Real-life evaluation of molecular multiplex IgE test methods in the diagnosis of pollen associated food allergy.

BACKGROUND: Diagnosis of food allergies is challenging, as combining information from specific IgE (sIgE)-sensitization pattern and skin prick tests (SPTs) with clinical history is necessary for a personalized management of allergic patients. The aim of this study was to compare two molecular tests, the ImmunoCAP ISAC (ISAC) and the Allergy Explorer, version 2 (ALEX2 ) in the context of pollen food syndrome (PFS) diagnosis in a real-life scenario, to assess the benefit of multiplex testing in PFS patients. METHODS: Diagnosis of food allergy was performed in 53 patients. Allergen-sIgE concentrations were measured with ISAC and ALEX2 . Results for sIgE were statistically compared with each other, with SPT results and with clinical presentation of the patients. RESULTS: Using ISAC as reference test for sIgE measurements, the average sensitivity of ALEX2 for PR-10 allergens was 83.2% and the average specificity 88.0%. If only low sIgE concentrations were included, the sensitivity was 60.8% and the specificity 91.1%. Apple and hazelnut sensitizations were confirmed in most patients by concordance of sIgE and SPT results. Significant correlations were shown between clinical symptoms and Mal d 1- and Gly m 4-sIgE levels measured by both tests and for Cor a 1-sIgE levels measured by ALEX2 . In eight patients, profilin related symptoms were supported by Hev b 8-sensitization. CONCLUSION: Multiplex testing is beneficial to understand patient-specific individual sensitization profiles and to providing personalized management recommendations. In the future, custom-designed test kits might enable reducing costs of multiplex testing for specific patient groups without compromising the diagnostic value.

Molecular allergology and its impact in specific allergy diagnosis and therapy.

Progressive knowledge of allergenic structures resulted in a broad availability of allergenic molecules for diagnosis. Component-resolved diagnosis allowed a better understanding of patient sensitization patterns, facilitating allergen immunotherapy decisions. In parallel to the discovery of allergenic molecules, there was a progressive development of a regulation framework that affected both in vitro diagnostics and Allergen Immunotherapy products. With a progressive understanding of underlying mechanisms associated to Allergen immunotherapy and an increasing experience of application of molecular diagnosis in daily life, we focus in analyzing the evidences of the value provided by molecular allergology in daily clinical practice, with a focus on Allergen Immunotherapy decisions.

The diagnosis and management of allergic reactions in patients sensitized to non-specific lipid transfer proteins.

Sensitization to one or more non-specific lipid transfer proteins (nsLTPs), initially thought to exist mainly in southern Europe, is becoming accepted as a cause of allergic reactions to plant foods across Europe and beyond. The peach nsLTP allergen Pru p 3 is a dominant sensitizing allergen and peaches a common food trigger, although multiple foods can be involved. A frequent feature of reactions is the requirement for a cofactor (exercise, alcohol, non-steroidal anti-inflammatory drugs, Cannabis sativa) to be present for a food to elicit a reaction. The variability in the food and cofactor triggers makes it essential to include an allergy-focused diet and clinical history in the diagnostic workup. Testing on suspected food triggers should also establish whether sensitization to nsLTP is present, using purified or recombinant nsLTP allergens such as Pru p 3. The avoidance of known trigger foods and advice on cofactors is currently the main management for this condition. Studies on immunotherapy are promising, but it is unknown whether such treatments will be useful in populations where Pru p 3 is not the primary sensitizing allergen. Future research should focus on the mechanisms of cofactors, improving diagnostic accuracy and establishing the efficacy of immunotherapy.

The TNF-like weak inducer of the apoptosis/fibroblast growth factor-inducible molecule 14 axis mediates histamine and platelet-activating factor-induced subcutaneous vascular leakage and anaphylactic shock.

BACKGROUND: Anaphylaxis includes mast cell (MC) activation, but less is known about downstream mechanisms (ie, vascular permeability controlled by endothelial cells [ECs]). The TNF-like weak inducer of apoptosis (TWEAK) and its sole receptor, fibroblast growth factor-inducible molecule 14 (Fn14), belong to the TNF superfamily and are involved in proinflammatory responses. OBJECTIVE: We sought to investigate the role of TWEAK/Fn14 axis in anaphylaxis. METHODS: In vivo vascular permeability and mouse models of passive systemic anaphylaxis (PSA) and active systemic anaphylaxis were applied to wild-type (WT), TWEAK- and Fn14-deficient mice (TWEAK-/- and Fn14-/-, respectively). Primary bone marrow-derived mast cells (BMMCs) and ECs from WT and Fn14-/- or TWEAK-/- mice were studied. The TWEAK/Fn14 axis was also investigated in human samples. RESULTS: Mice with PSA and active systemic anaphylaxis had increased Fn14 and TWEAK expression in lung tissues and increased serum soluble TWEAK concentrations. TWEAK and Fn14 deficiencies prevent PSA-related symptoms, resulting in resistance to decreased body temperature, less severe reactions, and maintained physical activity. Numbers of MCs after PSA are similar between genotypes in different tissue regions, such as ear skin and the trachea, tongue, peritoneum, lungs, and bone marrow. Moreover, in vitro studies revealed no differences in degranulation or mediator release between WT and Fn14-/- BMMCs after IgE-FcεRI stimulation. In vivo and in vitro histamine and platelet-activating factor administration increases Fn14 receptor expression in lungs and ECs. Moreover, Fn14 deficiency in ECs maintained in vitro impermeability when stimulated by mediators or activated BMMCs but not by TWEAK-/- BMMCs, indicating that Fn14 is crucial for endothelial barrier function. TWEAK/Fn14 deletion or TWEAK-blocking antibody prevented histamine/platelet-activating factor-induced vascular subcutaneous permeability. Circulating soluble TWEAK levels were increased in patients with anaphylaxis, and plasma from those patients increased Fn14 expression in ECs. CONCLUSION: The TWEAK/Fn14 axis participates in anaphylactic reactions. Inhibition of TWEAK/Fn14 interaction could be efficacious in anaphylaxis therapy.

New insights into the sensitization to nonspecific lipid transfer proteins from pollen and food: New role of allergen Ole e 7.

BACKGROUND: Ole e 7 is a nonspecific lipid transfer protein (nsLTP) from olive pollen, one of the main allergenic pollens worldwide. This allergenic nsLTP is responsible for severe symptoms in regions with high olive pollen exposure, where many Ole e 7-sensitized patients exhibit a co-sensitization to the peach nsLTP, Pru p 3. However, there is no evidence of cross-reactivity, which explains this observed co-sensitization. Therefore, the purpose of this study was to explore the relationship between Ole e 7 and Pru p 3. METHODS: A total of 48 patients sensitized to Ole e 7 and/or Pru p 3 were included in the study. Specific IgE serum levels were measured by ImmunoCAP 250 and ELISA. Inhibition assays were performed to determine the existence of cross-reactivity between both nsLTPs. Allergic response was analyzed ex vivo (basophil activation test) and in vitro (RBL-2H3 mast cell model). RESULTS: Common IgG and IgE epitopes were identified between both allergens. IgE-binding inhibition was detected in Ole e 7-monosensitized patients using rPru p 3 as inhibitor, reaching inhibition values of 25 and 100%. Ex vivo and in vitro assays revealed a response against rPru p 3 in four (31%) Ole e 7-monosensitized patients. CONCLUSIONS: Our results suggest that Ole e 7 could play a new role as primary sensitizer in regions with high olive pollen exposure, leading to the peach nsLTP sensitization. This co-sensitization process would occur because of the cross-reactivity between Ole e 7 and Pru p 3 observed in some allergic patients.

Performance of basophil activation test and specific IgG4 as diagnostic tools in nonspecific lipid transfer protein allergy: Antwerp-Barcelona comparison.

BACKGROUND: Recent studies show that nsLTP sensitization is not limited to the Mediterranean basin and can present diverse clinical phenotypes. It remains challenging to predict clinical outcome when specific IgE antibodies (sIgE) to nsLTPs are present. This study compares both clinical and in vitro allergy characteristics but also diagnostic performance of a basophil activation test (BAT) and sIgG4 in nsLTP-sensitized patients from Antwerp (ANT, Belgium) and Barcelona (BCN, Spain). METHODS: Adult subjects with positive sIgE rPru p 3 and/or rMal d 3 ≥ 0.10 kUA /L (n = 182) and healthy controls (n = 37) were included. NsLTP-sensitized individuals were stratified according to clinical symptoms with peach/apple, respectively. BAT rPru p 3 and rMal d 3 were performed and sIgG4 antibodies to both components quantified. RESULTS: In BCN, only ratios of sIgG4/sIgE rMal d 3 and BAT rMal d 3 (0.001 µg/mL) can identify clinically relevant Mal d 3 sensitization (sensitivity of 60%-63% and a specificity of 75%-67%, respectively). In ANT, only the sIgE/total IgE rPru p 3 ratio shows added value (sensitivity 60% and specificity 83%). Finally, it appears that symptomatic patients in BCN are more sensitive to lower allergen concentrations compared to ANT. In addition, it was shown that ANT patients were more often sensitized to pollen and that specific pollen sources differed between regions. CONCLUSIONS: NsLTP-related allergy profiles and diagnostic performance differ significantly between regions and are component-specific, which makes extrapolation of data difficult to do. In addition, it seems that basophil sensitivity might show geographical differences. Additional research is needed to confirm these findings.

Identification and molecular characterization of allergenic non-specific lipid-transfer protein from durum wheat (Triticum turgidum).

BACKGROUND: Common wheat (Triticum aestivum) and durum wheat (T. turgidum) are both involved in Baker's asthma (BA) and food allergy (FA) including wheat-dependent exercise-induced asthma (WDEIA). However, allergens in durum wheat have not been described, and the over-expression of T. turgidum non-specific lipid-transfer protein (nsLTPs) is considered to increase resistance to phytopathogens. OBJECTIVE: To identify and assess the allergenicity of nsLTP from T. turgidum. METHODS: Recombinant T. turgidum nsLTP Tri tu 14 was generated and tested for structural integrity (circular dichroism-spectroscopy) and purity (SDS-PAGE). Thirty-two wheat allergic patients were enrolled: 20 Spanish patients (BA) with positive bronchial challenge to wheat flour, and 12 Italian patients (wheat FA/WDEIA) with positive double-blind placebo-controlled food challenge/open food challenge (OFC) to pasta. IgE values to wheat, Tri tu 14, Tri a 14 (T. aestivum) and Pru p 3 (P. persica) were determined by ImmunoCAP testing. Allergenic potency (in vitro mediator release) and IgE cross-reactivity were investigated. RESULTS: Tri tu 14 was found to share 49% and 52% amino acid identity with Tri a 14 and Pru p 3, respectively. Among 25 Tri a 14 CAP positive sera, 23 (92%) were reactive to wheat extract, 22 (88%) to Tri tu 14 and 20 (80%) to Pru p 3. The correlation between Tri a 14 and Tri tu 14 specific IgE levels was r = 0.97 (BA) and r = 0.93 (FA/WDEIA), respectively. FA/WDEIA patients showed higher specific IgE values to Tri tu 14 and Pru p 3 than BA patients. Tri tu 14 displayed allergenic activity by mediator release from effector cells and IgE cross-reactivity with Pru p 3. The degree of IgE cross-reactivity between the two wheat nsLTPs varied between individual patients. CONCLUSIONS AND CLINICAL RELEVANCE: Sensitization to Tri tu 14 likely appears to be more important in wheat FA/WDEIA than in BA. Over-expression of Tri tu 14 in wheat would represent a risk for patients with nsLTP-mediated FA.

Interaction of Alt a 1 with SLC22A17 in the airway mucosa.

BACKGROUND: Despite all the efforts made up to now, the reasons that facilitate a protein becoming an allergen have not been elucidated yet. Alt a 1 protein is the major fungal allergen responsible for chronic asthma, but little is known about its immunological activity. Our main purpose was to investigate the ligand-dependent interactions of Alt a 1 in the human airway epithelium. METHODS: Alt a 1 with and without its ligand (holo- and apo- forms) was incubated with the pulmonary epithelial monolayer model, Calu-3 cells. Allergen transport and cytokine production were measured. Pull-down and immunofluorescence assays were employed to identify the receptor of Alt a 1 using the epithelial cell model and mouse tissues. Receptor-allergen-ligand interactions were analyzed by computational modeling. RESULTS: The holo-form could activate human monocytes, PBMCs, and polarized airway epithelial (Calu-3) cell lines. The allergen was also transported through the monolayer, without any alteration of the epithelial integrity (TEER). Alt a 1 also induced the production of proinflammatory IL8 and specific epithelial cytokines (IL33 and IL25) by Calu-3 cells. The interaction between epithelial cells and holo-Alt a 1 was found to be mediated by the SLC22A17 receptor, and its recognition of Alt a 1 was explained in structural terms. CONCLUSIONS: Our findings identified the Alt a 1 ligand as a central player in the interaction of the allergen with airway mucosa, shedding light into its potential role in the immunological response, while unveiling its potential as a new target for therapy intervention.

Peach Tree Pollen and Prunus persica 9 Sensitisation and Allergy in Children and Adolescents.

BACKGROUND: Although plant and fruit pollens are entomophilous and relevant in exposed workers, we have shown a high frequency of sensitisation and symptoms induction of peach tree pollen (PTP) and Prunus persica 9 (Pru p 9) in adults from areas of peach cultivars. METHODS: We studied the sensitisation and clinical relevance of PTP and Pru p 9 in a large group of children and adolescents aged 3-19 years. A detailed questionnaire plus skin prick testing to prevalent allergens, PTP, and Pru p 9 were carried out. The clinical relevance was established by nasal provocation test (NPT) and symptom score index. RESULTS: We evaluated 685 children (mean age 8.75 ± 3.3 years, median 9 years), 52% of them female. Sensitisation to PTP occurred in 20% of the cases following olive tree (33%) and Phleum pratense (26%). In a randomly selected subgroup of subjects sensitised to PTP, 30% were skin prick test-positive to Pru p 9. Most cases had rhinitis or rhinoconjunctivitis. NPT showed the relevance of PTP and Pru p 9 in the induction of symptoms. CONCLUSION: PTP and Pru p 9 are relevant in the induction of sensitisation and respiratory symptoms in children and adolescents. This allergen should be evaluated in children living in regions of peach tree cultivars.

Effect of pre- and post-weaning dietary supplementation with arginine and glutamine on rabbit performance and intestinal health.

BACKGROUND: The purpose of the present study was to assess if the exposure to glutamine (Gln), arginine (Arg) or their combination from pregnancy, through the maternal diet, to a post weaning supplemented diet, can stimulate litter performance, gut development and immune function. To this end does and their litters were fed the same basal diet no supplemented (control C), or supplemented with 0.4% Gln, 0.4% Arg, or 0.4 Gln + 0.4 Arg. Rabbits were weaned at 25 d of age and fed the same experimental diet as their mothers for 10 additional days (35 d of age). Bacterial translocation to mesenteric lymph nodes (MLN) at 6 d of age and intestinal histology, enzymatic activity, phenotypical and functional analysis of intraepithelial lymphocytes (IEL) from the appendix were determined at 6, 25 and 35 d of age. RESULTS: No significant differences on animal performance or mortality rates were observed among dietary treatments. However, kits from rabbit does supplemented with Gln tended (P ≤ 0.10) to reduce the translocation of total number of both aerobic and facultative anaerobic bacteria to the MLN. Also, rabbits fed the Gln supplemented diets maintained intestinal villous height at weaning compared to the non-supplemented diets (P < 0.05). The proportions of CD45+CD4+ and CD45+CD8+ IEL in the appendix were not affected by dietary means. However, in rabbits IEL at weaning dietary Gln significantly upregulated IL-2 and downregulated IL-6 expression. CONCLUSIONS: Despite a lack of effect on performance and mortality the inclusion of 0.4% Gln has a positive effect by maintaining intestinal villous height and modulating the cytokine profile at weaning. The supplementation with Arg or Arg + Gln at the selected doses in this study did not exert positive effects on rabbit intestinal health.

Energy Landscapes of Ligand Motion Inside the Tunnel-Like Cavity of Lipid Transfer Proteins: The Case of the Pru p 3 Allergen.

Allergies are a widespread problem in western countries, affecting a large part of the population, with levels of prevalence increasingly rising due to reasons still not understood. Evidence accumulated in recent years points to an essential role played by ligands of allergen proteins in the sensitization phase of allergies. In this regard, we recently identified the natural ligand of Pru p 3, a lipid transfer protein, a major allergen from peach fruit and a model of food allergy. The ligand of Pru p 3 has been shown to play a key role in the sensitization to peach and to other plant food sources that provoke cross-reactivity in a large proportion of patients allergic to peach. However, the question of which is the binding pose of this ligand in its carrier protein, and how it can be transferred to receptors of the immune system where it develops its function as a coadjuvant was not elucidated. In this work, different molecular dynamics simulations have been considered as starting points to study the properties of the ligand⁻protein system in solution. Besides, an energy landscape based on collective variables that describe the process of ligand motion within the cavity of Pru p 3 was obtained by using well-tempered metadynamics. The simulations revealed the differences between distinct binding modes, and also revealed important aspects of the motion of the ligand throughout its carrier protein, relevant to its binding⁻unbinding process. Our findings are potentially interesting for studying protein⁻ligand systems beyond the specific case of the allergen protein dealt with here.

Expression and Interaction Analysis among Saffron ALDHs and Crocetin Dialdehyde.

In saffron, the cleavage of zeaxanthin by means of CCD2 generates crocetin dialdehyde, which is then converted by an unknown aldehyde dehydrogenase to crocetin. A proteome from saffron stigma was released recently and, based on the expression pattern and correlation analyses, five aldehyde dehydrogenases (ALDHs) were suggested as possible candidates to generate crocetin from crocetin dialdehydes. We selected four of the suggested ALDHs and analyzed their expression in different tissues, determined their activity over crocetin dialdehyde, and performed structure modeling and docking calculation to find their specificity. All the ALDHs were able to convert crocetin dialdehyde to crocetin, but two of them were stigma tissue-specific. Structure modeling and docking analyses revealed that, in all cases, there was a high coverage of residues in the models. All of them showed a very close conformation, indicated by the low root-mean-square deviation (RMSD) values of backbone atoms, which indicate a high similarity among them. However, low affinity between the enzymes and the crocetin dialdehyde were observed. Phylogenetic analysis and binding affinities calculations, including some ALDHs from Gardenia jasmonoides, Crocus sieberi, and Buddleja species that accumulate crocetin and Bixa orellana synthetizing the apocarotenoid bixin selected on their expression pattern matching with the accumulation of either crocins or bixin, pointed out that family 2 C4 members might be involved in the conversion of crocetin dialdehyde to crocetin with high specificity.

A Comparative Study of Human Saposins.

Saposins are small proteins implicated in trafficking and loading of lipids onto Cluster of Differentiation 1 (CD1) receptor proteins that in turn present lipid antigens to T cells and a variety of T-cell receptors, thus playing a crucial role in innate and adaptive immune responses in humans. Despite their low sequence identity, the four types of human saposins share a similar folding pattern consisting of four helices linked by three conserved disulfide bridges. However, their lipid-binding abilities as well as their activities in extracting, transporting and loading onto CD1 molecules a variety of sphingo- and phospholipids in biological membranes display two striking characteristics: a strong pH-dependence and a structural change between a compact, closed conformation and an open conformation. In this work, we present a comparative computational study of structural, electrostatic, and dynamic features of human saposins based upon their available experimental structures. By means of structural alignments, surface analyses, calculation of pH-dependent protonation states, Poisson-Boltzmann electrostatic potentials, and molecular dynamics simulations at three pH values representative of biological media where saposins fulfill their function, our results shed light into their intrinsic features. The similarities and differences in this class of proteins depend on tiny variations of local structural details that allow saposins to be key players in triggering responses in the human immune system.

Identification of the ligand of Pru p 3, a peach LTP.

KEY MESSAGE: Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation. The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

A relevant IgE-reactive 28kDa protein identified from Salsola kali pollen extract by proteomics is a natural degradation product of an integral 47kDa polygalaturonase.

A highly prevalent IgE-binding protein band of 28kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein. The far-UV CD spectrum determined that rSal k 6 was folded. Immunostaining of the S. kali pollen protein extract with a rSal k 6-specific pAb and LC-MS/MS proteomic analyses confirmed the co-existence of the 28kDa band together with an allergenic band of about 47kDa in the pollen extract. Therefore, the 28kDa was assigned as a natural degradation product of the 47kDa integral polygalacturonase. The IgE-binding inhibition to S. kali pollen extract using rSal k 6 as inhibitor showed that signals directed to both protein bands of 28 and 47kDa were completely abrogated. The average prevalence of rSal k 6 among the three populations analyzed was 30%, with values correlating well with the levels of grains/m3 of Amaranthaceae pollen. Sal k 6 shares IgE epitopes with Oleaceae members (Fraxinus excelsior, Olea europaea and Syringa vulgaris), with IgE-inhibition values ranging from 20% to 60%, respectively. No IgE-inhibition was observed with plant-derived food extracts.

Detection of major food allergens in amniotic fluid: initial allergenic encounter during pregnancy.

BACKGROUND: Ingestion of food allergens present in maternal milk during breastfeeding has been hypothesized as a gateway to sensitization to food; however, this process could develop during pregnancy, as the maternal-fetal interface develops a Th2- and Treg-mediated environment to protect the fetus. We hypothesized that in these surroundings, unborn children are exposed to food allergens contained in the mother's diet, possibly giving rise to first sensitization. METHODS: The presence of allergens in utero was studied by analyzing amniotic fluid (AF) samples in two different stages of pregnancy: at 15-20 weeks and after delivery at term. An antibody microarray was developed to test for the most common food allergens. The array detects the presence of ten allergens from milk, fruit, egg, fish, nuts, and wheat. RESULTS: AF from 20 pregnant women was collected: eight after delivery at term and 12 from women who underwent diagnostic amniocentesis between weeks 15 and 20 of gestation. The presence of allergens was detected in all samples. Samples from amniocentesis had a higher allergen concentration than samples after delivery at term. CONCLUSIONS: We demonstrated the presence of intact major food allergens in AF samples. This early contact could explain subsequent sensitization to foods never eaten before.

Computational study of pH-dependent oligomerization and ligand binding in Alt a 1, a highly allergenic protein with a unique fold.

Alt a 1 is a highly allergenic protein from Alternaria fungi responsible for several respiratory diseases. Its crystal structure revealed a unique ß-barrel fold that defines a new family exclusive to fungi and forms a symmetrical dimer in a butterfly-like shape as well as tetramers. Its biological function is as yet unknown but its localization in cell wall of Alternaria spores and its interactions in the onset of allergy reactions point to a function to transport ligands. However, at odds with binding features in ß-barrel proteins, monomeric Alt a 1 seems unable to harbor ligands because the barrel is too narrow. Tetrameric Alt a 1 is able to bind the flavonoid quercetin, yet the stability of the aggregate and the own ligand binding are pH-dependent. At pH 6.5, which Alt a 1 would meet when secreted by spores in bronchial epithelium, tetramer-quercetin complex is stable. At pH 5.5, which Alt a 1 would meet in apoplast when infecting plants, the complex breaks down. By means of a combined computational study that includes docking calculations, empirical pKa estimates, Poisson-Boltzmann electrostatic potentials, and Molecular Dynamics simulations, we identified a putative binding site at the dimeric interface between subunits in tetramer. We propose an explanation on the pH-dependence of both oligomerization states and protein-ligand affinity of Alt a 1 in terms of electrostatic variations associated to distinct protonation states at different pHs. The uniqueness of this singular protein can thus be tracked in the combination of all these features.

Bet v 1 from birch pollen is a lipocalin-like protein acting as allergen only when devoid of iron by promoting Th2 lymphocytes.

It is hypothesized that allergens are at the borderline of self and non-self and, through as yet elusive circumstances, mount a Th2 response for allergic sensitization. The major birch pollen allergen Bet v 1 is considered the prototype for the PR-10 protein family causing respiratory allergy. Here, we give structural evidence that Bet v 1 is a lipocalin-like protein with a striking structural resemblance to human lipocalin 2. Lipocalin 2 is highly expressed in the lung where it exerts immunoregulatory functions dependent on being loaded with siderophore-bound iron (holo-form) or not (apo-form). We demonstrate that similar to lipocalin 2, Bet v 1 is capable of binding iron via catechol-based siderophores. Thereby, calculated Kd values of 66 nm surpassed affinities to known ligands nearly by a power of 10. Moreover, we give functional evidence of the immunomodulatory capacity of Bet v 1 being dependent on its iron-loaded state. When incubated to human immune cells, only the apo-form of Bet v 1, but not the holo-form, was able to promote Th2 cells secreting IL13. These results provide for the first time a functional understanding on the allergenicity of Bet v 1 and a basis for future allergen immunotherapies counteracting Th2 immune responses on a molecular basis.