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Les lipocalines
lundi 19 mars 2012, par ,
Cette famille de protéines est intéressante à plus d’un titre :
– elle abrite les allergènes les plus importants du chien, du cheval, des rongeurs, etc.. ainsi que les béta lactoglobulines des laits de mammifères
– elle est l’exemple type d’une famille moléculaire dont les membres ne croisent que très peu entre eux
– elle s’inscrit dans le cadre plus général d’une superfamille de protéines , les calycines
– elle pose la question d’une relation entre allergénicité et ressemblance avec les protéines équivalentes chez l’Homme 
– elle est un exemple supplémentaire de l’éventuelle relation entre allergénicité et composants non peptidiques des protéines, les lipocalines transportant des molécules hydrophobes .
Structure des lipocalines
Les lipocalines ont en commun une structure en tonnelet, 8 feuillets béta entourant une cavité capable de recevoir un ligand hydrophobe. De plus, de courtes séquences d’acides aminés sont retrouvées et caractérisent cette famille.
Selon le nombre de ces séquences, il est convenu de distinguer les lipocalines « centrales » (« core » en anglais) et lipocalines « périphériques » (« outlier »). Mais le positionnement des protéines dans ces catégories varie un peu d’un auteur à autre .
A quoi servent les lipocalines ?
Dans la mesure où ces protéines existent aussi bien chez les organismes eucaryotes que chez les procaryotes, on comprend que leurs rôles biochimiques puissent être divers :
– transport de molécules hydrophobes : phéromones (ex. l’aphrodisine), stéroïdes, rétinoïdes, acides gras (ex. béta lactoglobuline du lait)
– liaison avec d’autres protéines ou des récepteurs : retinol binding protein, C8 gamma (coagulation), apolipoprotéine D
– protéines colorantes chez les arthropodes (insecticyanine, crustacyanine)
– protéines de la salive d’insectes suceurs, avec action anti-coagulante (nitrophorines, pallidipines, procaline, triabine) , ou se liant à l’histamine (Arg r 1 de la tique du pigeon ). Les protéines D7 de la salive des diptères, comme les moustiques, présentant aussi des homologies avec des lipocalines comme les OBP (Odorant binding proteins) .
– les lipocalines peuvent aussi avoir une action d’inhibiteur d’enzyme : certaines inhibent les métallo-protéases, d’autres les cystéine protéases (ex. les VEGP sécrétées par les glandes de von Ebner )
– immuno-modulation : alpha 1 acide glycoprotéine, orosomucoïde
– rôle de défense chez les plantes
On le voit, les lipocalines ont de multiples rôles, beaucoup étant dévolus à des mécanismes de reconnaissance ou de signalement, c’est-à-dire proches de l’immunologie au sens large.
Quelles sont les principales lipocalines IgE-réactives ?
Chez les mammifères
– les béta lactoglobulines des laits de vache (Bos d 5), de jument, de chèvre, de brebis, etc..
– les allergènes des « phanères » du chien (Can f 1, Can f 2, Can f 4, Can f 6), du chat (Fel d 4, Fel d 7), du cheval (Equ c 1, Equ c 2), de la vache (Bos d 2), du lapin (Ory c 1, Ory c 2), du cobaye (Cav p 1, Cav p 2, Cav p 3), du sanglier , etc..
– les allergènes urinaires des rongeurs : Mus m 1 (souris), Rat n 1 (rat), ..
Chez les insectes
– Arg r 1 dans la salive de la tique du pigeon (Argas reflexus)
– Bla g 4 de la blatte germanique (Blatella germanica)
– les allergènes Aca s 13, Blo t 13, Der f 13, Der p 13, Lep d 13, Tyr p 13 de différents acariens
– des protéines D7 de la salive de moustique
– des protéines salivaires de triatomes, insectes suceurs de la famille des Réduvidés : procaline, triabine
La super-famille des calycines
Les lipocalines s’insèrent dans une classification plus large, la super-famille des calycines. Toutes ces protéines ont une structure en tonnelet et beaucoup d’entre elles ont une capacité de liaison/transport.
Les calycines regroupent, en plus des lipocalines, des protéines de transport des acides gras (dites FABP, pour fatty acid binding proteins), des avidines (dont la streptavidine), des inhibiteurs de métallo-protéases.
Sur le plan allergologique, on retiendra que Bla g 4 (blatte germanique) et le groupe 13 des acariens sont des FABP .
D’autres protéines sont listées dans la base Allergome comme FABP, notamment une protéine issue de la blatte américaine (Periplaneta americana), qui présente 43-47% d’identité avec le groupe 13 des acariens. Per a 4 de la blatte américaine est bien une lipocaline.
L’appartenance de ces FABP et des lipocalines à la même super-famille n’induit pas que ces allergènes ont des chances de croiser entre eux. La notion de super-famille a pour vocation de décrire des filiations entre protéines sur le plan structural et biochimique ; elle ne sous-entend rien du point de vue immunologique.
Au sein de certaines familles on peut trouver une assez bonne réactivité croisée (ex. LTP), laquelle ne s’étendra pas à d’autres familles de la même super-famille (ex. les gliadines qui, comme les LTP, font partie des prolamines).
Certaines bases de données (ex. Allfam [AllFam>http://www.meduniwien.ac.at/allergens/allfam/]) regroupent les allergènes par familles et super-familles. Ceci est d’une grande importance pour leur caractérisation biochimique, mais l’appartenance à une même famille ne sous-entend pas nécessairement une réactivité croisée.
Les lipocalines croisent-elles entre elles ?
Cette question revêt une grande importance pour deux raisons :
– un test diagnostique peut-il être positivé par une réactivité croisée n’ayant pas de relevance clinique ?
– l’allergie à un mammifère (ex. le chat) entraîne-t-elle un risque d’allergie à un autre mammifère (ex. le cheval) ?
Les chances de réactivité croisée entre protéines sont conditionnées par une homologie suffisante entre des zones limitées de ces protéines, a priori en surface de celles-ci. Il n’est pas nécessaire que l’homologie soit présente sur l’ensemble du polypeptide et, par exemple, on voit des réactions croisées entre la pro-hévéine du latex (une protéine barwin) et des protéines d’une autre famille les chitinases de classe 1 (ex. dans la banane ou l’avocat).
A contrario, à structure globale similaire peut correspondre une mauvaise ressemblance locale si les pourcentages d’identité entre protéines sont trop faibles.
C’est ainsi que la plupart des lipocalines ne peuvent croiser entre elles. Une étude des séquences polypeptidiques actuellement disponibles montre qu’il n’existe que 20 à 32% d’identité entre Can f 1 ou Can f 2 (chien), Equ c 1 (cheval), Rat n 1 (rat), Mus m 1 (souris), Fel d 4 (chat) et Bos d 2 ou Bos d5 (bœuf/vache).
Mais il existe aussi quelques exceptions :
– 57% entre Can f 1 et la VEGP humaine
– 63% entre Can f 1 et Fel d 7
– 67% entre Can f 6 et Fel d 4
– 57% entre Can f 6 et Equ c 1
– 67% entre Equ c 1 et Fel d 4
– 47% entre Equ c 1 et Rat n 1 ou Mus m 1
– 49% entre Fel d 4 et Mus m 1 et 55% entre Fel d 4 et Rat n 1
– 65% entre Rat n 1 et Mus m 1
Par ailleurs, tous ces aéroallergènes n’ont au mieux que 29% d’identité avec les béta lactoglobulines des laits de mammifères, tandis que ces dernières ont une bonne homologie entre elles : 93 à 97% d’identité au sein des bovidés (vache, brebis, chèvre) et 50-58% entre jument et bovidés.
En conséquence, on comprend :
1) qu’il n’y ait pas de réactivité croisée en règle générale entre lipocalines aéroportées, ni entre celles-ci et les lipocalines du lait
2) que les béta lactoglobulines croiseront aisément entre elles parmi les laits de bovidés, mais moins bien avec celle du lait de jument (et probablement d’ânesse)
3) que les rares observations de réactions croisées pour les phanères concernent les lipocalines les moins dissemblables, par exemple entre Can f 1 et VEGP humaine ou entre Equ c 1 et Mus m 1, comme l’a montré un récent travail de Saarelainen
4) que dans la relation chat-chien, l’homologie Fel d 4 – Can f 6 semble suffisante (67% d’identité) pour générer, par exemple, une réactivité au chien chez un patient allergique au chat.
Voir aussi Réactivités croisées entre phanères de mammifères
Si les lipocalines sont assez peu cross-réactives entre elles, sont-elles cross-immunogènes, c’est-à-dire croisantes au niveau des cellules T ? L’étude des épitopes T a montré une relative co-localisation des zones épitopiques le long de la chaîne peptidique pour Can f 1, Equ c 1, Bos d 2 et Rat n 1. Notamment au niveau de l’épitope T N-terminal, siège également du motif caractéristique des lipocalines .
Il n’est pas établi cependant que ces homologies soient suffisantes en soi pour faciliter, dans la réalité, une sensibilisation croisée, par exemple au cheval chez un patient ayant préalablement développé une sensibilisation au chien.
[1] - Virtanen T, Kinnunen T. Mammalian allergens. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 201-18 ↩
[2] - Malandain H. Non-peptide components of proteins and allergenicity: should we generalize the hapten concept? EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°285
Background: Why are some proteins allergens is still an unresolved question. Besides proteolytic and/or pro-inflammatory activities, recent data have shown that major allergens like Bet v 1 or Der p 2 bind lipid ligands.
Objective : To check if such biochemical and/or structural features are a frequent finding among proteins known as allergens.
Methods: Published literature and protein databases were studied. Airborne allergens were chosen because they directly reach immune system in native form, eg. not affected by digestion.
Results: We found a wide array of allergens displaying a lipophilic binding structure, such as a groove or a tunnel : lipid transfer proteins, lipocalins, Bet v 1 family, Bla g 2, mite groups 2, 13, and 14, pollen 2 EF-hand Ca binding proteins, ... Noticeably, most of these allergens were not glycosylated.
Conclusion: These observations suggest that protein-bound lipid moieties could play a role in conjunction with the peptide backbone for initiating a Th-2 response. One might hypothesize that surface receptors of surveillance immune cells, like Toll receptors, are programmed to detect non-self proteins as a whole : lipid + peptide, carbohydrate + peptide, and even metal ion + peptide. This hypothesis could also match the hapten concept of a self structure bearing a non-self ligand. Globally, such a mechanism would be similar to innate defense systems adopted by many eukaryotes, like hypersensitive reactions observed in plants in response to elicitins.
↩[3] - Pomés A. Intrinsic properties of allergens and environmental exposure as determinants of allergenicity. Allergy 2002;57:673-679 ↩
[4] - Flower DR, North ACT, Sansom CE. The lipocalin protein family: structural and sequence overview. Biochim Biophys Acta 2000;1482:9-24
Lipocalins are remarkably diverse at the sequence level yet have highly conserved structures. Most lipocalins share three characteristic conserved sequence motifs - the kernel lipocalins - while others are more divergent family members - the outlier lipocalins - typically sharing only one or two. This classification is a useful tool for analysing the family, and within these large sets are smaller groups sharing much higher levels of sequence similarity. The lipocalins are also part of a larger protein superfamily: the calycins, which includes the fatty acid binding proteins, avidins, a group of metalloproteinase inhibitors, and triabin. The superfamily is characterised by a similar structure (a repeated +1 topology beta-barrel) and by the conservation of a remarkable structural signature. [References: 58]
↩[5] - Gutierrez G, Ganfornina MD, Sanchez D. Evolution of the lipocalin family as inferred from a protein sequence phylogeny. Biochim Biophys Acta 2000;1482:35-45
The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles. It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses. We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family.
Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency. [References: 33]
↩[6] - Grzyb J, Latowski D, Strzalka K. Lipocalins - a family portrait. J Plant Physiol 2006;163:895-915
Lipocalins are a widely distributed group of proteins whose common feature is the presence of six-or eight-stranded beta-barrel in their tertiary structure and highly conservative motifs short conserved region, (SCR) in their amino acid sequences. The presence of three SCRs is typical for kernel lipocalins, while outlier lipocalins have only one or two such regions. Owing to their ability to bind and transport small, hydrophobic molecules, lipocalins participate in the distribution of such substances. However, the physiological significance of lipocalins is not limited to transfer processes. They play an important role in the regulation of immunological and developmental processes, and are also involved in the reactions of organisms to various stress factors and in the pathways of signal transduction. Of special interest is the enzymatic activity found in a few members of the lipocalin family, as well as the interaction with natural membranes, both directly with lipids and through membrane-localized protein receptors.
↩[7] - Flower DR. The lipocalin family: structure and function. Biochem J 1996;318:1-14
The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds.
↩[9] - Paddock CD, McKerrow JH, Hansell E, Foreman KW, Hsieh I, Marshall N. Identification, Cloning, and Recombinant Expression of Procalin, a Major Triatomine Allergen. J Immunol 2001;167:2694-2699
Among the most frequent anaphylactic reactions to insects are those attributed to reduviid bugs. We report the purification and identification of the major salivary allergen of these insects. This 20-kDa protein (procalin) is a member of the lipocalin family, which includes salivary allergens from other invertebrates and mammals. An expression system capable of producing reagent quantities of recombinant allergen was developed in Saccharomyces cerevisiae. Antisera produced against recombinant protein cross-reacts with ELISA with salivary allergen. Recombinant Ag is also shown to react with sera from an allergic patient but not with control sera. By immunolocalization, the source of the salivary Ag is the salivary gland epithelium and its secretions.
↩[10] - Paesen GC, Adams PL, Nuttall PA, Stuart DL. Tick histamine-binding proteins: lipocalins with a second binding cavity. Biochim Biophys Acta 2000;1482:92-101
Tick histamine-binding proteins (HBPs) are lipocalins with two binding pockets. One of these binds histamine with a high affinity and is found at the position expected from other lipocalins, adjacent to the omega-loop at the open-end of the beta-barrel. A second binding cavity, which is a low-affinity site for histamine in one of the HBPs, is located at the end of the barrel that is closed off in other lipocalins. In order to create the second site, the 'closed-end' region has undergone a major reconstruction. Typical lipocalin characteristics, such as the 3(10) helix and a structural cluster of highly conserved residues, have been lost, while an alpha-helix now shields the cavity from the exterior. The prominence of acidic residues in the binding pockets is another distinctive characteristic of HBPs. Whereas most lipocalins have highly hydrophobic binding cavities designed to bind lipophilic compounds, HBPs have evolved to trap cationic, hydrophilic molecules. [References: 26]
↩[12] - Valenzuela JG, Charlab R, Gonzalez EC, de Miranda-Santos IFK, Marinotti O, Francisschetti IMB, et al. The D7 family of salivary proteins in blood sucking diptera. Insect Mol Biol 2002;11:149-155
The D7 subfamily of salivary proteins is widespread in blood sucking Diptera and belongs to the superfamily of pheromone/odourant binding proteins. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. In the present work we report the sequence of seventeen novel D7 proteins, and propose an evolutionary scenario for the appearance of the several forms of this protein, based on a total of twenty-one sequences from Culex quinquefasciatus, Aedes aegypti, Anopheles gambiae, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.
↩[13] - van't Hof W, Blankenvoorde MFJ, Veerman ECI, Nieuw Amerongen AV. The salivary lipocalin von Ebner's gland protein is a cysteine protease inhibitor. J Biol Chem 1997;272:1837-1841
The lipocalins make up a heterogeneous superfamily of proteins. Although showing almost no sequence homology, they share very similar secondary and tertiary structures. Their ability to bind hydrophobic ligands is well established, but the physiological function of most lipocalins remains unclear. The lipocalin from the human Von Ebner's Gland of the tongue (VEGh) contains three sequence motifs corresponding with the papain-binding domains of cystatins, a family of naturally occurring cysteine proteinase inhibitors. We found that VEGh inhibited papain activity to a similar extent as salivary cystatin S. Furthermore, synthetic peptides derived from VEGh and cystatin C, comprising these three motifs, inhibited papain, too. We conclude that VEGh is a physiological inhibitor of cysteine proteinases and therefore can play a role in the control of inflammatory processes in oral and ocular tissues.
↩[14] - Frenette Charron JB, Breton G, Badawi M, Sarhan F. Molecular and structural analyses of a novel temperature stress-induced lipocalin from wheat and Arabidopsis. FEBS Lett 2002;517:129-132
Two cDNAs corresponding to a novel lipocalin were identified from wheat and Arabidopsis. The two cDNAs designated Tatil for Triticum aestivum L. temperature-induced lipocalin and Attil for Arabidopsis thaliana temperature-induced lipocalin encode polypeptides of 190 and 186 amino acids respectively. Structure analyses indicated the presence of the three structurally conserved regions that characterize lipocalins. Sequence analyses revealed that this novel class of plant lipocalin shares homology with three evolutionarily related lipocalins: the mammalian apolipoprotein D (ApoD), the bacterial lipocalin and the insect Lazarillo. The comparison of the putative tertiary structures of both the human ApoD and the wheat TaTIL suggest that the two proteins differ in membrane attachment and ligand interaction. Northern analyses demonstrated that Tatil and Attil transcripts are upregulated during cold acclimation and heat-shock treatment. The putative functions of this novel class of plant lipocalins during temperature stresses are discussed
↩[15] - Spinelli S, Vincent F, Pelosi P, Tegoni M, Cambillau C. Boar salivary lipocalin. Three-dimensional X-ray structure and androsterol/androstenone docking simulations. Eur J Biochem 2002;269:2449-2456
The X-ray structure of variant A of authentic boar salivary lipocalin (SAL), a pheromone-binding protein specifically expressed in the submaxillary glands of the boar, has been solved and refined at 2.1 A resolution. The structure displays a classical lipocalin fold with a nine-stranded sandwiched beta barrel and an alpha helix. A putative glycosylation site, at position 53, has been found to carry a GlcNAc sugar residue. In contrast with what was expected on the basis of mass spectroscopy reports, the internal cavity was found to be devoid of bound pheromonal compound (androstenone or androstenol). Instead, a small electron density volume could be satisfied by a glycerol molecule, a component of the cryoprotecting liquor. The internal cavity was revealed to be very small for steroid compound accommodation. Therefore, docking and molecular dynamics experiments were performed with both pheromonal compounds. These simulations clearly demonstrate a volume increase of the cavity upon steroid binding and the adaptation of the amino-acid side chains to the steroid molecules. This explains the higher affinity of SAL for both steroid molecules compared to other smaller molecules, although no specific interaction is established with either compound.
↩[16] - Hilger C, Paesen G, Pauli G, Hentges F. Arg r 1, the major allergen of the pigeon tick, is a histamine binding lipocalin. Allergy 2007;62(suppl. 83):96
Background: Arg r 1 is the major allergen of the European pigeon tick, Argas reflexus. This soft-backed tick is a parasite of wild pigeons and may bite occasionally human beings, inducing anaphylactic reactions in sensitzed patients. Arg r 1 shows about 30% identity to some tick salivary gland lipocalins binding histamine and/or serotonin. We intended to investigate the ligand binding properties of Arg r 1.
Methods: Recombinant Arg r 1 (rArg r 1) was expressed in E. coli. Native Arg r 1 was purified from whole tick extract by ion exchange chromatography. Circular dichroism spectroscopy was used to compare the three-dimensional structures of native and recombinant molecules. Radioligand binding and competitive binding assays as well as smooth muscle bioassays were performed with the recombinant molecule.
Results: rArg r 1 binds histamine with high affinity and specificity with a Kd of 7.7 ± 4.7 nM. Competition experiments show that synthetic H1 and H2 receptor antagonists do not compete at all, nor do epinephrine and norepinephrine. To deplace 50% of the histamine, 339 times more serotonin is needed. These results were confirmed in a biological assay by measuring the muscle contraction in isolated guinea pig ileum. Arg r 1 very efficiently inhibited histamine-induced contraction of guinea pig ileum. Circular dichroism spectra show highly identical curves in the 200 to 240 nm range for both native and recombinant Arg r 1. Addition of histamine induced a conformational change of the three-dimensional structure of Arg r 1.
Conclusion: Arg r 1, the major allergen of Argas reflexus(pigeon tick), has marked histamine binding properties. In this respect it is similar to other histamine and serotonin binding proteins found so far in salivary glands of ticks.
↩[18] - Malafronte RS, Calvo E, James AA, Marinotti O. The major salivary gland antigens of Culex quinquefasciatus are D7-related proteins. Insect Biochem Mol Biol 2003;33:63-71
The sera of persons with strong allergic responses to the bites of the mosquito, Culex quinquefasciatus, contained IgE antibodies reactive with two major salivary gland proteins with molecular weights of 35 and 28 kDa. These antigens were purified, their amino termini sequenced, and the sequences were used to search for similar sequences in public databases. Two cDNAs, CuQu-D7Clu1 and CuQu-D7Clu12, which encode D7-related proteins, were identified as containing predicted amino acid sequences identical to the 35 and 28 kDa antigens, respectively. These proteins are expressed specifically in adult female salivary glands and, their predicted tertiary structures are consistent with a role as carriers of hydrophobic molecules in mosquito saliva.
↩[19] - Paddock CD, McKerrow JH, Hansell E, Foreman KW, Hsieh I, Marshall N. Identification, Cloning, and Recombinant Expression of Procalin, a Major Triatomine Allergen. J Immunol 2001;167:2694-2699
Among the most frequent anaphylactic reactions to insects are those attributed to reduviid bugs. We report the purification and identification of the major salivary allergen of these insects. This 20-kDa protein (procalin) is a member of the lipocalin family, which includes salivary allergens from other invertebrates and mammals. An expression system capable of producing reagent quantities of recombinant allergen was developed in Saccharomyces cerevisiae. Antisera produced against recombinant protein cross-reacts with ELISA with salivary allergen. Recombinant Ag is also shown to react with sera from an allergic patient but not with control sera. By immunolocalization, the source of the salivary Ag is the salivary gland epithelium and its secretions.
↩[20] - Moffitt JE, Venarske D, Goddard J, Yates AB, deShazo RD. Allergic reactions to Triatoma bites. Ann Allergy Asthma Immunol 2003;91:122-128
BACKGROUND: Triatoma bugs are best known in the medical community as vectors of trypanosomiasis (Chagas disease). However, bites of Triatoma bugs are a cause of local cutaneous reactions and anaphylaxis, mainly in the western and southwestern United States. The reactions typically occur at night during sleep, and the bite may not be recognized. There is continuing public interest in medical complications of bites of these bugs, although the scope of the problem remains undefined. OBJECTIVE: To review the relevant medical literature, identify present knowledge, and determine future research goals for allergy to Triatoma. DATA SOURCES: Computerized databases were used to search the medical literature for articles in the English language on Triatoma bites, allergy and entomology, and Chagas disease. STUDY SELECTION: Almost all identified articles on Triatoma allergy were used. Only selected articles on Triatoma bites and entomology were pertinent to the objectives. Articles on Chagas disease were limited to cases in the United States.
RESULTS: Bites of Triatoma bugs have been known to cause anaphylaxis for more than a century. These insects inhabit a large area of the United States, but to date most reports of allergic reactions to their bites have originated in the West and Southwest. The reactions typically occur at night during sleep following a bite on uncovered skin and may be unrecognized. Procalin has been identified as the major salivary allergen of Triatoma protracta and was recently cloned and expressed through recombinant technique. Allergenic reactivity has been demonstrated to salivary gland extracts of 2 species. The extracts of these 2 species have not shown immunologic cross-reactivity. Immunotherapy using a salivary gland extract appeared to be beneficial in a small number of patients; however, no commercial testing or treatment allergen is available.
CONCLUSIONS: Triatoma bites appear to be an important cause of anaphylaxis, especially in the western and southwestern United States. Because exposure to these insects often occurs during sleep, the incidence of allergic reactions to them is unclear. An epidemiologic study should be performed to determine the incidence, prevalence, and range of allergic responses to the bites of these insects. The lack of commercial antigen limits diagnostic and treatment capabilities. The development of an allergen under the Orphan Drug Act should be encouraged.
↩[21] - Thomas WR, Smith WA, Hales BJ, Mills KL, O'Brien RM. Characterization and Immunobiology of House Dust Mite Allergens. Int Arch Allergy Immunol 2002;129:1-18
The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens has been recent, many of the allergens can be produced as high IgE-binding polypeptides. The tertiary structure of the group 2 allergens has been determined from recombinant proteins and they are an excellent model for the investigation of modified allergens. An unexpected property of the group 1, 2 and 3 allergens has been the high degree of polymorphism found by cDNA analysis. It has however been possible to identify sequences to represent the variation in the natural allergens. The group 7 and 14 allergens show secondary modifications which vary in different extracts creating batch variation. While some estimate of the importance of allergens can be obtained from IgE binding, few analyses of T-cell responses have been made and these regulate both the development of, and the protection from sensitization.
↩[22] - Arruda LK, Vailes LD, Hayden ML, Benjamin DC, Chapman MD. Cloning of cockroach allergen, Bla g 4, identifies ligand binding proteins (or calycins) as a cause of IgE antibody responses. J Biol Chem 1995;270:31196-31201
An allergen cloned from a Blattella germanica (German cockroach) cDNA library, encoded a 182-amino acid protein of 20,904 Da. This protein, designated B. germanica allergen 4 (Bla g 4), was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by affinity chromatography and high-performance liquid chromatography. The prevalence of serum IgE antibody to recombinant Bla g 4 in 73 cockroach allergic patients with asthma ranged from 40% (antigen binding radioimmunoassay) to 60% (plaque immunoassay). Cockroach allergic patients gave positive intradermal skin tests to recombinant Bla g 4 at concentrations of 10(-3)-10(-5) micrograms/ml, whereas non-allergic controls, or cockroach allergic patients with no detectable serum IgE antibody to Bla g 4, gave negative skin tests to 1 microgram/ml. Polymerase chain reaction and Southern analysis identified a 523-base pair DNA encoding Bla g 4 in both B. germanica and Periplaneta americana (American cockroach). However, Northern analysis showed that mRNA encoding Bla g 4 was transcribed in B. germanica but not in P. americana, suggesting that allergen expression was species specific. Sequence similarity searches showed that Bla g 4 was a ligand binding protein or calycin and unexpectedly revealed that this family contained several important allergens: beta-lactoglobulin, from cow milk, and rat and mouse urinary proteins. Although the overall sequence homology between these proteins was low (approximately 20%), macromolecular modeling techniques were used to generate two models of the tertiary structure of Bla g 4, based on comparisons with the x-ray crystal coordinates of bilin binding protein and rodent urinary proteins. The results show that members of the calycin protein family can cause IgE antibody responses by inhalation or ingestion and are associated with asthma and food hypersensitivity.
↩[23] - Saarelainen S, Rytkönen-Nissinen M, Rouvinen J, Taivainen A, Auriola S, Kauppinen A, et al. Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity. Clin Exp Allergy 2008;38:374-381
BACKGROUND: Although knowledge of the IgE cross-reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross-reactivity of animal allergens is poorly known .
OBJECTIVE: The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL) .
METHODS: The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition .
RESULTS: ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1 .
CONCLUSION: Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.
↩[24] - Hilger C, Swiontek K, Arumugam K, Lehners C, Hentges F. Identification of a new major dog allergen highly cross-reactive with Fel d 4 in a population of cat- and dog-sensitized patients. J Allergy Clin Immunol 2012;129:1149-1151 ↩
[25] - Kauppinen J, Zeiler T, Rautiainen J, Rytkönen-Nissinen M, Taivainen A, Mäntyjärvi R, et al. Mutant derivatives of the main respiratory allergen of cow are less allergenic than the intact molecule. Clin Exp Allergy 1999;29:989-996
BACKGROUND: Allergen immunotherapy offers an alternative for drug treatment in the management of allergic diseases. Because immunotherapy often induces side-effects, less allergenic preparations would be beneficial .
OBJECTIVE: The purpose of this study was to examine whether the allergenicity of a cow-derived lipocalin allergen, Bos d 2, could be diminished by substituting or deleting carboxy-terminal amino acids including the cysteine which forms a disulphide bond with a cysteine inside the molecule .
METHODS: Four recombinant mutants of Bos d 2 were created by substituting or deleting the four most carboxy-terminal amino acids. The immunological characteristics of the mutant preparations were compared with the unmodified rBos d 2 by Western blotting, ELISA inhibition, skin prick tests, and the proliferative responses of allergen-specific T-cell clones .
RESULTS: In Western blot, one of the two monoclonal antibodies showed reduced binding to the preparations without the terminal cysteine. In contrast, the other monoclonal antibody, human IgE and rabbit immune serum bound equally well to all the preparations. ELISA inhibition analyses revealed, however, that the preparations without the terminal cysteine bound antibody less efficiently. They were needed 15-38 times more than the unmodified rBos d 2 to cause the same level of inhibition. Surprisingly, one of the mutants with the terminal cysteine but a mutated adjacent amino acid turned out to be the weakest in inducing skin reactivity. All the preparations stimulated well allergen-specific T-cell clones .
CONCLUSIONS: The results show that the allergenicity of a lipocalin allergen, Bos d 2, can be diminished by modifying the carboxy-terminal end of the molecule. Modifications in the area which encompasses a disulphide bond impaired the antibody binding without affecting the T-cell stimulatory capacity. It was also shown that in vivo tests are necessary for determining the allergenicity of a modified allergen.
↩[26] - Immonen A, Kinnunen T, Sirven P, Taivainen A, Houitte D, Perasaari J, et al. The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes. Clin Exp Allergy 2007;37:939-947
Summary Background Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing. Objective Knowing the T cell epitopes in allergens is a prerequisite for developing novel preparations for allergen immunotherapy. Methods Specific T cell lines were generated with recombinant Equ c 1 from the peripheral blood mononuclear cells (PBMCs) of 10 horse-allergic subjects. For determining T cell epitopes, the lines were stimulated with 16mer synthetic Equ c 1 peptides overlapping by 14 amino acids. The binding capacity of Equ c 1 peptides to human leucocyte antigen class II molecules was determined by the competitive ELISA. Results The major horse allergen Equ c 1 resembles two other lipocalin allergens, the major cow allergen Bos d 2 and the major dog allergen Can f 1, in that it is weakly stimulatory for the PBMCs of sensitized subjects. Moreover, the T cell epitopes of Equ c 1 are clustered in a few regions along the molecule, as is the case with Bos d 2 and Can f 1. Similar to Bos d 2, Equ c 1 contains one immunodominant epitope region at the carboxy-terminal end of the molecule. The T cell lines of eight horse-allergic subjects out of 10 showed strong reactivity to one or both of the two overlapping peptides, p143-158 and p145-160, in this region. The region probably contains two overlapping epitopes. Conclusion The 18mer peptide p143-160 from the immunodominant region of Equ c 1 is a potential candidate for the peptide-based immunotherapy of horse-sensitized subjects
↩[27] - Jeal H, Draper A, Harris J, Newman Taylor A, Cullinan P, Jones M. Determination of the T cell epitopes of the lipocalin allergen, Rat n 1. Clin Exp Allergy 2004;34:1919-1925
BACKGROUND: Laboratory animal allergy (LAA) is an important cause of occupational sensitization and asthma. Rats are a frequent cause of LAA and the major rat allergen, Rat n 1, is a member of the lipocalin protein family, which includes several other animal allergens such as the cow allergen, Bos d 2. To date, Bos d 2 is the only mammalian lipocalin allergen to have been studied in detail .
OBJECTIVE: We undertook a cross-sectional study of a large population of individuals exposed to laboratory rats to determine the proliferative responses of peripheral blood mononuclear cells (PBMCs) to the major rat allergen, Rat n 1 .
METHODS: Eighty-three cases (defined by a positive skin prick test (SPT) > or =3 mm and/or a positive RAST > or =2% binding) and 274 referents without specific IgE to rats were tested for their proliferative responses of PBMCs to rat allergen. Cytokine release to rat urinary protein was examined in 28 sensitized and 42 non-sensitized exposed individuals .
RESULTS: Proliferation to rat urinary protein was weak in all individuals. Four regions within Rat n 1 were identified as containing potential immunodominant T cell epitopes and three of these co-localized within the conserved regions of the lipocalin molecule. All four regions within Rat n 1 overlapped considerably with the characterized epitopes of the lipocalin allergen, Bos d 2. IL-5 and ratios of IL-5/IFN-gamma were significantly increased in cases .
CONCLUSION: The response to Rat n 1 is remarkably similar to the cow lipocalin allergen Bos d 2. T cell epitopes within lipocalins appear to co-localize with the conserved regions of the molecule. LAA is characterized by an increased production of IL-5. Investigation of other lipocalin allergens will provide further information about the allergenicity of this group of proteins.
↩[28] - Immonen A, Farci S, Taivainen A, Partanen J, Pouvelle-Moratille S, Narvanen A, et al. T Cell Epitope-Containing Peptides of the Major Dog Allergen Can f 1 as Candidates for Allergen Immunotherapy. J Immunol 2005;175:3614-3620
Imprimer la bibliographieOne prerequisite for developing peptide-based allergen immunotherapy is knowing the T cell epitopes of an allergen. In this study, human T cell reactivity against the major dog allergen Can f 1 was investigated to determine peptides suitable for immunotherapy. Seven T cell epitope regions (A-G) were found in Can f 1 with specific T cell lines and clones. The localization of the epitope regions shows similarities with those of the epitopes found in Bos d 2 and Rat n 1. On average, individuals recognized three epitopes in Can f 1. Our results suggest that seven 16-mer peptides (p15-30, p33-48, p49-64, p73-88, p107-122, p123-138, and p141-156), each from one of the epitope regions, show widespread T cell reactivity in the population studied, and they bind efficiently to seven HLA-DRB1 molecules (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominant in Caucasian populations. Therefore, these peptides are potential candidates for immunotherapy of dog allergy.
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