Mechanisms underlying induction of allergic sensitization by Pru p 3.

BACKGROUND: 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. OBJECTIVE: We sought to evaluate the role of the Pru p 3 lipid-ligand in the immunogenic activity of Pru p 3. METHODS: In vitro cultures of different cell types (monocyte-derived dendritic cells [moDCs], PBMCs [peripheral blood mononuclear cells] and epithelial and iNKT-hybridoma cell lines) have been used to determine the immunological capacity of the ligand, by measuring cell proliferation, maturation markers and cytokine production. To study the capacity of the lipid-ligand to promote sensitization to Pru p 3 in vivo, a mouse model of anaphylaxis to peach has been produced and changes in the humoral and basophil responses have been analysed. RESULTS: The lipid-ligand of Pru p 3 induced maturation of moDCsc and proliferation of PBMCs. Its immunological activity resided in the phytosphingosine tail of the ligand. The adjuvant activity of the ligand was also confirmed in vivo, where the complex of Pru p 3-ligand induced higher levels of IgE than Pru p 3 alone. The immunological capacity of the Pru p 3 ligand was mediated by CD1d, as maturation of moDCs was inhibited by anti-CD1d antibodies and Pru p 3-ligand co-localized with CD1d on epithelial cells. Finally, Pru p 3-ligand presented by CD1d was able to interact with iNKTs. CONCLUSIONS AND CLINICAL RELEVANCE: The Pru p 3 lipid-ligand could act as an adjuvant to promote sensitization to Pru p 3, through its recognition by CD1d receptors. This intrinsic adjuvant activity of the accompanying lipid cargo could be a general essential feature of the mechanism underlying the phenomenon of allergenicity.

Structural similarities of human and mammalian lipocalins, and their function in innate immunity and allergy.

Owners and their domestic animals via skin shedding and secretions, mutually exchange microbiomes, potential pathogens and innate immune molecules. Among the latter especially lipocalins are multifaceted: they may have an immunomodulatory function and, furthermore, they represent one of the most important animal allergen families. The amino acid identities, as well as their structures by superposition modeling were compared among human lipocalins, hLCN1 and hLCN2, and most important animal lipocalin allergens, such as Can f 1, Can f 2 and Can f 4 from dog, Fel d 4 from cats, Bos d 5 from cow's milk, Equ c 1 from horses, and Mus m 1 from mice, all of them representing major allergens. The ß-barrel fold with a central molecular pocket is similar among human and animal lipocalins. Thereby, lipocalins are able to transport a variety of biological ligands in their highly conserved calyx-like cavity, among them siderophores with the strongest known capability to complex iron (Fe(3+) ). Levels of human lipocalins are elevated in nonallergic inflammation and cancer, associated with innate immunoregulatory functions that critically depend on ligand load. Accordingly, deficient loading of lipocalin allergens establishes their capacity to induce Th2 hypersensitivity. Our similarity analysis of human and mammalian lipocalins highlights their function in innate immunity and allergy.

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 theoretical study on the formation of iodine oxide aggregates and monohydrates.

Biotic and abiotic emissions of molecular iodine and iodocarbons from the sea or the ice surface and the intertidal zone to the coastal/polar marine boundary layer lead to the formation of iodine oxides, which subsequently nucleate forming iodine oxide particles (IOPs). Although the link between coastal iodine emissions and ultrafine aerosol bursts is well established, the details of the nucleation mechanism have not yet been elucidated. In this paper, results of a theoretical study of a range of potentially relevant aggregation reactions of different iodine oxides, as well as complexation with water molecules, are reported. Thermochemical properties of these reactions are obtained from high level ab initio correlated calculations including spin-orbit corrections. The results show that the nucleation path most likely proceeds through dimerisation of I2O4. It is also shown that water can hinder gas-to-particle conversion to some extent, although complexation with key iodine oxides does not remove enough of these to stop IOP formation. A consistent picture of this process emerges from the theoretical study presented here and the findings of a new laboratory study reported in the accompanying paper (Gomez Martin et al., 2013).

Characterization of IgE epitopes of Cuc m 2, the major melon allergen, and their role in cross-reactivity with pollen profilins.

BACKGROUND: Plant profilins are described as minor allergens, although with some exceptions in foods such as melon, watermelon or orange. In fact, they could be responsible for many cross-reactions among distantly related species. This is likely to be a consequence of the presence of common epitopes. OBJECTIVE: To characterize the B epitopes of Cuc m 2, a model of plant food profilin, using phage display techniques and to compare with other profilins, such as those of timothy grass and birch pollen, and human I profilin, to understand the mechanism of cross-reaction among members of this family. METHODS: IgE of melon-allergic patients was used to select clones from a phage display 12 mer peptide library. After two rounds of screening, Cuc m 2-specific clones were eluted and the DNA insertion sequenced. The residues of each clone were mapped on the Cuc m 2 surface to define a mimotope, which was also localized on the three-dimensional surfaces of other profilins. RESULTS: Seventeen melon-allergic patients were selected. Sera from each of them recognized the melon profilin, Cuc m 2, but the majority also recognized Phl p 12 or Bet v 2, timothy grass-, and birch-pollen profilins, respectively. A Cuc m 2 mimotope was defined and mapped onto its surface giving the following sequence: S(2)W(3)A(5)Y(6)D(9)H(10)T(111)P(112)G(113)Q(114)N(116)M(117)R(121)L(122). The homologous residues in Phl p 12 and Bet v 2 had almost identical sequences. By contrast, the homologous sequence in human profilin showed many differences. CONCLUSIONS: The identified mimotope could be involved in cross-reactions among food and pollen profilins. Many of these cross-reactions observed in the clinical realm could be explained by the presence of a common epitope found in food and pollen allergens. A new strategy of immunotherapy based on this IgE region could be used in alternative immunotherapy strategies.

Variation of geometries and electron properties along proton transfer in strong hydrogen-bond complexes.

Proton transfer in hydrogen-bond systems formed by 4-methylimidazole in both neutral and protonated cationic forms and by acetate anion are studied by means of MP26-311++G(d,p) ab initio calculations. These two complexes model the histidine (neutral and protonated)-aspartate diad present in the active sites of enzymes the catalytic mechanism of which involves the formation of strong hydrogen bonds. We investigate the evolution of geometries, natural bond orbital populations of bonds and electron lone pairs, topological descriptors of the electron density, and spatial distributions of the electron localization function along the process N-H...O-->N...H...O-->N...H-O, which represents the stages of the H-transfer. Except for a sudden change in the population of electron lone pairs in N and O at the middle N...H...O stage, all the properties analyzed show a smooth continuous behavior along the covalent --> hydrogen bond transit inherent to the transfer, without any discontinuity that could identify a formation or breaking of the hydrogen bond. This way, the distinction between covalent or hydrogen-bonding features is associated to subtle electron rearrangement at the intermolecular space.

Distinct molecular surfaces and hydrophobicity of amino acid residues in proteins.

Hydrophobicity is a useful concept to rationalize the role played by amino acid residues in terms of buried or exposed conformation with regard to the aqueous environment in proteins. The relationship of this concept with distinct approaches to represent the molecular surface is analyzed by computing reliable surface areas for three definitions namely the van der Waals, solvent-accessible, and solvent-excluded molecular surfaces. The surface areas are obtained for all of the naturally occurring amino acids by first setting a proper reference standard state and then calculating their values for a database of proteins containing a total of 4297 residues. Despite the great differences in these molecular surfaces, proper indexes are here defined for handling the information of interest to study the hydrophobic behavior of amino acids provided by such surfaces.

Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds.

Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the structure-activity relationships of these enzymes. Here we report the identification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein modeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins were then generated by site-directed mutagenesis, expressed in Escherichia coli , and characterized for their chitinolytic activity. Glu124 and Glu146, the only carboxylic residues properly located into the active site cleft to participate in catalysis, were both mutated to Gln and Asp. Our results suggest that Glu124 functions as the general acid catalyst whereas Glu146 is likely to act as a general base. Other mutations involving three highly conserved active site residues, Gln173, Thr175, and Asn254, also impaired the chitinolytic activity of Ch3. The effects of these variants on the fungus Trichoderma viride revealed that catalysis is not necessary for antifungal activity. Similarly to its homologous nonenzymatic polypeptides hevein and stinging nettle lectin, the N-terminal chitin-binding domain of Ch3 appears to interfere itself with hyphal growth.