beta interaction is significantly weaker, exhibiting an association constant of (3.0 +/- 0.6) x 10(6) M(-1). The Ca(2+)-binding constants for beta/beta are likewise diminished, at (1.0 +/- 0.1) x 10(5) and (2.3 +/- 0.2) x 10(4) M(-1). The magnitude of the apparent DeltaDeltaG(degree)' for Ca(2+) binding by alpha/alpha and beta/beta, at 3.4 kcal/mol, approaches that measured for the intact proteins (3.6 kcal/mol) and is substantially larger than the 1.5 kcal/mol value previously measured for the isolated CD-EF domains. This result suggests that the AB domain can modulate the Ca(2+) affinities of the CD and EF sites. Interestingly, the heterologous alpha/beta complex displays a larger association constant [(6.6 +/- 0.4) x 10(6) M(-1)] than the homologous beta/beta complex and heightened Ca(2+) affinity [binding constants of (1.3 +/- 0.1) x 10(6) and (8.8 +/- 0.2) x 10(4) M(-1)]. By contrast, beta/alpha associates more weakly than alpha/alpha and exhibits sharply reduced affinity for Ca(2+). Thus, the interaction between the beta AB domain and beta CD-EF domain may act to attenuate Ca(2+) affinity in the intact protein.
Article
Plant food allergens belong to a rather limited number of protein families and are also characterized by a number of biochemical and physicochemical properties, many of which are also shared by food allergens of animal origin. These include thermal stability and resistance to proteolysis, which are enhanced by an ability to bind ligands, such as metal ions, lipids, or steroids. Other types of lipid interaction, including membranes or other lipid structures, represent another feature that might promote the allergenic properties of certain food proteins. A structural feature clearly related to stability is intramolecular disulfide bonds alongside posttranslational modifications, such as N-glycosylation. Some plant food allergens, such as the cereal seed storage prolamins, are rheomorphic proteins with polypeptide chains that adopt an ensemble of secondary structures resembling unfolded or partially folded proteins. Other plant food allergens are characterized by the presence of repetitive structures, the ability to form oligomers, and the tendency to aggregate. A summary of our current knowledge regarding the molecular properties of food allergens is presented. Although we cannot as yet predict the allergenicity of a given food protein, understanding of the molecular properties that might predispose them to becoming allergens is an important first step and will undoubtedly contribute to the integrative allergenic risk assessment process being adopted by regulators.
Article
To address data management and data exchange problems in the nuclear magnetic resonance (NMR) community, the Collaborative Computing Project for the NMR community (CCPN) created a "Data Model" that describes all the different types of information needed in an NMR structural study, from molecular structure and NMR parameters to coordinates. This paper describes the development of a set of software applications that use the Data Model and its associated libraries, thus validating the approach. These applications are freely available and provide a pipeline for high-throughput analysis of NMR data. Three programs work directly with the Data Model: CcpNmr Analysis, an entirely new analysis and interactive display program, the CcpNmr FormatConverter, which allows transfer of data from programs commonly used in NMR to and from the Data Model, and the CLOUDS software for automated structure calculation and assignment (Carnegie Mellon University), which was rewritten to interact directly with the Data Model. The ARIA 2.0 software for structure calculation (Institut Pasteur) and the QUEEN program for validation of restraints (University of Nijmegen) were extended to provide conversion of their data to the Data Model. During these developments the Data Model has been thoroughly tested and used, demonstrating that applications can successfully exchange data via the Data Model. The software architecture developed by CCPN is now ready for new developments, such as integration with additional software applications and extensions of the Data Model into other areas of research.
Article
Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients.
Article
Fish-hypersensitive patients can probably tolerate some fish species while being allergic to others. To determine the allergenic cross-reactivity between 9 commonly edible fish: cod, salmon, pollack, mackerel, tuna, herring, wolffish, halibut, and flounder. Sera from 10 patients allergic to fish and rabbit antisera against 3 parvalbumins (Gad c 1, Sal s 1, and The c 1) were used. Cross-reactivity was investigated by SDS/PAGE and IgE immunoblotting, IgG ELISA, IgE ELISA inhibition, and skin prick test (SPT). Cod (Gad c 1), salmon (Sal s 1), pollack (The c 1), herring, and wolffish share antigenic and allergenic determinants as shown by immunoblots and IgE ELISA, whereas halibut, flounder, tuna, and mackerel displayed lowest cross-reactivities. The highest mean IgE ELISA inhibition percent of 10 sera was obtained by Gad c 1, followed by The c 1, herring, Sal s 1, wolffish, halibut, flounder, tuna, and mackerel with the least inhibition. Nine of the 10 patients showed positive SPT to cod, salmon, and pollack; 8 patients reacted to recombinant (r) Sal s 1. Positive SPTs to rGad c 1 and rThe c 1 were demonstrated in 1 patient. Gad c 1, Sal s 1, The c 1, herring, and wolffish contained the most potent cross-reacting allergens, whereas halibut, flounder, tuna, and mackerel were the least allergenic in the current study. The latter could probably be tolerated by some of the tested patients.
Article
As conventional immunotherapy is less efficacious in patients with allergic multi-sensitivities compared with mono-sensitized subjects, new intervention strategies are needed. Therefore, an allergen chimer was genetically engineered for treatment of multi-sensitization with birch and grass pollen on the basis of mucosal tolerance induction. The major birch pollen allergen Bet v 1 served as a scaffold for N- and C-terminal linkage of the immunodominant peptides of the grass pollen allergens Phl p 1 and Phl p 5 and this new construct was cloned and expressed in Escherichia coli. After purification, physicochemical and immunological characterization the chimer was used for intranasal tolerance induction prior to poly-sensitization with Bet v 1, Phl p 1 and Phl p 5. The immunological characterization revealed that the conformation of Bet v 1 within the chimer was comparable to that of natural as well as recombinant Bet v 1. The chimer was immunogenic in mice for T and B cell responses to the three allergens. Intranasal application of the chimer prior to poly-sensitization significantly suppressed humoral and cellular allergen-specific Th2 responses and prevented development of airway inflammation upon allergen challenge. Moreover, local allergen-specific IgA antibodies were induced by the chimer. The mechanisms of poly-tolerance induction seemed to be mediated by regulatory cytokines, since TGF-beta and IL-10 mRNA in splenocytes were upregulated and tolerance was transferable with these cells. The data indicate that such allergen chimers harboring several unrelated allergens or allergen peptides could serve as mucosal polyvalent vaccines for prevention of multi-sensitivities.
Article
The major allergens of trees belonging to the Fagales order are collectively known as the Bet v 1 family. Members of the Fagales order have distinct geographic distribution, and it is expected that depending on the exposure pattern of the individual, inclusion of other Bet v 1 family members might increase the efficacy of the treatment. We aimed to generate molecules that are suitable for specific immunotherapy not only against birch pollen allergy but also against allergies caused by other cross-reactive tree pollens. Fourteen genes of the Bet v 1 family were randomly recombined in vitro by means of DNA shuffling. This library of chimeric proteins was screened for molecules displaying low capacity to induce release of inflammatory mediators but with T-cell immunogenicity higher than that of the parental allergens. Two chimeric proteins were selected from the library of shuffled clones displaying low allergenicity and high immunogenicity, as determined in in vitro assays using human and animal cells and antibodies, as well as in vivo in animal models of allergy. Our results show that it is possible to randomly recombine in vitro T- and B-cell epitopes of a family of related allergens and to select chimeric proteins that perfectly match the criteria presently thought to be relevant for improving allergen-specific immunotherapy. The hypoallergenic chimeras described here recombine epitopes of the major Fagales pollen allergens and thus can efficiently substitute a mixture of extracts used for treating patients with tree pollen-induced spring pollinosis worldwide.
Article
In silico analysis of allergens can identify putative relationships among protein sequence, structure, and allergenic properties. Such systematic analysis reveals that most plant food allergens belong to a restricted number of protein superfamilies, with pollen allergens behaving similarly. We have investigated the structural relationships of animal food allergens and their evolutionary relatedness to human homologs to define how closely a protein must resemble a human counterpart to lose its allergenic potential. Profile-based sequence homology methods were used to classify animal food allergens into Pfam families, and in silico analyses of their evolutionary and structural relationships were performed. Animal food allergens could be classified into 3 main families--tropomyosins, EF-hand proteins, and caseins--along with 14 minor families each composed of 1 to 3 allergens. The evolutionary relationships of each of these allergen superfamilies showed that in general, proteins with a sequence identity to a human homolog above approximately 62% were rarely allergenic. Single substitutions in otherwise highly conserved regions containing IgE epitopes in EF-hand parvalbumins may modulate allergenicity. These data support the premise that certain protein structures are more allergenic than others. Contrasting with plant food allergens, animal allergens, such as the highly conserved tropomyosins, challenge the capability of the human immune system to discriminate between foreign and self-proteins. Such immune responses run close to becoming autoimmune responses. Exploiting the closeness between animal allergens and their human homologs in the development of recombinant allergens for immunotherapy will need to consider the potential for developing unanticipated autoimmune responses.
Article
Allergic reaction following fish consumption can trigger life-threatening reactions in predisposed individuals. Parvalbumins from different species have been identified as the major fish allergens. There are two distinct phylogenetic lineages of parvalbumins, alpha and beta. Most allergic reactions are caused by beta-parvalbumins. We cloned and expressed cDNAs encoding cod (Gadus morhua) and carp (Cyprinus carpio) beta-parvalbumins and purified natural cod beta-parvalbumin. CD spectra of the purified proteins showed that their overall secondary structure contents were very similar. No differences in thermal stability were monitored in the calcium-bound or calcium-depleted form of natural cod parvalbumin. IgE reactivity was assessed using 26 sera of fish allergic patients from Spain, The Netherlands, and Greece in immunoblot and ELISA experiments. Twenty-five of the 26 patients with IgE reactivity to native and recombinant cod parvalbumin also reacted to the recombinant carp parvalbumin. IgE inhibition assays were performed using cod and carp extracts and purified recombinant parvalbumin of cod and carp. High crossreactivity among cod and carp parvalbumins was observed in immunoblots as well as in fluid phase assays. Natural and recombinant parvalbumins gave comparable results when performing various in vitro diagnostic assays.
Automated assignment of ambiguous nuclear overhauser effects with ARIA
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Impact of hydrostatic pressure on an intrinsically disordered protein: high-pressure NMR study of a-synuclein
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A general strategy for generation of hypoallergenic molecules for immunotherapy of fish allergy
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Swoboda I, Balic N, Klug C, Focke M, Weber M, Spitzauer S, Neubauer A, Quirce S, Douladiris N, Papadopoulus N, Valenta R. A general strategy for generation of hypoallergenic molecules for immunotherapy of fish allergy. J Allergy Clin Immunol 2013;132:979–981.