Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen

Inhalation of allergens produced by the American cockroach (Periplaneta americana) induces IgE Ab production and the development of asthma in genetically predisposed individuals. The cloning and expression in Escherichia coli of P. americana tropomyosin allergen have been achieved. The protein shares high homology with other arthropod tropomyosins (80% identity) but less homology with vertebrate ones (50% identity). The recombinant allergen was produced in E. coli as a nonfusion protein with a yield of 9 mg/l of bacterial culture. Both natural and recombinant tropomyosins were purified by isoelectric precipitation. P. americana allergen 1 (Per a 1) and Per a 7 (tropomyosin) are to date the only cross-reacting allergens found in cockroaches. ELISA and Western blot inhibition experiments, using natural and recombinant purified tropomyosins from shrimp and cockroach, showed that tropomyosin induced cross-reactivity of IgE from patients allergic to these allergens, suggesting that this molecule could be a common allergen among invertebrates.     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.     

Allergenic tropomyosins and their cross-reactivities

The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock. A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen. Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates. Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.     

New insights into the allergenicity of tropomyosin: a bioinformatics approach

The invertebrate panallergen tropomyosin is a protein with an extremely simple folding. This makes it a perfect target for investigating structural differences between invertebrate and vertebrate tropomyosins, which are not considered allergenic. Phylogenetic and sequence analyses were conducted in order to explore the differences in primary structure between several tropomyosins and to promote an experimental development in the field of food allergy, based on the study of tropomyosin. The phylogenetic analyses showed that tropomyosin is a useful evolutionary marker. The phylogenetic trees obtained with tropomyosin were not always phylogenetically correct, but they might be useful for allergen avoidance by tropomyosin allergic individuals. Sequence analyses revealed that the probability of alpha helix folding in invertebrate tropomyosins was lower than in all the studied vertebrate ones, except for the Atlantic bluefin tuna Thunnus thynnus tropomyosin. This suggested that the lack of alpha helix folding may be involved in the immunogenicity of tropomyosins. More specifically, the regions adjacent to the positions 133–135 and 201 of the invertebrate tropomyosins, presented lower probability of alpha helix folding than those of vertebrates and are candidates to be responsible for their allergenicity.     

Comparative analysis of barnacle tropomyosin: divergence from decapod tropomyosins and role as a potential allergen

Tropomyosin, a myofibrillar protein of 35-38 kDa, represents a major and cross-reactive allergen in decapod crustaceans. This study was initiated to clarify whether decapod-allergic patients also recognize tropomyosins of barnacles, crustaceans phylogenetically remote from decapods, which are locally consumed as a delicacy. On SDS-PAGE, a 37 kDa protein was observed in all the heated extracts prepared from two species of decapods (American lobster Homarus americanus and black tiger prawn Penaeus monodon) and two species of barnacles (acorn barnacle Balanus rostratus and goose barnacle Capitulum mitella). In immunoblotting, the 37 kDa protein was found to react with monoclonal antibodies against American lobster tropomyosin and hence identified as tropomyosin. The patient sera reacted to tropomyosins from both decapods and barnacles and the reactivity was abolished by preincubation with American lobster tropomyosin, demonstrating that barnacle tropomyosins are allergens cross-reactive with decapod tropomyosins. However, the amino acid sequence of acorn barnacle tropomyosin, deduced by cDNA cloning experiments, shares higher sequence identity with abalone tropomyosins than with decapod tropomyosins. In accordance with this, the phylogenetic tree made for tropomyosins from various animals showed that the acorn barnacle tropomyosin is evolutionally classified not into the decapod tropomyosin family but into the molluscan tropomyosin family.     

美洲大蠊Per a7基因的克隆、表达及免疫学鉴定

根据原肌球蛋白基因序列设计引物,以我国南方地区美洲大蠊Periplaneta Americana RNA为模板,用RT-PCR方法扩增出852 bp的全长编码片段,经序列分析发现该基因与NCBI公布的Per a7有3个核苷酸发生改变。将目的片段克隆到pET24a(+)表达载体中,在大肠杆菌BL21 Star获得表达,融合蛋白的分子量约为33 kD。利用蟑螂过敏性患者血清对表达产物进行Western blotting检测,出现明显的识别条带,说明表达产物具有IgE结合活性。     

锯缘青蟹主要过敏原的纯化与鉴定

以锯缘青蟹为研究对象,从免疫鉴定、分离纯化、抗体制备和免疫学分析等方面对其主要过敏原进行研究。首先利用过敏者血清的免疫印迹法,确定锯缘青蟹的主要过敏原为分子量约38kD的蛋白。然后通过制备丙酮粉、等电点沉淀、硫酸铵沉淀及加热处理对分子量为38kD的主要过敏蛋白进行了高度纯化。该蛋白的pI约为4.5,与虾的原肌球蛋白Pena1性质相近,证实了锯缘青蟹的主要过敏原为原肌球蛋白。通过免疫新西兰大白兔,制备了原肌球蛋白的抗血清,采用Protein A Sepharose亲和层析柱对动物抗体进行了纯化。该抗血清效价高,经4×105倍稀释后仍能与抗原进行反应。该抗体与甲壳类动物及软体动物的原肌球蛋白具有较强的免疫交叉反应,可用于食品过敏原检测。       

Animal models of type I allergy using recombinant allergens

Various animal models including guinea pigs, monkeys, dogs, rats, and mice have been established in an attempt to provide insights into the complex immunological and pathophysiological mechanisms of human type I allergic diseases. The detailed knowledge of the murine genome, the various components of the murine immune system, and the generation of engineered mice has made the murine system the most attractive among all animal models. The availability of multitude technologies and reagents to characterize and manipulate immunological pathways and mediators adds to the outstanding opportunities to assess the pathology of allergic diseases and to develop novel therapeutic strategies in mice. Numerous sensitization protocols with food and aero-allergens are used to establish an allergic/asthma-like phenotype in mice. Requirements for an appropriate murine model include a close resemblance to the pathology of the disease in humans, the objective measurement of the physiologic parameters, as well as reliability and reproducibility of the experimental data. With respect to reproducible experimental conditions, it has been recognized that extract preparations from natural allergen sources can vary in their allergen-content and -composition. This might influence the degree of sensitization or the outcome of treatment strategies in dependence of the applied extract preparation. The use of recombinant allergens in experimental in vivo and in vitro systems can overcome these problems. Another aspect, that has become obvious from the experimental studies, is that allergens can differ in their immunogenicity as well as in their capacity to act as tolerogens. Therefore, it seems important that the efficacy of the different allergen-molecules to act as therapeutic agents is individually examined. In this review, examples of animal models are described, in which recombinant allergens have been used for sensitization and/or treatment of allergic responses and how they have been used to enhance our understanding of the pathology of allergic diseases.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments and second structure information of Fag s 1: Fagales allergen from <Emphasis Type="Italic">Fagus sylvatica</Emphasis>

Fagales allergens belonging to the Bet v 1 family account responsible for the majority of spring pollinosis in the temperate climate zones in the Northern hemisphere. Among them, Fag s 1 from beech pollen is an important trigger of Fagales pollen associated allergic reactions. The protein shares high similarity with birch pollen Bet v 1, the best-characterized member of this allergen family. Of note, recent work on Bet v 1 and its homologues found in Fagales pollen demonstrated that not all allergenic members of this family have the capacity to induce allergic sensitization. Fag s 1 was shown to bind pre-existing IgE antibodies most likely primarily directed against other members of this multi-allergen family. Therefore, it is especially interesting to compare the structures of Bet v 1-like pollen allergens, which have the potential to induce allergic sensitization with allergens that are mainly cross-reactive. This in the end will help to identify allergy eliciting molecular pattern on Bet v 1-like allergens. In this work, we report the ¹H, ¹⁵N and ¹³C NMR assignment of beech pollen Fag s 1 as well as the secondary structure information based on backbone chemical shifts.     

糖蛋白过敏原N-糖链与过敏的相关性研究

糖蛋白是一种含有寡糖链的蛋白质,糖链与蛋白质之间以共价键相连。N-糖蛋白为常见过敏原之一,主要来源于食物、吸入物、昆虫毒素等,能够引起过敏反应。N-糖蛋白过敏原的N-糖链结构影响过敏原与IgE的结合,影响抗原提呈细胞（APC）对过敏原的识别和提呈。本文在介绍与过敏相关的N-糖蛋白、常见N-糖蛋白过敏原的N-糖链结构及与过敏相关的糖基化酶的基础上,进一步分析过敏原N-糖链影响过敏的机制,为临床预防与治疗过敏性疾病提供新的思路。     

Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.     

House dust mite sensitization drives cross-reactive immune responses to homologous helminth proteins

The establishment of type 2 responses driven by allergic sensitization prior to exposure to helminth parasites has demonstrated how tissue-specific responses can protect against migrating larval stages, but, as a consequence, allow for immune-mediated, parasite/allergy-associated morbidity. In this way, whether helminth cross-reacting allergen-specific antibodies are produced and play a role during the helminth infection, or exacerbate the allergic outcome awaits elucidation. Thus, the main objective of the study was to investigate whether house dust mite (HDM) sensitization triggers allergen-specific antibodies that interact with Ascaris antigens and mediate antibody-dependent deleterious effects on these parasites as well as, to assess the capacity of cross-reactive helminth proteins to trigger allergic inflammation in house dust mite presensitized mice. Here, we show that the sensitization with HDM-extract drives marked IgE and IgG1 antibody responses that cross-react with Ascaris larval antigens. Proteomic analysis of Ascaris larval antigens recognized by these HDM-specific antibodies identified Ascaris tropomyosin and enolase as the 2 major HDM homologues based on high sequence and structural similarity. Moreover, the helminth tropomyosin could drive Type-2 associated pulmonary inflammation similar to HDM following HDM tropomyosin sensitization. The HDM-triggered IgE cross-reactive antibodies were found to be functional as they mediated immediate hypersensitivity responses in skin testing. Finally, we demonstrated that HDM sensitization in either B cells or FcγRIII alpha-chain deficient mice indicated that the allergen driven cell-mediated larval killing is not antibody-dependent. Taken together, our data suggest that aeroallergen sensitization drives helminth reactive antibodies through molecular and structural similarity between HDM and Ascaris antigens suggesting that cross-reactive immune responses help drive allergic inflammation.     

Multiple Amb a I allergens demonstrate specific reactivity with IgE and T cells from ragweed-allergic patients.

The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels.     

Biochemical and molecular biological aspects of silverfish allergens

Insects and insect-derived materials have been implicated as a risk factor for sensitization and subsequent elicitation of allergic rhinitis and allergic bronchial asthma. During the last decades, insects other than those known as allergenic, were investigated for their potential role in inducing and triggering an IgE immune response. Among these, the silverfish, an insect belonging to the Thysanura order, appeared to be of particular interest. Silverfish (Lepisma saccharina) is the most primitive living insect, and represents a descendent of the ancestral wingless insects. They are 3-12 mm long, have three tail feelers and are covered with shiny scales. They shun light and need a humid environment and their diet consists of carbohydrate materials such as paper and book-binding glue, crumbs of bread and flour. Because of these features, silverfish finds an optimal habitat both in dwellings and workplaces and in spite of its antiquity, silverfish has succeeded in exploiting the new opportunity created by man. Although its importance significantly increased when it has been demonstrated that house dust contains significant silverfish levels even in houses where the inhabitants were unaware of its presence, no silverfish extract for diagnosis of allergic diseases is commercially available yet. Identification of optimal extraction conditions and characterization of allergenic extracts are the first steps to obtain an effective allergen preparation suitable for diagnosis and therapy, and will be useful as a reference preparation for assessing silverfish exposure in different indoor environments. It has been cloned and characterized a silverfish tropomyosin, named Lep s 1, which represents the first allergen identified in silverfish extract and can be regarded as a molecule cross-reactive among inhalant and edible invertebrates allergenic sources. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.     

House-dust-mite allergens: A review

The house-dust mites,Dermatophagoides farinae, D. pheronyssinus andEuroglyphus maynei are cosmopolitan inhabitants of the homes of humans worldwide. These mites are the sources of multiple potent allergens that trigger allergic reactions in house-dust-mite-sensitive individuals. Many laboratories using widely varied mite materials and allergic sera, and biochemical and immunological assays, have isolated and characterized, to varying degrees, some of the allergens produced by these mites. The resulting large body of literature is difficult to interpret and relate. This review briefly summarizes the progress made in isolating and characterizing mite-derived antigens and allergens, the relationship between antigens isolated in different laboratories, and the patients' reactivity to these allergens. A brief summary of the allergic reaction and the role of IgE are provided as background.     

A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease

The respiratory allergens that induce experimental Th cell type 2-dependent allergic lung inflammation may be grouped into two functional classes. One class of allergens, in this study termed type I, requires priming with adjuvants remote from the lung to overcome airway tolerogenic mechanisms that ordinarily preclude allergic responses to inhaled Ags. In contrast, the other, or type II, allergen class requires neither remote priming nor additional adjuvants to overcome airway tolerance and elicit robust allergic lung disease. In this study, we show in an experimental model that diverse type II allergens share in common proteolytic activity that is both necessary and sufficient for overcoming airway tolerance and induction of pulmonary allergic disease. Inactivated protease and protease-free Ag fragments showed no allergenic potency, demonstrating that only active protease acting on endogenous substrates was essential. Furthermore, induction of airway tolerance could be aborted and allergic lung disease established by simply adding purified protease to a type I allergen. Thus, exogenous proteases are common to type II allergens and may be generally required to overcome the innate resistance of the airway to Th cell type 2 activation and allergic inflammation, raising concern for their potential contribution to diseases such as asthma.     

Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.

Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.     

A nematode immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2 inflammation

Helminth parasites modulate the immune system by complex mechanisms to ensure persistence in the host. Released immunomodulatory parasite components lead to a beneficial environment for the parasite by targeting different host cells and in parallel to a modulation of unrelated inflammatory responses in the host, such as allergy. The aim of this study was to investigate the effect of the potent helminth immunomodulator, filarial cystatin, in a murine model of airway inflammation and hyperreactivity induced by a clinically relevant aeroallergen (timothy grass (Phleum pratense) pollen) and on the function of peripheral blood mononuclear cells (PBMCs) from timothy grass pollen allergic patients. BALB/c mice were systemically sensitised with a recombinant major allergen of timothy grass pollen (rPhl p 5b) and then challenged with timothy grass pollen extract (GPE) via the airways. Filarial cystatin was applied i.p. during the sensitisation phase. Airway hyperresponsiveness to methacholine challenges, inflammation of airways, inflammatory cell recruitment, cytokine production and lung histopathology were investigated. In a translational approach, PBMCs from allergic subjects and healthy controls were treated in vitro with cystatin prior to stimulation with GPE. Administration of filarial cystatin suppressed rPhl p 5b-induced allergen-specific Th2-responses and airway inflammation, inhibited local recruitment of eosinophils, reduced levels of allergen-specific IgE and down-regulated IL-5 and IL-13 in the bronchoalveolar lavage (BAL). Ex vivo restimulation with cystatin of spleen cells from cystatin-treated mice induced the production of IL-10, while cystatin inhibited allergen-specific IL-5 and IL-13 levels. Human PBMCs from timothy grass pollen allergic patients displayed a shift towards a Th1 response after treatment with cystatin. These results show that filarial cystatin ameliorates allergic inflammation and disease in a clinically relevant model of allergy. This data indicate that filarial cystatin has a modulatory effect on grass pollen-specific responses warranting further investigation of potential preventive and therapeutic options in the treatment of allergies.