Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE–cross-reactivity

Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.

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THE ANTIGENIC DETERMINANT OF THE LH-RELEASING HORMONE FOR THREE DIFFERENT ANTISERA

Acta Endocrinologica ◽

10.1530/acta.0.0780222 ◽

1975 ◽

Vol 78 (1) ◽

pp. 222-231 ◽

Cited By ~ 21

Author(s):

A. Arimura ◽

H. Sato ◽

D. H. Coy ◽

R. B. Worobec ◽

A. V. Schally ◽

...

Keyword(s):

Serum Albumin ◽

Rabbit Antiserum ◽

Cross Reactivity ◽

Amide Group ◽

Antigenic Determinants ◽

Releasing Hormone ◽

C Terminus ◽

Lh Rh ◽

Lh Releasing Hormone ◽

Antigen Antibody

ABSTRACT Antigenic determinants of LH-releasing hormone (LH-RH) were investigated by testing the cross-reaction of LH-RH analogues and fragments in LH-RH radioimmunoassay (RIA) systems using 3 different antisera against the LH-RH decapeptide. Rabbit antiserum No. 419 was generated against LH-RH adsorbed on polyvinylpyrrolidone (PVP). Antisera Nos. 710 and 742 were produced by immunizing rabbits with LH-RH conjugated either with bovine serum albumin through its C-terminus, or with human serum albumin through the N-terminus, respectively. For antiserum No. 419, the N-terminal (pyro)-glutamic acid and/or histidine in positions 1 and 2 of LH-RH, respectively, were found to enhance the antigen-antibody interaction, but were not indispensable for it. Similarly, the C-terminal amide and glycine-NH2 did not play a major role in these interactions. The LH-RH heptapeptide fragment, corresponding to amino acid sequence from positions 3 to 9, showed a cross-reactivity in this RIA system with LH-RH, although greater amounts than those of cold LH-RH were required for a comparable inhibition of binding of labelled LH-RH. For antiserum No. 710, the LH-RH hexapeptide fragment corresponding to positions 2 to 7 showed considerable cross-reac tivity. Histidine in position 2 played an important role but neither the amide group nor the glycine amide group at the C-terminus were essential. For antiserum No. 742, the C-terminal tetrapeptide-amide fragment of LH-RH showed considerable cross-reactivity in the LH-RH, the amide moiety itself being of crucial importance. These antisera may be useful in investigating peptides related to LH-RH in biological materials.

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Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014243 ◽

2020 ◽

Vol 295 (51) ◽

pp. 17398-17410 ◽

Cited By ~ 1

Author(s):

Martina Di Muzio ◽

Sabrina Wildner ◽

Sara Huber ◽

Michael Hauser ◽

Eva Vejvar ◽

...

Keyword(s):

Lipid Transfer Protein ◽

Cross Reactivity ◽

Antibody Binding ◽

Deuterium Exchange ◽

Hydrogen Deuterium Exchange ◽

Lipid Transfer ◽

Ige Reactivity ◽

C Terminus ◽

Immunological Mechanisms ◽

Hydrogen Deuterium

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

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Food antigen-specific IgE in dogs with suspected food hypersensitivity

Tierärztliche Praxis Ausgabe K Kleintiere / Heimtiere ◽

10.1055/a-1274-9210 ◽

2020 ◽

Vol 48 (06) ◽

pp. 395-402

Author(s):

Sandra A. Baumann ◽

Cornelius Fritz ◽

Ralf S. Mueller

Keyword(s):

Clinical Relevance ◽

Specific Ige ◽

Elimination Diet ◽

Cross Reactivity ◽

Food Hypersensitivity ◽

Strong Correlations ◽

Odds Ratios ◽

Cross Reactions ◽

Ige Reactivity ◽

Food Specific Ige

Abstract Objective Knowledge of cross-reactions in food-sensitive dogs will influence the choice of elimination diets and the long-term management of those patients. The objective of this study was to evaluate food allergen-specific IgE tests of suspected allergic dogs for concurrent positive reactions as possible evidence for cross reactions between allergens. Material and methods Results of serum IgE tests from 760 suspected allergic dogs submitted to 2 laboratories were evaluated statistically. After the tested allergens were grouped by their phylogenetic relationship, odds ratios as well as a sensitivity analysis of the odds ratios were performed to evaluate if concurrent positive IgE results to 2 allergens occurred more often than expected. Results Within related allergen pairs 27% (laboratory 1) and 72% (laboratory 2) of the pairs could be considered as associated. For the unrelated allergen pairs only 6.8% and 10.6% of the analyzed pairs were considered associated respectively. Strong correlations were shown in the group of ruminant allergens, especially beef and lamb, and grain allergens. High rates of concurrent reactions were also detected in the poultry group, especially between chicken and duck, as well as between pork and ruminant allergens, and soy and grain allergens. Conclusion As our results showed not only correlations within related but also between non-related allergens, the possible relevance of carbohydrate moieties as well as panallergens for canine hypersensitivities warrants further study. Further investigations are necessary to distinguish co-sensitization from cross-reactions and determine the clinical relevance of food-specific IgE reactivity. Clinical relevance Due to possible cross reactivity related allergens, especially beef and lamb as well as grain allergens, should not be used for an elimination diet to avoid false results.

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Chenopodium album pollen profilin (Che a 2): homology modeling and evaluation of cross-reactivity with allergenic profilins based on predicted potential IgE epitopes and IgE reactivity analysis

Molecular Biology Reports ◽

10.1007/s11033-010-0398-2 ◽

2010 ◽

Vol 38 (4) ◽

pp. 2579-2587 ◽

Cited By ~ 5

Author(s):

Akram Amini ◽

Mojtaba sankian ◽

Mohammad Ali Assarehzadegan ◽

Fatemeh Vahedi ◽

AbdolReza Varasteh

Keyword(s):

Homology Modeling ◽

Chenopodium Album ◽

Cross Reactivity ◽

Ige Reactivity

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Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2157 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2157-2167 ◽

Cited By ~ 78

Author(s):

J D Saide ◽

S Chin-Bow ◽

J Hogan-Sheldon ◽

L Busquets-Turner ◽

J O Vigoreaux ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Monoclonal Antibodies ◽

Flight Muscle ◽

Cross Reactivity ◽

Antigenic Determinants ◽

Immunogold Staining ◽

Thick Filaments ◽

The Matrix ◽

Flight Muscles

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.

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IMMUNOLOGIC STUDIES OF WATER-SOLUBLE HUMAN AMYLOID FIBRILS

Journal of Experimental Medicine ◽

10.1084/jem.130.4.797 ◽

1969 ◽

Vol 130 (4) ◽

pp. 797-808 ◽

Cited By ~ 29

Author(s):

Edward C. Franklin ◽

Mordechai Pras

Keyword(s):

Human Serum ◽

Comparative Studies ◽

Amyloid Fibrils ◽

Complement Fixation ◽

Cross Reactivity ◽

Water Soluble ◽

Antigenic Determinants ◽

Normal Tissues ◽

Absorption Studies ◽

Normal Human

Eight preparations of soluble amyloid and degraded amyloid (DAM) were compared immunologically. Unlike amyloid fibrils, six of eight preparations of DAM proved to be relatively strong immunogens. Antisera to DAM reacted weakly or not at all with normal human serum or extracts of normal tissues, but were specifically reactive with amyloid fibrils or DAM. Comparative studies of DAM'S from eight different subjects showed some degree of cross-reactivity among them, yet demonstrated that they were not identical. Similar conclusions were obtained by quantitative precipitin and complement fixation analyses. Comparison of the amyloid fibrils with the homologous DAM by complement fixation and absorption studies demonstrated the existence in DAM of antigenic determinants that were lacking or inaccessible in the native fibrils. A search for amyloid precursors and antibodies to amyloid in the sera of 12 patients proved unsuccessful.

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Cross-reactivity between mammalian myoglobins: Linear vs spatial antigenic determinants

Immunochemistry ◽

10.1016/0019-2791(77)90251-8 ◽

1977 ◽

Vol 14 (4) ◽

pp. 283-288 ◽

Cited By ~ 27

Author(s):

John G.R. Hurrell ◽

John A. Smith ◽

Pamela E. Todd ◽

Sydney J. Leach

Keyword(s):

Cross Reactivity ◽

Antigenic Determinants

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Cross-reactivity between mammalian myoglobins: Linear vs spatial antigenic determinants

Molecular Immunology ◽

10.1016/0161-5890(77)90132-8 ◽

1977 ◽

Vol 14 (4) ◽

pp. 283-288

Author(s):

J Hurrell

Keyword(s):

Cross Reactivity ◽

Antigenic Determinants

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Detection and identification of allergens from Canadian mustard varieties of Sinapis alba and Brassica juncea

Biomolecules ◽

10.3390/biom9090489 ◽

2019 ◽

Vol 9 (9) ◽

pp. 489 ◽

Cited By ~ 5

Author(s):

L’Hocine ◽

Pitre ◽

Achouri

Keyword(s):

Brassica Juncea ◽

Protein Extraction ◽

Immunoglobulin E ◽

Sinapis Alba ◽

Cross Reactivity ◽

Protein Profiling ◽

Ige Reactivity ◽

Ige Binding ◽

Reducing Conditions ◽

Detection And Identification

Currently, information on the allergens profiles of different mustard varieties is rather scarce. Therefore, the objective of this study was to assess protein profiles and immunoglobulin E (IgE)-binding patterns of selected Canadian mustard varieties. Optimization of a non-denaturing protein extraction from the seeds of selected mustard varieties was first undertaken, and the various extracts were quantitatively and qualitatively analyzed by means of protein recovery determination and protein profiling. The IgE-binding patterns of selected mustard seeds extracts were assessed by immunoblotting using sera from mustard sensitized and allergic individuals. In addition to the known mustard allergens—Sin a 2 (11S globulins), Sin a 1, and Bra j 1 (2S albumins)—the presence of other new IgE-binding protein bands was revealed from both Sinapis alba and Brassica juncea varieties. Mass spectrometry (MS) analysis of the in-gel digested IgE-reactive bands identified the unknown ones as being oleosin, β-glucosidase, enolase, and glutathione-S transferase proteins. A bioinformatic comparison of the amino acid sequence of the new IgE-binding mustard proteins with those of know allergens revealed a number of strong homologies that are highly relevant for potential allergic cross-reactivity. Moreover, it was found that Sin a 1, Bra j 1, and cruciferin polypeptides exhibited a stronger IgE reactivity under non-reducing conditions in comparison to reducing conditions, demonstrating the recognition of conformational epitopes. These results further support the utilization of non-denaturing extraction and analysis conditions, as denaturing conditions may lead to failure in the detection of important immunoreactive epitopes.

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