Identification of an IgE-binding determinant of the major allergen Myr pI from the venom of the Australian jumper ant Myrmecia pilosula

The structure of the single allergenic determinant of the major ant venom allergen, Myr, p I from the Australian jumper ant Myrmecia pilosula has been determined by inhibition studies with synthetic peptides. A 14 amino-acid C-terminal peptide sequence has been shown to constitute this determinant. Half-maximal inhibition of binding of ant venom-specific IgE antibodies to the native venom was obtained with this peptide at a concentration of 5 · 10⁻⁸M. This allergenic determinant was invariant for all ant venom-allergic subjects tested whose IgE antibodies recognized this allergen.     

Molecular cloning,characterization, and expression of Cuc m 2, a major allergen in Cucumis melo

Background:

Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon.

Methods:

Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli.

Results:

Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins.

Conclusion:

Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins. Key Words: Melon, Profilin, Cross-reactivity, Fruit allergy, Recombinant allergen     

Rationally designed hypoallergenic mutant variants of the house dust mite allergen Der p 21

BackgroundAllergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant.MethodsHerein, we used a structural bioinformatics and immunoinformatics approach to design hypoallergenic mutant variants of the Der p 21 allergen of Dermatophagoides pteronyssinus, which were then recombinantly expressed in bacteria and tested for their IgE-reactivities. For this, we scanned the wild-type Der p 21 protein for all possible single amino acid substitutions in key IgE-binding regions that could render destabilization of the major epitope regions.ResultsFour main substitutions (D82P, K110G, E77G, and E87S) were selected to build mutant variants of the Der p 21 allergen, which were produced in their recombinant forms; two of these variants showed reduced reactivity with IgE. Molecular dynamic simulations and immune simulations demonstrated the overall effects of these mutations on the structural stability of the Der p 21 allergen and on the profile of immune response induced through immunotherapy.ConclusionsWhen produced in their recombinant forms, two of the Der p 21 mutant variants, namely proteins K110G and E87S, showed significantly reduced IgE reactivities against sera from HDM-allergic individuals (n = 20; p < 0.001).General significanceThis study successfully translated a rational in silico mutagenesis design into low IgE-binding mutant variants of the allergen rDer p 21. These novel hypoallergens are promising to compose next-generation allergen-immunotherapy formulations in near future.     

Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.).

The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).     

Lys89, Lys90, and Phe91 are critical core amino acid residues of the Pen ch 18 major fungal allergen recognized by human IgE antibodies

A vacuolar serine protease (Pen ch 18) has been identified as a major allergen of Penicillium chrysogenum. The molecular features of antigenic determinant(s) on Pen ch 18 recognized by human IgE antibodies, however, have remained unclear. Here, we show that a dominant IgE epitope on the N-terminally processed Pen ch 18 allergen was narrowed down to residues 83-91. In addition, Lys89, Lys90, and possibly Phe91 were identified as the core residues. Substitution of Lys89, Lys90, or Phe91 with alanine can significantly reduce IgE-binding to Pen ch 18. Immunoblot inhibition confirmed that Lys89 and Phe91 played a significant role in IgE-binding against Pen ch 18. Molecular modeling suggests they are located on a loop-like structure at or near the surface of the major fungal allergen.     

Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae

cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum. The sequences cover the complete open reading frame encoding the prepro-form. The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues. Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding. The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy. In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding. The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.     

The Transaldolase,a Novel Allergen of Fusarium proliferatum,Demonstrates IgE Cross-Reactivity with Its Human Analogue

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.     

Molecular Cloning and Sequencing of the Extracellular Pectate Lyase II Gene from Erwinia carotovora Er

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for peetate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1 % and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca2+-binding proteins

Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca²⁺-binding sites of Ca²⁺-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca²⁺-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the IgE of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species.     

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.     

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus)

Background:

The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen.

Methods:

The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA.

Section Title

The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1.

Conclusion:

We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb''s-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron.Key Words: Allergen, cDNA cloning, Cro s 1, Occupational allergy, Saffron pollen     

丹参变应原基因（Sam a1）的克隆及其原核表达

Ⅰ型变态反应是小分子植物蛋白结合IgE抗体引起的一种疾病。本文首次从丹参中克隆出变应原蛋白基因,命名为：Sam a1（GenBank注册号：EU122383）。Sam a1包含483 bp的开放读码框,编码160个氨基酸残基。其推导氨基酸（命名为：Sam a1）具有Bet v1结构域,属于病原菌相关的Bet v1蛋白家族,与苹果中变应原mal d1类似蛋白同源性高达66％。三级结构预测模型显示Sam a1含有T细胞主要抗原决定簇的C端α螺旋。实时定量PCR结果显示：黄瓜细菌性角斑病菌和NaCl溶液诱导下Sam a1的表达呈现上调趋势,表明Sam a1可能与病程相关蛋白和盐胁迫相关。Sam a1转入大肠杆菌M15后,表达出与预测蛋白大小相当的目标蛋白,IPTG诱导后5～7 h目标蛋白可大量表达。本研究为从植物分子机制上了解中药变态反应作用机制奠定了基础,尤其对中药产业的发展具有重要的意义。     

Identification of critical amino acids in the IgE epitopes of Ric c 1 and Ric c 3 and the application of glutamic acid as an IgE blocker

Background

The allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae) is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis.

Methodology/Principal Findings

The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction.

Conclusions/Significance

The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units     

Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP)

Background

Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass.

Methods

A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay.

Results

Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120–170 and 224–244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction.

Conclusion

Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.     

Reactivity of German Cockroach Allergen, Bla g 2, Peptide Fragments to IgE Antibodies in Patients' Sera

Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichia-expressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients'' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.     

Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II

The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p II was determined by automated Edman degradation of the protein and selected fragments. Cleavage of the protein by enzymatic and chemical techniques established an unambiguous sequence for the protein. Lol p II contains 97 amino acid residues, with a calculated molecular weight of 10,882. The protein lacks cysteine and glutamine and shows no evidence of glycosylation. Theoretical predictions by Fraga's (Fraga, S. (1982) Can. J. Chem. 60, 2606-2610) and Hopp and Woods' (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828) methods indicate the presence of four hydrophilic regions, which may contribute to sequential or parts of conformational B-cell epitopes. Analysis of amphipathic regions by Berzofsky's method indicates the presence of a highly amphipathic region, which may contain, or contribute to, an Ia/T-cell epitope. This latter segment of Lol p II was found to be highly homologous with an antibody-binding segment of the major rye allergen Lol p I and may explain why immune responsiveness to both the allergens is associated with HLA-DR3.     

Patient-tailored cloning of allergens by phage display: Peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10¹¹ cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-( -proline)-Sepharose and yielded 30 mg l⁻¹ culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.     

Identification of critical amino acids in an immunodominant IgE epitope of Pen c 13, a major allergen from Penicillium citrinum

Background

Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13.

Methodology/Principal Findings

Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A¹⁴⁸–E¹⁶⁶) and S22 (A²⁴³–K²⁷⁴) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T²⁶¹–K²⁷⁴), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity.

Conclusions/Significance

Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases.     

NMR Structure and IgE Epitopes of Blo t 21, a Major Dust Mite Allergen from Blomia tropicalis

Blo t 21 is a paralogue of the group 5 allergen, Blo t 5, a major allergen from the dust mite Blomia tropicalis. Blo t 21 has moderate sequence identity (40.7%) to Blo t 5 and low to moderate cross-reactivity to Blo t 5. In B. tropicalis, the most prevalent and allergenic allergens are in the order of Blo t 21, Blo t 5, and Blo t 7. Here, we determined the NMR solution structure of Blo t 21, which represents the first structure of the group 21 dust mite allergen. The structure of Blo t 21 closely resembles the structures of Blo t 5 and Der p 5, comprising three anti-parallel α-helices arranged in a helical bundle. Using site-directed mutagenesis and specific IgE binding ELISA, Blo t 21 was found to contain both conserved and unique charged IgE epitope residues at the L2 loop region and on helix α3. Cross-inhibition assays confirmed that Blo t 21 has a low to moderate cross-reactivity with Blo t 5 and Der p 5 and represents a novel group of major allergen in B. tropicalis. In addition to group 5 allergens, Blo t 21 has also a low to moderate cross-reactivity with group 21 allergens from Dermatophagoides mites, confirming that B. tropicalis is a major and distinct source of dust mite allergens.