Effect of Point Mutations on Structural and Allergenic Properties of the Lentil Allergen Len c 3

Plant lipid transfer proteins (LTPs) are known to be clinically significant allergens capable of binding various lipid ligands. Recent data showed that lipid ligands affected the allergenic properties of plant LTPs. In this work, we checked the assumption that specific amino acid residues in the Len c 3 structure can play a key role both in the interaction with lipid ligands and IgE-binding capacity of the allergen. The recombinant analogues of Len c 3 with the single or double substitutions of Thr41, Arg45 and/or Tyr80 were obtained by site-directed mutagenesis. All these amino acid residues are located near the “bottom” entrance to the hydrophobic cavity of Len c 3 and are likely included in the IgE-binding epitope of the allergen. Using a bioinformatic approach, circular dichroism and fluorescence spectroscopies, ELISA, and experiments mimicking the allergen Len c 3 gastroduodenal digestion we showed that the substitution of all the three amino acid residues significantly affected structural organization of this region and led both to a change of the ligand-binding capacity and the allergenic potential of Len c 3.

Crosstalk Between the Immune System and Plant-Derived Nanovesicles: A Study of Allergen Transporting

Background: Nanometer-sized membrane-surrounded vesicles from different parts of plants including fruits are gaining increasing attention due to their anti-inflammatory and anticancer effects demonstrated by in vitro and in vivo studies, and as nanovectors for molecular delivery of exogenous substances. These nanomaterials are very complex and contain a diverse arsenal of bioactive molecules, such as nucleic acids, proteins, and lipids. Our knowledge about the transport of allergens in vesicles isolated from plant food is limited today.Methods: Here, to investigate the allergenicity of strawberry-derived microvesicles (MVs), nanovesicles (NVs), and subpopulations of NV, we have set up a multidisciplinary approach. The strategy combines proteomics-based protein identification, immunological investigations, bioinformatics, and data mining to gain biological insights useful to evaluate the presence of potential allergens and the immunoglobulin E (IgE) inhibitory activity of vesicle preparations.Results: Immunological test showed that several proteins of strawberry-derived vesicles compete for IgE binding with allergens spotted on the FABER biochip. This includes the known strawberry allergens Fra a 1, Fra a 3, and Fra a 4, and also other IgE-binding proteins not yet described as allergens in this food, such as gibberellin-regulated proteins, 2S albumin, pectate lyase, and trypsin inhibitors. Proteomics identified homologous sequences of the three strawberry allergens and their isoforms in total protein extract (TPE) but only Fra a 1 and Fra a 4 in the vesicle samples. Label-free quantitative proteomic analysis revealed no significant enrichment of these proteins in strawberry vesicles with respect to TPE.Conclusion: Immunological tests and bioinformatics analysis of proteomics data sets revealed that MVs and NVs isolated from strawberries can carry functional allergens their isoforms as well as proteins potentially allergenic based on their structural features. This should be considered when these new nanomaterials are used for human nutraceutical or biomedical applications.

Mapping Sequential IgE-Binding Epitopes on Major and Minor Egg Allergens

<b><i>Introduction:</i></b> Molecular studies of hen’s egg allergens help define allergic phenotypes, with IgE to sequential (linear) epitopes on the ovomucoid (OVM) protein associated with a persistent disease. Epitope profiles of other egg allergens are largely unknown. The objective of this study was to construct an epitope library spanning across 7 allergens and further evaluate sequential epitope-specific (<i>ses-</i>)IgE and <i>ses-</i>IgG₄ among baked-egg reactive or tolerant children. <b><i>Methods:</i></b> A Bead-Based Epitope Assay was used to identify informative IgE epitopes from 15-mer overlapping peptides covering the entire OVM and ovalbumin (OVA) proteins in 38 egg allergic children. An amalgamation of 12 B-cell epitope prediction tools was developed using experimentally identified epitopes. This ensemble was used to predict epitopes from ovotransferrin, lysozyme, serum albumin, vitellogenin-II fragment, and vitellogenin-1 precursor. <i>Ses-</i>IgE and <i>ses-</i>IgG₄ repertoires of 135 egg allergic children (82 reactive to baked-egg, the remaining 52 tolerant), 46 atopic controls, and 11 healthy subjects were compared. <b><i>Results:</i></b> 183 peptides from OVM and OVA were screened and used to create an aggregate algorithm, improving predictions of 12 individual tools. A final library of 65 sequential epitopes from 7 proteins was constructed. Egg allergic children had higher <i>ses-</i>IgE and lower <i>ses-</i>IgG₄ to predominantly OVM epitopes than both atopic and healthy controls. Baked-egg reactive children had similar <i>ses-</i>IgG₄ but greater <i>ses-</i>IgE than tolerant group. A combination of OVA-sIgE with <i>ses-</i>IgEs to OVM-023 and OVA-028 was the best predictor of reactive phenotype. <b><i>Conclusion:</i></b> We have created a comprehensive epitope library and showed that <i>ses-</i>IgE is a potential biomarker of baked-egg reactivity.

The first reported case of Blaptica dubia cockroach allergy

Background Periplaneta americana and Blattella germanica cockroaches are widespread, and risk of sensitization increases in urban environments where these roaches thrive as household pests. There are no prior reports of Blaptica dubia cockroach allergy, though human exposure to B. dubia is increasing through commercial breeding as feeder insects. Case presentation A 50-year-old B. dubia cockroach breeder presented with progressively worsening upper and lower respiratory symptoms in recent years. Symptoms were worse with exposure to her B. dubia roach colony. Skin prick testing (SPT) to B. dubia cast skin, internal organs, and feces was performed in both the subject and a human control. Testing for P. americana and B. germanica sensitization was also performed in the subject. SDS–Polyacrylamide gel electrophoresis (PAGE), immunoblots, and enzyme-linked immunosorbent assays (ELISA) studies were performed using the subject and control serums to explore for specific IgE binding to B. dubia as well as P. americana. Our results showed SPT was positive to B. dubia internal organs in the subject and negative in the control. In the subject, SPT was negative to P. americana though intradermal (ID) testing was positive and serum specific IgE (sIgE) testing was negative to B. germanica. Immunoblotting of the subject's serum to B. dubia internal organ extract showed several distinct bands of IgE binding at 47 kilodaltons (kD), 68 kD, 74 kD, 83 kD, and 118 kD. The strongest band was at 118 kD on B. dubia immunoblotting, which was absent in P. americana on SDS-PAGE. ELISA studies showed an increased IgE response to both B. dubia and P. americana in the subject versus the control. Conclusions This case confirmed the first reported allergy to B. dubia cockroaches. There may be cross-reactivity between B. dubia and P. americana, though our case suggests SPT and sIgE testing using P. americana and B. germanica extract has potential to miss a B. dubia cockroach allergy. This allergy is likely underreported, and further study is needed to explore the natural history of B. dubia cockroach allergy.

The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPOHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.

Occupational Allergic Sensitization Among Workers Processing King Crab (Paralithodes camtschaticus) and Edible Crab (Cancer pagurus) in Norway and Identification of Novel Putative Allergenic Proteins

Introduction: Asthma and allergy occur frequently among seafood processing workers, with the highest prevalence seen in the crustacean processing industry. In this study we established for the first time the prevalence of allergic sensitization in the Norwegian king- and edible crab processing industry and characterized the IgE-reactive proteins.Materials and Methods: Two populations of crab processing workers participated; 119 king crab and 65 edible crab workers. The investigation included information on work tasks and health through a detailed questionnaire. Allergic sensitization was investigated by crab-specific IgE quantification and skin prick tests (SPT) to four in-house prepared crab extracts; raw meat, cooked meat, raw intestines and raw shell. Allergen-specific IgE binding patterns were analyzed by IgE immunoblotting to the four allergen extracts using worker serum samples. Total proteins in crab SPT extracts and immunoblot-based IgE binding proteins were identified by mass spectrometric analysis.Results: Positive SPTs were established in 17.5% of king- and 18.1% of edible crab workers, while elevated IgE to crab were demonstrated in 8.9% of king- and 12.2% of edible crab processing workers. There was no significant difference between the king and edible crab workers with respect to self-reported respiratory symptoms, elevated specific IgE to crab or SPT results. Individual workers exhibited differential IgE binding patterns to different crab extracts, with most frequent binding to tropomyosin and arginine kinase and two novel IgE binding proteins, hemocyanin and enolase, identified as king- and edible crab allergens.Conclusions: Occupational exposure to king- and edible crabs may frequently cause IgE mediated allergic sensitization. Future investigations addressing the diagnostic value of crab allergens including tropomyosin and arginine kinase and the less well-known IgE-binding proteins hemocyanin and enolase in a component-resolved diagnostic approach to crab allergy should be encouraged.

Fel d1 Blocking Antibodies: A Novel Method to Reduce IgE-Mediated Allergy to Cats

Fel d1 is an important allergen produced by cats that causes IgE reactions in up to 95% of cat-allergic adults. Immunotherapy to reduce human allergy to cats has demonstrated that people have the capacity to produce allergen-specific neutralizing antibodies that block IgE-mediated allergic responses. We wished to determine if “blocking” antibodies could be used to reduce the IgE binding ability of cat allergens prior to their exposure to humans. Here, we describe the characterization of Fel d1-specific antibodies. We demonstrated the efficacy of a rabbit polyclonal and an allergen-specific chicken IgY to bind to Fel d1 in cat saliva and block Fel d1-IgE binding and IgE-mediated basophil degranulation. Fel d1 blocking antibodies offer a new and exciting approach to the neutralization of cat allergens.