Major Fish Allergen Research Articles

Effect of tyrosinase and caffeic acid crosslinking of turbot parvalbumin on the digestibility, and release of mediators and cytokines from activated RBL-2H3 cells

Turbot can induce allergy in susceptible individuals due to the presence of parvalbumin (PV), a major fish allergen. This study aimed at evaluating the digestibility and the ability of PV to elicit the release of cellular degranulation, following treatment with tyrosinase (PV-Tyr), caffeic acid (PV-CA) and in combination (PV-Tyr/CA), using in vitro digestion and RBL-2H3 (passive rat basophil leukemia) cell line. The digestion assay products revealed that the stability of PV in simulated gastric fluid (SGF) was stronger, while in simulated intestinal fluid (SIF) was rather weak. Western blot analysis revealed that the IgG-binding abilities of the cross-linked PV were markedly reduced. Moreover, crosslinking hampered the release of cellular degranulation process in RBL-2H3 cell lines. PV-Tyr/CA showed highly significant reduction in the release rate of β-hexosaminidase (66.02%), histamine (35.01%), tryptase (29.25%), cysteinyl leukotrienes (29.72%), prostaglandin D2 (34.96%), IL-4 (43.99%) and IL-13 (38.93%) and shown potential in developing hypoallergenic fish products.

Rational design of a hypoallergenic Phl p 7 variant for immunotherapy of polcalcin-sensitized patients

Polcalcins are important respiratory panallergens, whose IgE-binding capacity depends on the presence of calcium. Since specific immunotherapy is not yet available for the treatment of polcalcin-sensitized patients, we aimed to develop a molecule for efficient and safe immunotherapy. We generated a hypoallergenic variant of the grass pollen polcalcin Phl p 7 by introducing specific point mutations into the allergen’s calcium-binding regions. We thereby followed a mutation strategy that had previously resulted in a hypoallergenic mutant of a calcium-binding food allergen, the major fish allergen parvalbumin. Dot blot assays performed with sera from Phl p 7-sensitized patients showed a drastically reduced IgE reactivity of the Phl p 7 mutant in comparison to wildtype Phl p 7, and basophil activation assays indicated a significantly reduced allergenic activity. Rabbit IgG directed against mutant rPhl p 7 blocked patients’ IgE binding to wildtype Phl p 7, indicating the mutant’s potential applicability for immunotherapy. Mass spectrometry and circular dichroism experiments showed that the mutant had lost the calcium-binding capacity, but still represented a folded protein. In silico analyses revealed that the hypoallergenicity might be due to fewer negative charges on the molecule’s surface and an increased molecular flexibility. We thus generated a hypoallergenic Phl p 7 variant that could be used for immunotherapy of polcalcin-sensitized individuals.

Variability of allergens in commercial fish extracts for skin prick testing.

Commercial allergen extracts for allergy skin prick testing (SPT) are widely used for diagnosing fish allergy. However, there is currently no regulatory requirement for standardization of protein and allergen content, potentially impacting the diagnostic reliability of SPTs. We therefore sought to analyse commercial fish extracts for the presence and concentration of fish proteins and invitro IgE reactivity using serum from fish-allergic patients. Twenty-six commercial fish extracts from five different manufacturers were examined. The protein concentrations were determined, protein compositions analysed by mass spectrometry, followed by SDS-PAGE and subsequent immunoblotting with antibodies detecting4 fish allergens (parvalbumin, tropomyosin, aldolase and collagen). IgE-reactive proteins were identified using serum from 16 children with confirmed IgE-mediated fish allergy, with focus on cod, tuna and salmon extracts. The total protein, allergen concentration and IgE reactivity of the commercial extracts varied over 10-fold between different manufacturers and fish species. The major fish allergen parvalbumin was not detected by immunoblotting in 6/26 extracts. In 7/12 extracts, five known fish allergens were detected by mass spectrometry. For cod and tuna, almost 70% of patients demonstrated the strongest IgE reactivity to collagen, tropomyosin, aldolase A or β-enolase but not parvalbumin. Commercial fish extracts often contain insufficient amounts of important allergens including parvalbumin and collagen, resulting in low IgE reactivity. A comprehensive proteomic approach for the evaluation of SPT extracts for their utility in allergy diagnostics is presented. There is an urgent need for standardized allergen extracts, which will improve the diagnosis and management of fish allergy.

Molecular characterization of B-cell epitopes for the major fish allergen, parvalbumin, by shotgun proteomics, protein-based bioinformatics and IgE-reactive approaches

Parvalbumins beta (β-PRVBs) are the main fish allergens. The only proven and effective treatment for this type of hypersensitivity is to consume a diet free of fish. We present the molecular characterization of B-cell epitopes by shotgun proteomics of different β-PRVBs combined with protein-based bioinformatics and IgE-reactive approaches. The final goal of this work is to identify potential peptide vaccine candidates for fish allergy. Purified β-PRVBs from the main fifteen different fish species that cause allergy were analyzed by shotgun proteomics. Identified β-PRVBs peptide sequences and ninety-eight β-PRVB protein sequences from UniProtKB were combined, aligned and analyzed to determine B-cell epitopes using the Kolaskar and Tongaonkar algorithm. The highest rated predicted B-cell peptide epitopes were evaluated by ELISA using the corresponding synthetic peptides and sera from healthy and fish allergic patients. A total of 35 peptides were identified as B-cell epitopes. The top B-cell peptide epitopes (LKLFLQV, ACAHLCK, FAVLVKQ and LFLQNFV) that may induce protective immune responses were selected as potential peptide vaccine candidates. The 3D model of these peptides were located in the surface of the protein. This study provides the global characterization of B-cell epitopes for all β-PRVBs sequences that will facilitate the design of new potential immunotherapies. SignificanceThis work provides the global characterization of B-cell epitopes for all β-PRVBs sequences by Shotgun Proteomics combined with Protein-based Bioinformatics and IgE-reactive approaches. This study will increase our understanding of the molecular mechanisms whereby fish allergens elicit allergic reactions and will facilitate the design of new potential peptide vaccine candidates.

S100 proteins: Diagnostic and prognostic biomarkers in laboratory medicine

S100 proteins are members of the superfamily of Ca2+-binding proteins characterized by the specific Ca2+-binding motif, the EF-hand. Proteins of this superfamily are of clinical use as important diagnostic and prognostic biomarkers in adult and pediatric Laboratory Medicine. For example, measurements of troponin are nowadays the ‘gold standard’ in the diagnosis of patients with acute coronary syndrome. Parvalbumins were identified as major fish allergens and blocking antibodies, induced by immunization with a hypoallergenic parvalbumin mutant, were shown to reduce allergic symptoms. Mutations in calmodulin are linked to inherited ventricular tachycardia, and cardiac arrhythmias. S100 proteins, the largest sub-group within the EF-hand protein family, are closely associated with cardiovascular diseases, various types of cancer, inflammation and autoimmune pathologies and brain diseases. The intention of this review is to focus on the clinical use of S100 proteins as biomarkers and potential drug targets helping to improve the diagnosis of these human diseases in children and adults leading to more selective therapeutic interventions.

Purification, Characterization, and Crystal Structure of Parvalbumins, the Major Allergens in Mustelus griseus.

Fish play important roles in human nutrition and health, but also trigger allergic reactions in some population. Parvalbumin (PV) represents the major allergen of fish. While IgE cross-reactivity to PV in various bony fish species has been well characterized, little information is available about allergens in cartilaginous fish. In this study, two shark PV isoforms (named as SPV-I and SPV-II) from Mustelus griseus were purified. Their identities were further confirmed by mass spectroscopic analysis. IgE immunoblot analysis showed that sera from fish-allergic patients reacted to both SPV-I and SPV-II, but the majority of sera reacted more intensely to SPV-I than SPV-II. Thermal denaturation monitored by CD spectrum showed that both of the SPV allergens are highly thermostable. SPV-I maintained its IgE-binding capability after heat denaturation, while the IgE-binding capability of SPV-II was reduced. The results of crystal structure showed that SPV-I and SPV-II were similar in their overall tertiary structure, but their amino acid sequences shared lower similarities, indicating that the differences in the IgE-binding capabilities of SPV-I and SPV-II might be due to differential antigen epitopes in these two isoforms.

Identification of Linear IgE Epitopes for a Major Allergen–Type I Collagen in Fish (Rainbow Trout)

Type I collagen was described as a major allergen in fish. The purpose of this study was to screen and identify the linear IgE epitopes of type I collagen α1 and α2 subunits in rainbow trout. Five bioinformatics tools were used to predict the potential epitopes and the resultant epitopes were confirmed by LAD2 cells degranulation assay with sera from fish allergic patients. As the result, 10 peptides of α1 and α2 subunits were predicted, respectively, and these peptides were assembled by solid-phase synthesis. 14 epitopes were identified by LAD2 cells degranulation assay, among which, peptide 2, 5–7 were identified as linear epitope of α1 and peptide 11–20 were identified as linear epitope of α2. Moreover, for α1 and α2 subunits, the similarity of sequences was greater than 79%, suggesting the cross-reactivity of fish collagen. The findings of this study provided a strong support for further research of reduction of the collagen allergenicity.

Characterization of Rask1 a novel major allergen in Indian mackerel and identification of parvalbumin as the major fish allergen in 33 Asia-Pacific fish species.

Fish is a well-recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under-investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat-processed fish from Vietnam. The aim of this study was to identify the major heat-stable allergens from frequently exported Asia-Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta). Heated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV-specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy. Heat-stable IgE-reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D-gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species. Different PVs were identified as the major heat-stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world-wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Rask1. Improved diagnostics for fish allergy against Asia-Pacific species should be developed with focus on PV.

Solution structure of the major fish allergen parvalbumin Sco j 1 derived from the Pacific mackerel

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.

Severe anaphylaxis due to crocodile-meat allergy exhibiting wide cross-reactivity with fish allergens

Fish is one of the 8 most important elicitors of food-allergic reactions alongside egg, peanuts, tree nuts, and shellfish.1 Parvalbumins have been shown to be major fish allergens. It has been suggested that the allergic response to this protein is related to the quantity of the allergen present in the muscle of different fish species.2

In search for globally disordered apo-parvalbumins: Case of parvalbumin β-1 from coho salmon

Parvalbumin (PA) is a classical EF-hand calcium-binding protein of muscle, neuronal, and other tissues, and a major fish allergen. Although certain apo-PAs lack tertiary structure, functional implications of that feature and its structural prerequisites remain unclear. In a search for unstable PAs, we probed conformational stability of parvalbumin β-1 from coho salmon (csPA), a cold water fish species, using circular dichroism, scanning calorimetry, hydrophobic probe fluorescence, limited proteolysis, chemical crosslinking and dynamic light scattering techniques. Apo-csPA is shown to be mainly monomeric protein with markedly disorganized secondary structure and lack of rigid tertiary structure. Examination of per-residue propensity for intrinsic disorder in the PA groups with either folded or unfolded apo-form using the average PONDR® VSL2P profiles revealed that the N-terminal region that includes α-helix A, AB-loop and N-terminal half of α-helix B is predicted to be less ordered in PAs with disordered apo-state. Application of the structural criteria developed for discrimination of disordered PAs indicate that the latter comprise about 16–19% of all PAs. We show that structural instability of apo-β-PA serves as a hallmark of elevated calcium affinity of the protein. Therefore, the successful predictions of unstable apo-PAs might facilitate search for PAs with maximal calcium affinity and possibly serving as calcium sensors.

Sensitivity of Moroccans to sardine parvalbumin and effect of heating and enzymatic treatments

ABSTRACTThe aim of this study was to evaluate the IgE sensitivity to sardine parvalbumin (SP) in a Moroccan population from Fez region, and then to study the effect of temperature and enzymatic digestion on the allergenicity of SP. This work was conducted with a questionnaire completed by a sera-bank, obtained from 1008 patients recruited from Fez Hospitals. Their sera were analyzed for specific IgE against SP. From the questionnaire, 8.1% reported adverse reactions to fish and shellfish, where sardine was the most common species causing adverse reactions in patients. Evaluation of specific IgE showed that 7.8% of patients present higher values. Further indirect ELISA and dot-blot results indicated that SP, a major fish allergen, showed a reduction in the human IgE binding under heating and pepsin hydrolysis. These results demonstrate that this population was sensitive to SP and the sensitivity could be reduced by heating and more where it was digested by pepsin.

Optimisation of an extraction technique of fish allergens suitable for detection and diagnosis

An optimised protocol for the extraction of allergenic proteins in fish was developed. Twelve existing or modified extraction buffers were evaluated based on the amount and quality of obtained 12 kDa IgG-binding protein (major fish allergen parvalbumin) by SDS-PAGE, immunoblotting, and ELISA. The results indicated that using DL-Dithiothreitol during the extraction process, the stability and functionality of the final extract were significantly increased. Solution 3 (containing Tris-HCl, Glycine, and DL-Dithiothreitol) yielded the highest amount of parvalbumin. The extract also induced a higher allergenic reactivity to the three tested human sera by immunoblotting. The results indicated that solution 3 provided better results in fish allergen detection and diagnosis. In conclusion, our study discovered and established solution 3 (Tris-HCl + Glycine + DTT) as the most potential buffer to overcome the conventional allergen extraction buffer and therefore it is highly recommended as a substitute optimised extraction buffer.

Allergenicity of bony and cartilaginous fish - molecular and immunological properties.

Allergy to bony fish is common and probably increasing world-wide. 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 47-54%. 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.

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy

BackgroundFish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed.ObjectivesThis study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice.MethodsC3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1–specific basophil degranulation, and Cyp c 1–induced allergic symptoms in the mouse model.ResultsA mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1–induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice.ConclusionsAntibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy.

Effect of Maillard reaction on the structural and immunological properties of recombinant silver carp parvalbumin

The purpose of this study was to investigate the influence of Maillard reaction on the structural and immunological properties of parvalbumin (PV), the major allergen in fish. Recombinant silver carp PV (rPV) was employed and incubated with glucose at 60 °C for 72 h. The IgG/IgE binding properties of rPV were weakened after Maillard reaction as demonstrated by dot blotting. Allergenicity decrease of rPV by Maillard reaction was further confirmed on sensitized RBL-2H3 cell with the decreasing release of β-hexosaminidase, histamine and suppressing the production of interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α). Comparison of the glycation sites with documented epitopes suggested the direct blocking of conjugated carbohydrates (K88, K97, and K108) on IgE-binding epitopes. Glycated rPV (G-rPV) exhibited slightly more resistance against pepsin digestion in vitro, which probably due to the formation of aggregation and the increase of hydrophobicity of G-rPV.

Allergy to fish collagen: Thermostability of collagen and IgE reactivity of patients' sera with extracts of 11 species of bony and cartilaginous fish

BackgroundParvalbumin was identified as a major fish allergen, and has been well investigated. Collagen was identified as a second allergen; however, its allergenic properties remain uncharacterized. Although fish is an important staple in coastal countries, its thermostability is unknown. Therefore, we aimed to determine the thermostability of fish collagen as an allergen. MethodsMeat of seven bony and four cartilaginous fishes was heated at various temperatures and times, and extracts were analyzed using SDS-PAGE, IgE-ELISA, and SPTs. ResultsCollagen was dissolved from heated meat of Pacific mackerel into a crude extract. Collagen in the extracts was degraded at a high heating load—140 °C (10 min) or 100 °C (320 min). However, ELISA revealed the IgE reactivities of patients' sera with the extracts were unchanged even after heating the samples. Patients strongly reacted to extract proteins of other bony fish, which were detected by patients' IgE even after heating at 100 °C (320 min). In contrast, reactivities of the extracts of cartilaginous fish were lower than those of bony fish. SPTs in one patient revealed that all bony and cartilaginous fish extracts prepared from heated meat elicited allergic reactions. ConclusionsThe IgE reactivity of patients' sera to fish collagen in extracts was retained even when fish meat was treated by a high heating load. As for the fish collagen, the IgE reactivities to cartilaginous fish were lower than that to bony fish. Reducing IgE reactivity to fish meat using heat is difficult, and other modalities will be required to produce hypoallergenic fish meat.

Reduction in IgE reactivity of Pacific mackerel parvalbumin by heat treatment

Parvalbumin, a major fish allergen, has been reported to be highly thermostable. However, little is known as to whether parvalbumin is stable at more than 100°C. Thermostability of the Pacific mackerel parvalbumin was examined by subjecting heated (20–140°C) muscle extracts to SDS–PAGE, western blotting and ELISA. As judged by SDS–PAGE and western blotting with the anti-parvalbumin antiserum recognizing the primary structure, the parvalbumin was not degraded even under severe heating conditions. However, western blotting analysis with the monoclonal antibody recognizing the stereoscopic structure revealed that the parvalbumin undergoes conformational changes in a heating load-dependent manner. Importantly, the IgE reactivity of the parvalbumin determined by ELISA using patient sera was also reduced in a heating load-dependent manner; complete loss of IgE reactivity was induced by heating at 140°C. This study showed that the allergenicity of the Pacific mackerel parvalbumin is considerably less thermostable than assumed for other fish parvalbumins.

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

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.

Quantification of major allergen parvalbumin in 22 species of fish by SDS–PAGE

Fish is an important causative material of food allergy. Although the allergenicity of fish is considered to correlate with the content of parvalbumin, the major fish allergen, available information about the parvalbumin content in fish is limited. In this study, a simple and reliable quantification method for fish parvalbumin by SDS–PAGE was first established. Application of the SDS–PAGE method to 22 species of fish revealed a marked variation in parvalbumin content among fish. Furthermore, the parvalbumin content was found to be higher in dorsal white muscle than in ventral white muscle, in rostral part of white muscle than in caudal part of white muscle and in white muscle than in dark muscle. IgE reactivity of fish was roughly proportional to parvalbumin content. Interestingly, large-sized migratory fish, such as salmon, swordfish and tuna, were commonly very low in both parvalbumin content and IgE reactivity.