María Garrido-Arandia

Transport of Pru p 3 across gastrointestinal epithelium – an essential step towards the induction of food allergy?

Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated.

Alt a 1 from Alternaria interacts with PR5 thaumatin-like proteins

Alt a 1 is a protein found in Alternaria alternata spores related to virulence and pathogenicity and considered to be responsible for chronic asthma in children. We found that spores of Alternaria inoculated on the outer surface of kiwifruits did not develop hyphae. Nevertheless, the expression of Alt a 1 gene was upregulated, and the protein was detected in the pulp where it co‐localized with kiwi PR5. Pull‐down assays demonstrated experimentally that the two proteins interact in such a way that Alt a 1 inhibits the enzymatic activity of PR5. These results are relevant not only for plant defense, but also for human health as patients with chronic asthma could suffer from an allergic reaction when they eat fruit contaminated with Alternaria.

Two nonspecific lipid transfer proteins (nsLTPs) from tomato seeds are associated to severe symptoms of tomato-allergic patients.

SCOPE Tomato has become one of the most consumed vegetables worldwide, but its intake is often accompanied by an increasing risk of inducing allergic symptoms. The aim of the work was the identification of new seed-specific allergens associated with severe symptoms in patients allergic to this edible vegetable. METHODS AND RESULTS We used 22 sera from well-defined tomato-allergic patients. Tomato seed proteins were purified and analyzed for biochemical and immunological characterization. A mixture of two associated IgE-binding nsLTPs was purified from tomato seeds. Both allergens, Sola l 7 and Sola l 6, displayed primary structure differences with respect to their counterpart, Sola l 3, from tomato pulp/peel. They retained the ability to bind IgE from 71.4% of patients with severe symptoms. The purified proteins induced positive basophil activation test and skin prick test, and displayed cross-reactivity with homologous allergens from peanut and sunflower seeds, among others. CONCLUSION We herein described two novel allergens from tomato seeds that belong to the nonspecific lipid transfer protein family classes 1 and 2, respectively. This is the first work associating IgE reactivity to these proteins with severe symptoms of certain tomato-allergic patients. Therefore, they are optimal candidates for clarifying the diagnosis of the tomato allergy.

Molecular Dynamics of Major Allergens from Alternaria, Birch Pollen and Peach

In the search for factors that make a protein allergenic (an issue that remains so far elusive) some common features of allergens such as small size, high stability and lipid binding are recognized in spite of their structural diversity. Other relevant but still poorly understood feature is their capability to form homodimers. We investigated by means of Molecular Dynamics (MD) calculations the stability in solution of several dimers of three major allergens from Alternaria mold, birch pollen, and peach fruit known to play essential roles in allergic disease. By running 20 ns MD simulations we found essential properties on solution that provide information of interest on their dimerization, stability of their epitopes and dynamical features of ligand binding cavities. Our results show that three essential allergen proteins display a distinct behavior on their trends to form homodimers in solution.

Characterisation of a flavonoid ligand of the fungal protein Alt a 1

Spores of pathogenic fungi are virtually ubiquitous and cause human disease and severe losses in crops. The endophytic fungi Alternaria species produce host-selective phytotoxins. Alt a 1 is a strongly allergenic protein found in A. alternata that causes severe asthma. Despite the well-established pathogenicity of Alt a 1, the molecular mechanisms underlying its action and physiological function remain largely unknown. To gain insight into the role played by this protein in the pathogenicity of the fungus, we studied production of Alt a 1 and its activity in spores. We found that Alt a 1 accumulates inside spores and that its release with a ligand is pH-dependent, with optimum production in the 5.0–6.5 interval. The Alt a 1 ligand was identified as a methylated flavonoid that inhibits plant root growth and detoxifies reactive oxygen species. We also found that Alt a 1 changes its oligomerization state depending on the pH of the surrounding medium and that these changes facilitate the release of the ligand. Based on these results, we propose that release of Alt a 1 should be a pathogenic target in approaches used to block plant defenses and consequently to favor fungal entry into the plant.

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

Key messagePru 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.AbstractThe 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.

Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients.

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.

Endothelial Regulator of Calcineurin 1 Promotes Barrier Integrity and Modulates Histamine-Induced Barrier Dysfunction in Anaphylaxis

Anaphylaxis, the most serious and life-threatening allergic reaction, produces the release of inflammatory mediators by mast cells and basophils. Regulator of calcineurin 1 (Rcan1) is a negative regulator of mast-cell degranulation. The action of mediators leads to vasodilation and an increase in vascular permeability, causing great loss of intravascular volume in a short time. Nevertheless, the molecular basis remains unexplored on the vascular level. We investigated Rcan1 expression induced by histamine, platelet-activating factor (PAF), and epinephrine in primary human vein (HV)-/artery (HA)-derived endothelial cells (ECs) and human dermal microvascular ECs (HMVEC-D). Vascular permeability was analyzed in vitro in human ECs with forced Rcan1 expression using Transwell migration assays and in vivo using Rcan1 knockout mice. Histamine, but neither PAF nor epinephrine, induced Rcan1-4 mRNA and protein expression in primary HV-ECs, HA-ECs, and HMVEC-D through histamine receptor 1 (H1R). These effects were prevented by pharmacological inhibition of calcineurin with cyclosporine A. Moreover, intravenous histamine administration increased Rcan1 expression in lung tissues of mice undergoing experimental anaphylaxis. Functional in vitro assays showed that overexpression of Rcan1 promotes barrier integrity, suggesting a role played by this molecule in vascular permeability. Consistent with these findings, in vivo models of subcutaneous and intravenous histamine-mediated fluid extravasation showed increased response in skin, aorta, and lungs of Rcan1-deficient mice compared with wild-type animals. These findings reveal that endothelial Rcan1 is synthesized in response to histamine through a calcineurin-sensitive pathway and may reduce barrier breakdown, thus contributing to the strengthening of the endothelium and resistance to anaphylaxis. These new insights underscore its potential role as a regulator of sensitivity to anaphylaxis in humans.

Mechanisms underlying induction of allergic sensitization by Pru p 3

Recently, the nature of the lipid‐ligand of Pru p 3, one of the most common plant food allergens in southern Europe, has been identified as a derivative of the alkaloid camptothecin bound to phytosphingosine. However, the origin of its immunological activity is still unknown.