Parvalbumin Isoforms Research Articles

Mercury metalloproteomic profile in muscle tissue of Arapaima gigas from the Brazilian Amazon.

Metalloproteomics is an innovative methodology for identifying of protein-associated mercury. Thus, we analyzed the muscle proteome of Arapaima gigas (pirarucu), collected in the Madeira River of the Brazilian Amazon, to identify protein-associated mercury, with the aim of identifying possible mercury biomarkers in fish muscle tissue. After obtaining the protein pellet, we conducted two-dimensional electrophoresis (2D PAGE) to fractionate the muscle proteome. Total mercury in muscle tissue and protein pellets and mapping of mercury content in protein spots of the 2D PAGE gels was determined using graphite furnace atomic absorption spectrometry (GFAAS). The protein-associated mercury identification was performed using liquid chromatography coupled with sequence mass spectrometry (LC‒MS/MS). Total mercury determinations by GFAAS indicated concentrations on the order of 153 ± 1.90mgkg-1 and 142 ± 1.50mgkg-1 (total precipitation of protein fraction) and 139 ± 1.45mgkg-1 (fractional precipitation of protein fraction) in muscle tissue and protein pellets, respectively. Mercury concentrations in the range of 48 ± 0.90 to 165 ± 3.00mgkg-1 were found in twelve protein spots. Among the 2D PAGE protein spots, eleven Hg-binding proteins were identified using LC‒MS/MS, which showed characteristics of mercury exposure biomarkers for important metabolic functions, such as five parvalbumin isoforms, triosephosphate isomerase, cofilin 2 (muscle), and fructose-bisphosphate aldolases.

The first reptilian allergen and major allergen for fish-allergic patients: Crocodile β-parvalbumin.

BackgroundClinical cross‐reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish‐allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE‐cross‐binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE‐binding proteins in crocodile meat with a focus on PV and their clinical relevance.MethodsProteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish‐allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE‐binding proteins were identified and quantified by SDS‐PAGE, mass spectrometric analyses, and immunoblotting using commercial and in‐house antibodies, as well as individual and pooled patients’ serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay.ResultsOf the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish‐allergic patients. Seventy percent (31 of 44) of fish PV‐sensitized patients demonstrated IgE‐binding to crocodile PV. Crocodile β‐PV was the major IgE‐binding protein but 20‐fold less abundant than α‐PV. Cellular reactivity was demonstrated for β‐PV and epitopes predicted, explaining frequent IgE‐cross‐binding of β‐PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (β‐PV as Cro p 1 and α‐PV as Cro p 2).ConclusionFish‐allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.

Structural Changes beyond the EF-Hand Contribute to Apparent Calcium Binding Affinities: Insights from Parvalbumins.

Members of the parvalbumin (PV) family of calcium (Ca2+) binding proteins (CBPs) share a relatively high level of sequence similarity. However, their Ca2+ affinities and selectivities against competing ions like Mg2+ can widely vary. We conducted molecular dynamics simulations of several α-parvalbumin (αPV) constructs with micromolar to nanomolar Ca2+ affinities to identify structural and dynamic features that contribute to their binding of ions. Specifically, we examined a D94S/G98E construct with a lower Ca2+ affinity (≈-18 kcal/mol) relative to the wild type (WT) (≈-22 kcal/mol) and an S55D/E59D variant with enhanced affinity (≈-24 kcal/mol). Additionally, we also examined the binding of Mg2+ to these isoforms, which is much weaker than Ca2+. We used mean spherical approximation (MSA) theory to evaluate ion binding thermodynamics within the proteins' EF-hand domains to account for the impact of ions' finite sizes and the surrounding electrolyte composition. While the MSA scores differentiated Mg2+ from Ca2+, they did not indicate that Ca2+ binding affinities at the binding loop differed between the PV isoforms. Instead, molecular mechanics generalized Born surface area (MM/GBSA) approximation energies, which we used to quantify the thermodynamic cost of structural rearrangement of the proteins upon binding ions, indicated that S55D/E59D αPV favored Ca2+ binding by -20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E αPV. Meanwhile, Mg2+ binding was favored for the S55D/E59D αPV and D94S/G98E αPV variants by -18.32 and -1.65 kcal/mol, respectively. These energies implicate significant contributions to ion binding beyond oxygen coordination at the binding loop, which stemmed from changes in α-helicity, β-sheet character, and hydrogen bonding. Hence, Ca2+ affinity and selectivity against Mg2+ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions elsewhere. Our findings broaden our understanding of the molecular bases governing αPV ion binding that are likely shared by members of the broad family of CBPs.

Molecular and immunological characterization of grass carp (Ctenopharyngodon idella) parvalbumin Cten i 1: A major fish allergen in Hong Kong.

Grass carp is the most commonly consumed fish species in Hong Kong. The allergenicity of grass carp and its allergen content are yet to be reported. This study characterized the major allergen in grass carp and investigated its allergenicity. Sixty-nine subjects with history of IgE-mediated allergic reaction to grass carp were recruited. The protein content in steamed grass carp extract was resolved by SDS-PAGE, and the major allergen was identified by immunoblotting with serum from subjects allergic to grass carp. The identity of allergen was elucidated by mass spectrometry and amino acid sequence obtained by amplifying the specific gene from cDNA library of grass carp. The cross-reactivity between parvalbumins from grass carp and other phylogenetically close (common carp) or commercially important (cod and salmon) species was investigated by competitive inhibition ELISA. A major IgE-binding protein was found at approximately 9kDa and identified as parvalbumin by immunoblotting and mass spectrometry. Grass carp parvalbumin was more allergenic than common carp, salmon, and cod parvalbumins despite sharing high sequence homology. This newly identified major allergenic parvalbumin isoform from grass carp was registered as Cten i 1 in the World Health Organization and International Union of Immunological Societies allergen database. Grass carp parvalbumin is identified as the major fish allergen in Hong Kong. The strong allergenicity of Cten i 1 contributes to the high IgE reactivity of grass carp. Grass carp, among other fish species, should be considered when managing fish-allergic patients.

Characteristics of Chinese fish-allergic patients: Findings from double-blind placebo-controlled food challenges

The use of fish extracts in diagnosis is often held back by low specificity,1 varying allergen content in fish extracts,2 and the presence of cross-reactive hypoallergenic parvalbumin isoforms.3 Use of component-resolved diagnostic with Gad c 1 as the generic fish allergy marker overlooks the varying pattern of fish consumption across continents. Perez-Gordo et al4 showed that sole and whiff parvalbumins were the major fish allergens in a Spanish population. Barramundi, salmon, and tuna were the major causative species of fish allergy in Australia,5 whereas mackerel scad and anchovy were major culprits in the Philippines.

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.

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.

Identification of protein biomarkers of mercury toxicity in fish

Bioaccumulative metals such as mercury are found in increasing amounts in fish and their consumers. In the region of the Madeira River, in the Brazilian Amazon, mercury (Hg) is a predominant contaminant in the aquatic ecosystem. There is therefore a need to find specific biomarkers of mercury toxicity in fish to monitor contaminations. Here, mercury-bound proteins were identified in the liver tissues of fishes Mylossoma duriventre and Brachyplatystoma rousseauxii. Mercury was quantified in liver tissue, pellets and protein spots by graphite furnace atomic absorption. Proteins were fractionated by two-dimensional polyacrylamide gel electrophoresis and identified by mass spectrometry with electrospray ionization. We identified nine proteins linked to mercury and that presented biomarker characteristics of mercury. Among the proteins identified, isoforms of parvalbumin, ubiquitin-40S ribosomal protein S27a, brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 and betaine–homocysteine S-methyltransferase 1 are notable for having the molecular function of binding to metallic ions.

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.

Rapid Muscle Relaxation in Siniperca chuatsi is Coordinated by Parvalbumin (PVALB) and MiR-181a.

Parvalbumins (PVALBs) are particularly abundant in the fast-contracting muscles and correlate positively with muscle relaxation speed in amphibians and fishes. MiRNAs play important roles in diverse biologic processes via binding to the 3' untranslated region (3'UTR) of the target mRNAs. In the study, four PVALB isoforms, named as PVALB1, 2, 3, and 4, were identified in the mandarin fish (Siniperca chuatsi) fast muscle and PVALB4 exhibited the highest expression level among them. By bioinformatics analysis, a putative miR-181a binding site in PVALB4 was detected and the direct interaction between miR-181a and PVALB4 was confirmed with the luciferase reporter assay. Further, when miR-181a was inhibited, it substantially increased PVALB4 mRNA expression level and the muscle relaxation rate in vivo. Taken together, the obtained results suggest that miRNA-181a/PVALB4 is an evolutionarily conserved miRNAtarget pair and their interaction is correlated with muscle relaxation rate in the mandarin fish. Therefore, the study revealed a novel molecular mechanism in the regulation of skeletal muscle relaxation in fish.

Differentiation between fresh and frozen–thawed sea bass (Dicentrarchus labrax) fillets using two-dimensional gel electrophoresis

This study aimed to identify a protein marker that can differentiate between fresh skinless and frozen–thawed sea bass (Dicentrarchus labrax) fillets using the two-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Distinct gel patterns, due to proteins with low molecular weight and low isoelectric points, distinguished fresh fillets from frozen–thawed ones. Frozen–thawed fillets showed two specific protein spots as early as the first day of the study. However, these spots were not observed in fresh fillets until at least 13days of storage between 0 and 4°C, fillets were judged, beyond this period, fish were unfit for human consumption as revealed by complementary studies on fish spoilage indicators namely total volatile basic nitrogen and biogenic amines. Mass spectrometry identified the specific proteins as parvalbumin isoforms. Parvalbumins may thus be useful markers of differentiation between fresh and frozen–thawed sea bass fillets.

Differential IgE binding to isoallergens from Asian seabass (Lates calcarifer) in children and adults

Fish allergy is a common food allergy, with prevalence rates in the general population ranging between 0.2% and 2.3%. In both adults and children fish ranks in the top eight foods known to cause IgE mediated food allergy. Fish allergy is rarely outgrown and individuals with fish allergy may be allergic to some but not all species of fish. Whilst fish allergy occurs around the world, the characterization of allergenic components of individual species of fish has been largely confined to Northern hemisphere and European fish species. To date allergy to commonly consumed fish in the Asian-Pacific region including barramundi (Asian seabass; Lates calcarifer) have been less well investigated.The aim of this study was to identify and characterize allergenic proteins from barramundi in both fish allergic adult and pediatric patients. Serum from 17 fish allergic adults and children from Australia were characterized by immunoblotting and enzyme linked immunosorbent assays (ELISA) against raw and heated barramundi. Molecular analysis of identified allergens included genetic sequencing and generation of recombinant isoallergens. Two novel parvalbumin isoforms of the β-type were identified as the only allergens in barramundi and subsequently designated as Lat c 1.0101 and Lat c 1.0201 by the International Union of Immunological Societies. These two isoallergens do not differ in their ability to bind IgE antibodies, but are differentially expressed in barramundi tissue. This study characterized two novel heat stable parvalbumin allergens from barramundi, with differential IgE binding capacity between adults and pediatric patients.

Fish allergens at a glance: variable allergenicity of parvalbumins, the major fish allergens.

Fish is a common trigger of severe, food-allergic reactions. Only a limited number of proteins induce specific IgE-mediated immune reactions. The major fish allergens are the parvalbumins. They are members of the calcium-binding EF-hand protein family characterized by a conserved protein structure. They represent highly cross-reactive allergens for patients with specific IgE to conserved epitopes. These patients might experience clinical reactions with various fish species. On the other hand, some individuals have IgE antibodies directed against unique, species-specific parvalbumin epitopes, and these patients show clinical symptoms only with certain fish species. Furthermore, different parvalbumin isoforms and isoallergens are present in the same fish and might display variable allergenicity. This was shown for salmon homologs, where only a single parvalbumin (beta-1) isoform was identified as allergen in specific patients. In addition to the parvalbumins, several other fish proteins, enolases, aldolases, and fish gelatin, seem to be important allergens. New clinical and molecular insights advanced the knowledge and understanding of fish allergy in the last years. These findings were useful for the advancement of the IgE-based diagnosis and also for the management of fish allergies consisting of advice and treatment of fish-allergic patients.

Sequence microheterogeneity of parvalbumin, the major fish allergen

The microheterogeneity of amino acid sequence observed in various allergens may affect immune response, but incidence of sequence microheterogeneity in allergens and its relation to their allergenicity are unclear. The occurrence of sequence microheterogeneity in major fish allergen, parvalbumin (PA), has been explored using bioinformatics approaches. 44% of 111 species with known PA sequence have PA isoforms. 41% of these species exhibit from 1 to 4 cases of PA sequence microheterogeneity, i.e. unique pairs of PA isoforms with sequence identity above 90%. 29% of 210 PA sequences studied are characterized by microheterogeneity. The occurrence of allergens among them is 2.5-fold higher than among other PAs. The incidence of sequence microheterogeneity within more allergenic β isoform of PA is 2.0-fold lower than that for its less allergenic α isoform. 39 residues affected by PA microheterogeneity are concentrated in the region of helices A, B, F, while helices D and E are the most conservative region. 44% and 11% of the microheterogeneous substitutions are located in the species-specific and cross-reactive IgE-binding epitopes of PAs, respectively. 45% and 48% of the substitution cases are predicted to cause notable changes in protein disorder propensity and protein stability, respectively. Hence, the increased allergenicity rate among PAs having microheterogeneous isoforms can be related to differences in their IgE-binding caused directly or in an allosteric manner. Overall, sequence microheterogeneity is shown to be inherent to many of PAs and likely contributes to PA allergenicity.

Metal-specific structural changes in parvalbumin

Parvalbumin is a small protein of EF-hand family whose main role is considered to be metal buffering. Recent evidences indicate that parvalbumin also fulfills more complicated functions, which may be determined by the diversity in structural changes in response to the binding of different metal cations. In the present work the conformations of α and β isoforms of pike parvalbumin in the Ca2+- and Mg2+-loaded state were studied by intrinsic fluorescence, circular dichroism and bis-ANS extrinsic fluorescence. We have determined the structural region causing different spectral response on the binding of Mg2+- and Ca2+ ions in pike β-parvalbumin. Our data reveal similarity of the metal-bound forms of α-parvalbumin. In contrast, those of β isoform differ significantly in the tyrosine spectral range. We also discuss the possible physiological consequences of the structural rearrangements accompanied Mg2+/Ca2+ exchange in pike β-parvalbumin.

A proteomics strategy for determining the synthesis and degradation rates of individual proteins in fish

In order to study the protein dynamics in the tissues of fish we have developed a proteomics-based strategy to determine the rates of synthesis and degradation of individual proteins. We have demonstrated the feasibility of this approach by measuring the turnover of multiple isoforms of parvalbumin (β1-7) in the skeletal muscle of common carp (Cyprinus carpio). A stable isotope-labelled amino acid ([(2)H(7)] l-leucine) was administered to the carp via the diet and its incorporation into the isoforms of parvalbumin in muscle over time was monitored by LC-MS analysis of signature peptides. The relative isotope abundance was calculated and used to deconvolute the data. The β7 parvalbumin isoform had a rate of synthesis that was greater than the rate of degradation. In contrast the rate of degradation of the β5 isoform exceeded its rate of synthesis, whilst the analysis revealed that the other parvalbumin β-isoforms (β1, β2, β3, β4 and β6) had a rate of synthesis that was equal to the rate of degradation. This work has addressed a number of technical challenges and represents the first study to use proteomic approaches to measure the turnover of individual proteins in fish.

Comparative Analyses of Allergens between Landlocked and Anadromous Species of Masu Salmon Oncorhynchus masou masou

Although the difference in allergenicity between landlocked and anadromous salmon is little understood, only anadromous salmon are recommended to be labeled in the current allergen labeling system. This study was designed to examine the allergenic potency of landlocked species (yamame) and anadromous species (sakuramasu) of masu salmon Oncorhynchus masou masou, with special reference to parvalbumin, a known major fish allergen. Analysis of the heated extracts by SDS-PAGE suggested that yamame contains parvalbumin in the muscle at considerably higher levels, as compared with sakuramasu. In accordance with this, the parvalbumin content in the muscle of yamame (1.8-7.8 mg/g), determined by visible-light ELISA, was significantly higher than that of sakuramasu (0.28-0.52 mg/g). Furthermore, fluorescence ELISA experiments showed that the heated extract from yamame reacts with serum from fish-allergic patients more strongly than that from sakuramasu. Three parvalbumin isoforms (PA-I, -II and -III) were individually purified from yamame and sakuramasu by gel filtration and reverse-phase HPLC. Based on the retention times in reverse-phase HPLC and the molecular weights estimated by MALDI/TOF-MS, PA-I, -II and -III from yamame were judged to be identical with PA-I, -II and -III from sakuramasu, respectively. Taken together, our data indicate that landlocked masu salmon (yamame) is more allergenic than anadromous salmon (sakuramasu).

Molecular cloning and sequences analysis of parvalbumin gene in crucian carp and mandarin fish

Parvalbumin(PV) is the major allergen of fish species.So far,research concerning fish allergy has been focused mainly on marine fish.China is the largest consumer and producer of freshwater fish in the world,and many people suffered from allergy when consuming freshwater fish.Therefore,figuring out the molecular characters of PV from freshwater fish would be important.In this study,four gene sequences of PV isoforms were amplified and cloned from crucian carp and mandarin fish by RT-PCR,with two genes from each species.All the four genes were 330 bp in length,encoding 109 amino acid residues.The deduced molecular weight of them was approximately 11.6 ku,and isoelectric points were 4.45-4.69.The existence of amino acid residues of Ala-14,Leu-16,Cys-19,Phe-67,Gln-69 and Thr-79 suggested that all the sequences belonged to β-PV.The identity between the two sequences of crucian carp was 80.73 %,and was 83.49 % in mandarin fish.The identity between PVs from different fish species was higher than 75 % and the sequences of Ca2+binding sites were highly conserved.Phylogenetic tree based on some PV amino acid residues revealed that PVs are clustered into two major clades,teleost fish and other vertebrates.PVs of teleost were clustered into orthologous groups,suggesting duplication of the PV genes before the speciation of teleost while after the speciation of fish.The tertiary structures of these four PVs were constructed using SWISS-MODEL.They all contain three helix-loop-helix motifs named EF-hand.Two domains next to the C terminal were Ca2+ binding sites.These results may provide a molecular basis for further study on the allergen of fish PVs.

Purification, immunological properties and molecular cloning of two allergenic parvalbumins from the crimson sea bream, Evynnis japonica

Parvalbumin, a calcium-binding sarcoplasmic protein of 12kDa, represents a major allergen of fish. Previous immunoblotting experiments suggested the presence of a 14kDa allergen, together with parvalbumin, in sea breams. In this study, the 14kDa allergen (PA I) and parvalbumin (PA II) were purified from the white muscle of crimson sea bream, Evynnis japonica, by gel filtration and reverse-phase HPLC. Amino acid sequencing of lysylendopeptidase peptide fragments demonstrated that both PA I and PA II were isoforms of parvalbumin. As analysed by ELISA, PA I showed weaker reactivities with IgG (monoclonal or polyclonal antibody) and serum IgE in fish-allergic patients than did other fish parvalbumins, including PA II. The amino acid sequences of PA I and PA II were elucidated by cDNA cloning. PA I was found to have more specific amino acid residues at several positions compared to the other fish parvalbumins.

Purification and Characterization of Parvalbumins, the Major Allergens in Red Stingray (Dasyatis akajei)

Fish has received increasing attention because it induces IgE-mediated food allergy. Parvalbumin (PV) represents the major allergen of fish, and IgE cross-reactivity to PV in various teleost fish species has been shown, while little information is available about allergens in elasmobranch fish. In this study, two PV isoforms (named as PV-I and PV-II) from red stingray (Dasyatis akajei) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose and Sephacryl S-200. Purified PVs revealed a single band on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of PV-I and PV-II were 12.29 and 11.95 kDa, respectively, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot using antifrog PV monoclonal antibody (PARV-19) showed positive reactions to the two proteins, confirming that they were PVs, although their immunological reactivities were weaker than those of PV from silver carp. The N-terminal amino acid sequence of PV-I was determined, and comparison with PVs from other fish species showed low homology between teleost and elasmobranch fish. The isoelectric points of PV-I and PV-II were 5.4 and 5.0, respectively, as determined by two-dimensional electrophoresis (2-DE), suggesting that both isoforms belong to the α-group. IgE immunoblotting analysis showed that sera from fish-allergic patients reacted to both PV-I and PV-II from red stingray. Thermal stability revealed that PV-I easily formed oligomers than PV-II, which might contribute to the maintenance of its allerginicity during heat processing.