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Les parvalbumines
mardi 27 janvier 2009, par
Les parvalbumines ont une place à part parmi les familles de protéines contenant des allergènes : c’est en effet une parvalbumine, celle de morue (Gad c 1) qui a été le premier allergène moléculaire caractérisé (Aas K., 1966).
- Les parvalbumines sont des protéines de faible masse (environ 12 kD), présentes chez les vertébrés dans les muscles et, accessoirement, dans d’autres organes (système nerveux central, reins, certaines glandes).
- Ces protéines ont un rôle de régulation du calcium. Elles lient les ions Ca2+ et/ou Mg2+ au niveau de boucles particulières dites « EF-hand » et font partie de la superfamille des « calcium-binding proteins ».
- On connaît d’autres allergènes appartenant à cette superfamille, comme les polcalcines des pollens (2 EF-hand).
- Les parvalbumines contiennent 3 EF-hands dont une est non fonctionnelle.
- La présence/absence du Ca2+ influe sur la conformation et la stabilité de la protéine : l’IgE-réactivité est inférieure en l’absence de Ca2+.
On distingue deux sortes de parvalbumines : les alpha et les bêta parvalbumines.
- Si les alpha ont des pourcentages d’identité de 50-60% avec Cyp c 1 (une bêta parvalbumine de carpe), les bêta parvalbumines n’ont pas une très forte identité entre elles-mêmes : entre 60 et 90%
(cf. tableau ci-après). Un pourcentage d’identité de seulement 64-67% a été trouvé, par exemple, entre les parvalbumines de morue Gad c 1 et Gad m 1 provenant pourtant de deux espèces proches . - Ces différences sont compliquées par la présence d’isoformes qui sont, elles aussi, modérément homologues : par exemple 67% d’identité entre deux isoformes de l’allergène The c 1 du lieu d’Alaska (Theragra chalcogramma) .
Pourcentages d’identité avec la béta parvalbumine de carpe Cyp c 1.01, en gardant le meilleur % dans le cas de plusieurs séquences pour le même organisme.
(Source : (BlastP effectué le 25/01/09 sur www. ebi.ac.uk, et )
| Allergènes | Parvalbumines | |||
|---|---|---|---|---|
| béta | alpha | |||
| Thunnus albacares | Thon | 88 | ||
| Lates calcarifer | Perche | 87 | ||
| Tetraodon spp. | Fugu | 86 | ||
| Merluccius bilinearis | Merlu | 85 | ||
| Cyp c 1.02 | Cyprinus carpio | Carpe | 84 | |
| Ang j 1 | Anguilla japonica | Anguille | 84 | |
| Lutjanus argentimaculatus | Vivaneau | 83 | ||
| Par ol 1 | Paralichthys olivaceus | Cardeau | 82 | |
| Opsanus tau | Poisson-crapaud | 82 | ||
| Tra j 1 | Trachurus japonicus | Carangue | 81 | |
| Evy j 1 | Evynnis japonica | Pagre | 81 | |
| Ore a 1 | Oreomchromis aureus | Tilapia | 81 | |
| Gad m 1 | Gadus morhua | Morue | 80 | |
| Ict p 1 | Ictalurus punctatus | Poisson-chat | 80 | |
| Merlangius merlangus | Merlan | 80 | ||
| Sparus aurata | Dorade | 79 | ||
| Esox lucius | Brochet | 76 | 59 | |
| Sco j 1 | Scomber japonicus | Maquereau | 76 | |
| Clupea harengus | Hareng | 76 | ||
| Merluccius merluccius | Merluche | 75 | ||
| Coregonus lavaretus | Cisco | 75 | ||
| Kat p 1 | Katsuwonus pelamis | Bonite | 75 | |
| Sebastes marinus | Sébaste | 75 | ||
| The c 1 | Theragra chalcogramma | Lieu | 74 | |
| Sal s 1 | Salmo salar | Saumon | 78 | |
| Sar sa 1 | Sardinops sagax | Sardine | 73 | |
| Graptemys geographica | Tortue | 73 | ||
| Bufo marinus | Crapaud | 69 | ||
| Boa constrictor | Boa | 69 | ||
| Gad c 1 | Gadus callarias | Morue | 68 | |
| Lep w 1 | Lepidorhombus whiffiagonis | Cardine | 68 | |
| Ran e 2 / e 1 | Rana esculenta | Grenouille | 68 | 50 |
| Lim m 1 | Rana macrodon | Grenouille | 64 | |
| Gallus gallus | Poulet | 54 | ||
| Felis domesticus | Chat | 58 | ||
| Mus misculus | Souris | 56 | ||
| Homo sapiens | Homme | 56 | ||
| Raja clavata | Raie | 55 | ||
| Bos taurus | Bœuf | 54 | ||
| Oryctolagus cuniculus | Lapin | 54 | ||
| Triakis semifasciata | Requin | 53 | ||
Les parvalbumines peuvent former des dimères (ou plus) , ce qui pourrait compliquer leur identification en blot.
Pour l’instant, les bêta parvalbumines sont considérées comme les allergènes prépondérants des poissons. Mais il existe des alpha parvalbumines dans les poissons cartilagineux (raies, requins, roussette) et une IgE-réactivité pour ces alpha parvalbumines est possible, ayant déjà été montrée chez des patients ayant présenté une allergie alimentaire à la grenouille .
Les alpha parvalbumines étaient aussi en cause dans une observation de réactions anaphylactiques à la viande de poulet ou de dinde . Ce patient présentait également un syndrome oral avec le saumon et le thon, mais était négatif pour les béta parvalbumines. Il n’est pas exclu que l’allergie au poisson ait été le reflet d’une réactivité croisée entre alpha parvalbumines de poulet et de poisson (54% d’identité entre poulet et morue).
Les contenus en parvalbumines ne sont pas homogènes, ni entre les poissons, ni à l’intérieur d’un même poisson :
- les muscles rouges, utiles pour la nage continue, sont nettement plus pauvres en parvalbumine que les muscles de la chair blanche, utiles pour les courtes accélérations .
- Il paraît cependant difficile de distinguer l’allergènicité des poissons actifs (ex. thon, maquereau, sardine) de celle des poissons peu actifs (ex. morue) sur le seul contenu en parvalbumines.
[1] - Swoboda I, Bugajska-Schretter A, Valenta R, Spitzauer S. Recombinant fish parvalbumins: candidates for diagnosis and treatment of fish allergy. Allergy 2002;57(Suppl. 72):94-96
Fish and fish products represent one of the most important causes of IgE-mediated food hypersensitivity. In sensitized individuals contact with and consumption of fish can lead to severe health problems, ranging from urticaria and dermatitis to angiedema, diarrhoea, asthma and, at worst, systemic anaphylactic reactions and death. Parvalbumin, a small calcium-binding protein present in the muscles of vertebrates, was identified as the major fish allergen. We describe the isolation and characterization of cDNA clones coding for carp parvalbumin by IgE immunoscreening of a carp muscle expression library. These clones will be the basis for the production of recombinant carp parvalbumin, a useful tool for in vitro and in vivo diagnosis of fish allergy.
↩[2] - Das Dores S, Chopin C, Villaume C, Fleurence J, Guéant JL. A new oligomeric parvalbumin allergen of Atlantic cod (Gad mI) encoded by a gene distinct from that of Gad cI. Allergy 2002;57(Suppl. 72):79-83
BACKGROUND: The major allergen of Baltic cod (Gadus callarias) is a 12.3-kDa parvalbumin with two calcium-binding sites corresponding to EF-hand motifs. Our group found a 24-kDa IgE-reactive band that was also recognized by a monoclonal antiparvalbumin antibody in Atlantic cod (Gadus morhua). Our purpose was to purify and to determine the cDNA deduced sequence of this new cod allergen .
METHODS: Proteins from pre rigor mortis Atlantic cod were separated by gel filtration and the eluted peaks were analysed by SDS-PAGE and Western blotting with sera of sensitized patients and with antiparvalbumin. Protein bands were microsequenced, RNA transcripts were amplified by reverse transcription and polymerase chain reaction (RT-PCR) using primer combinations overlapping the open reading frame .
RESULTS: Four IgE and antiparvalbumin reactive proteins(12.5, 24, 38 and 51 kDa) were detected in gel filtration eluate. The cDNA deduced sequence of the 24 kDa protein had 109 amino acid residues with a molecular weight of 11.5 kDa and a theoretical pI of 4.34. The 24 kDa band corresponded therefore to a dimer of a beta-parvalbumin. Its homology was higher with Sal sI than with Gad cI. This new allergen was named Gad mI .
CONCLUSION: We have characterized a new parvalbumin allergen in Gadus morhua. This protein formed oligomers in native and in reducing conditions. Gad mI and Gad cI may correspond to two distinct genes of Gadus species.
↩[3] - Van Do T, Hordvik I, Endresen C, Elsayed S. Characterization of parvalbumin, the major allergen in Alaska pollack, and comparison with codfish Allergen M. Mol Immunol 2005;42:345-353
Increased fish consumption has led to frequent reporting of fish allergy and adverse reactions. Alaska pollack (Theragra chalcogramma) is a globally important commercial fish species, belonging to the Gadidae family. This family of fish also includes cod whose parvalbumin, Allergen M (Gad c 1), has been thoroughly studied and considered as a reference to sensitization in fish allergy. In the present study, parvalbumin from Alaska pollack, designated The c 1, was purified by use of anion exchange chromatography. To demonstrate the homogeneity of the purified protein, reverse phase high performance liquid chromatography was performed and showed two distinct fractions which had similar IgG and IgE binding capacities. Accordingly, cDNA cloning revealed two isotypic parvalbumin transcripts in pollack muscle. Recombinant parvalbumins of pollack exhibited low IgG and IgE binding capacities, in contrast to the native counterparts, which were almost as potent as cod Gad c 1. The allergenicity of The c 1 was assayed by ELISA inhibition, and compared to cod, the concentration required for obtaining 50% ELISA inhibition (C 50%) was only 18% higher for The c 1.
↩[4] - Untersmayr E, Szalai K, Riemer AB, Hemmer W, Swoboda I, Hantusch B, et al. Mimotopes identify conformational epitopes on parvalbumin, the major fish allergen. Mol Immunol 2006;43:1454-1461
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.
↩[5] - Lee SJ, Ju CC, Chu SL, Chien MS, Chan TH, Liao WL. Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus. J Exp Zoolog A Comp Exp Biol 2007;307:51-61
The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia.
↩[6] - Das Dores S, Chopin C, Villaume C, Fleurence J, Guéant JL. A new oligomeric parvalbumin allergen of Atlantic cod (Gad mI) encoded by a gene distinct from that of Gad cI. Allergy 2002;57(Suppl. 72):79-83
BACKGROUND: The major allergen of Baltic cod (Gadus callarias) is a 12.3-kDa parvalbumin with two calcium-binding sites corresponding to EF-hand motifs. Our group found a 24-kDa IgE-reactive band that was also recognized by a monoclonal antiparvalbumin antibody in Atlantic cod (Gadus morhua). Our purpose was to purify and to determine the cDNA deduced sequence of this new cod allergen .
METHODS: Proteins from pre rigor mortis Atlantic cod were separated by gel filtration and the eluted peaks were analysed by SDS-PAGE and Western blotting with sera of sensitized patients and with antiparvalbumin. Protein bands were microsequenced, RNA transcripts were amplified by reverse transcription and polymerase chain reaction (RT-PCR) using primer combinations overlapping the open reading frame .
RESULTS: Four IgE and antiparvalbumin reactive proteins(12.5, 24, 38 and 51 kDa) were detected in gel filtration eluate. The cDNA deduced sequence of the 24 kDa protein had 109 amino acid residues with a molecular weight of 11.5 kDa and a theoretical pI of 4.34. The 24 kDa band corresponded therefore to a dimer of a beta-parvalbumin. Its homology was higher with Sal sI than with Gad cI. This new allergen was named Gad mI .
CONCLUSION: We have characterized a new parvalbumin allergen in Gadus morhua. This protein formed oligomers in native and in reducing conditions. Gad mI and Gad cI may correspond to two distinct genes of Gadus species.
↩[7] - Van Do T, Hordvik I, Endresen C, Elsayed S. Characterization of parvalbumin, the major allergen in Alaska pollack, and comparison with codfish Allergen M. Mol Immunol 2005;42:345-353
Increased fish consumption has led to frequent reporting of fish allergy and adverse reactions. Alaska pollack (Theragra chalcogramma) is a globally important commercial fish species, belonging to the Gadidae family. This family of fish also includes cod whose parvalbumin, Allergen M (Gad c 1), has been thoroughly studied and considered as a reference to sensitization in fish allergy. In the present study, parvalbumin from Alaska pollack, designated The c 1, was purified by use of anion exchange chromatography. To demonstrate the homogeneity of the purified protein, reverse phase high performance liquid chromatography was performed and showed two distinct fractions which had similar IgG and IgE binding capacities. Accordingly, cDNA cloning revealed two isotypic parvalbumin transcripts in pollack muscle. Recombinant parvalbumins of pollack exhibited low IgG and IgE binding capacities, in contrast to the native counterparts, which were almost as potent as cod Gad c 1. The allergenicity of The c 1 was assayed by ELISA inhibition, and compared to cod, the concentration required for obtaining 50% ELISA inhibition (C 50%) was only 18% higher for The c 1.
↩[8] - Kuehn A, Felten P, Hilger C, Hentges F. Cloning, characterization and expression of cDNAs encoding the fish allergen parvalbumin from tuna, trout, redfish and herring. Allergy 2007;62(suppl. 83):329
Background: Fish allergy is a widespread concern of clinical importance. In different culture areas, the market share of consumed fish species varies. From cod, carp, mackerel and salmon, the major allergen parvalbumin (parv) was previously characterized in detail. Indeed, other species like Yellowfin tuna (Thunnus albacares), rainbow trout (Oncorhynchus mykiss), redfish (Sebastus marinus) and herring (Clupea harengus) are also of importance for European markets. The aim in this study was to further analyze the native parvalbumins of these fishes and to clone the cDNAs in view of recombinant expression.
Methods: Protein extracts were prepared from fish tissues, proteins separated by SDS PAGE and analyzed for the presence of parvalbumins. Purified, native parvalbumins were obtained from heat treatment and GFC (gel filtration chromatography). Cloning of the cDNA sequences was performed by 3'- and 5'-RACE PCR strategy using degenerated, but parvalbumin-specific oligonucleotides. Recombinant parvalbumins were expressed in E. coli and purified by IMAC (immobilized metal ion affinity chromatography). Protein identity and antigenicity was confirmed by Western immunoblotting.
Results: Eight different cDNAs encoding full-length parv were cloned: 1 from tuna (T1), 2 from rainbow trout (O1, O2), 2 from redfish (R1, R2) and 3 from herring (H1, H2, H3). The translated sequences (108 - 110 aa) belong to the beta-lineage of parv. Compared with carp parv Cyp c1, protein identities range from 71 % to 89 %. Recombinant parv from tuna, rainbow trout, redfish and herring were expressed as soluble proteins in E. coli M15 with a yield of 5 - 15 mg/ 500 ml of bacterial culture. The recombinant proteins were compared with native parvalbumins in regard of their IgE antibody binding characteristic.
Conclusion: Parv from four fish species, which are commonly consumed in Europe, were investigated. For the first time, recombinant parvalbumins are available from tuna, rainbow trout, redfish and herring. The recombinant proteins showed good IgE binding properties. They could be used for species-specific sub-typing of anti-parvalbumin IgE reactivity in fish allergic patients.
↩[9] - Das Dores S, Chopin C, Villaume C, Fleurence J, Guéant JL. A new oligomeric parvalbumin allergen of Atlantic cod (Gad mI) encoded by a gene distinct from that of Gad cI. Allergy 2002;57(Suppl. 72):79-83
BACKGROUND: The major allergen of Baltic cod (Gadus callarias) is a 12.3-kDa parvalbumin with two calcium-binding sites corresponding to EF-hand motifs. Our group found a 24-kDa IgE-reactive band that was also recognized by a monoclonal antiparvalbumin antibody in Atlantic cod (Gadus morhua). Our purpose was to purify and to determine the cDNA deduced sequence of this new cod allergen .
METHODS: Proteins from pre rigor mortis Atlantic cod were separated by gel filtration and the eluted peaks were analysed by SDS-PAGE and Western blotting with sera of sensitized patients and with antiparvalbumin. Protein bands were microsequenced, RNA transcripts were amplified by reverse transcription and polymerase chain reaction (RT-PCR) using primer combinations overlapping the open reading frame .
RESULTS: Four IgE and antiparvalbumin reactive proteins(12.5, 24, 38 and 51 kDa) were detected in gel filtration eluate. The cDNA deduced sequence of the 24 kDa protein had 109 amino acid residues with a molecular weight of 11.5 kDa and a theoretical pI of 4.34. The 24 kDa band corresponded therefore to a dimer of a beta-parvalbumin. Its homology was higher with Sal sI than with Gad cI. This new allergen was named Gad mI .
CONCLUSION: We have characterized a new parvalbumin allergen in Gadus morhua. This protein formed oligomers in native and in reducing conditions. Gad mI and Gad cI may correspond to two distinct genes of Gadus species.
↩[10] - Hilger C, Thill L, Grigioni F, Lehners C, Falagiani P, Ferrara A, et al. IgE antibodies of fish allergic patients cross-react with frog parvalbumin. Allergy 2004;59:653-660
BACKGROUND: The major allergens in fish are parvalbumins. Important immunoglobulin (Ig)E cross-recognition of parvalbumins from different fish species has been shown. Recently frog parvalbumin alpha has been found to be responsible for a case of IgE-mediated anaphylaxis triggered by the ingestion of frog meat. The aim of this study was to investigate whether IgE antibodies of fish allergic persons cross-react with frog parvalbumin and to appreciate its clinical relevance .
METHODS: The sera of 15 fish allergic patients and one fish and frog allergic patient were tested by IgE-immunoblotting against frog muscle extract. Sera were tested against recombinant parvalbumin alpha and beta from Rana esculenta. Skin prick tests were performed in selected patients with recombinant frog parvalbumin. Ca(2+) depletion experiments and inhibition studies with purified cod and frog recombinant parvalbumin were done to characterize the cross-reactive pattern .
RESULTS: Fourteen of the sera tested had IgE antibodies recognizing low molecular weight components in frog muscle extract. Calcium depletion experiments or inhibition of patient sera with purified cod parvalbumin led to a significant or complete decrease in IgE binding. When tested against recombinant parvalbumins, three of 13 sera reacted with alpha parvalbumin and 11 of 12 reacted with beta parvalbumin from R. esculenta. Skin prick tests performed with recombinant frog parvalbumin were positive in fish allergic patients. Inhibition studies showed that a fish and frog allergic patient was primarily sensitized to fish parvalbumin .
CONCLUSION: Cod parvalbumin, a major cross-reactive allergen among different fish species, shares IgE binding epitopes with frog parvalbumin. This in vitro cross-reactivity seems to be also clinically relevant. Parvalbumins probably represent a new family of cross-reactive allergens.
↩[11] - Kuehn A, Lehners C, Hilger C, Hentges F. Food allergy to chicken meat with IgE reactivity to muscle alpha-parvalbumin. Allergy 2009;64:1557-1558 ↩
[12] - Schein CH, Ivanciuc O, Braun W. Common physical-chemical properties correlate with similar structure of the IgE epitopes of peanut allergens. J Agric Food Chem 2005;53:8752-8759
Imprimer la bibliographieAlthough many sequences and linear IgE epitopes of allergenic proteins have been identified and archived in databases, structural and physicochemical discriminators that define their specific properties are lacking. Current bioinformatics tools for predicting the potential allergenicity of a novel protein use methods that were not designed to compare peptides. Novel tools to determine the quantitative sequence and three-dimensional (3D) relationships between IgE epitopes of major allergens from peanut and other foods have been implemented in the Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/). These peptide comparison tools are based on five-dimensional physicochemical property (PCP) vectors. Sequences from SDAP proteins similar in their physicochemical properties to known epitopes of Ara h 1 and Ara h 2 were identified by calculating property distance (PD) values. A 3D model of Ara h 1 was generated to visualize the 3D structure and surface exposure of the epitope regions and peptides with a low PD value to them. Many sequences similar to the known epitopes were identified in related nut allergens, and others were within the sequences of Ara h 1 and Ara h 2. Some of the sequences with low PD values correspond to other known epitopes. Regions with low PD values to one another in Ara h 1 had similar predicted structure, on opposite sides of the internal dimer axis. The PD scale detected epitope pairs that are similar in structure and/or reactivity with patient IgE. The high immunogenicity and IgE reactivity of peanut allergen proteins might be due to the proteins' arrays of similar antigenic regions on opposite sides of a single protein structure.
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