ENEA, a peach and apricot IgE-binding protein cross-reacting with the latex major allergen Hev b 5.

Peach and apricot can cause allergic reactions with symptoms ranging from mild to very severe, including anaphylaxis. Sometimes subjects allergic to fruits of the Prunus genus have been reported to be also allergic to rubber latex products. The objective of this study is the characterization of a newly identified peach and apricot protein showing similarities with the allergens Hev b 5 from rubber latex and Man e 5 from manioc. This protein has been named ENEA on the basis of the single letter amino acid code of the first four N-terminal residues of the isolated molecule. It has been found in very variable amounts in different peach cultivars and batches. ENEA was isolated from peach pulp extracts by chromatographic separations and identified by direct protein sequencing. At that time, the full length sequence was available only for the homologous protein of the taxonomically closely related apricot, which was produced as a recombinant molecule in Escherichia coli. The following availability of the full length sequence of peach ENEA revealed a very high identity (97%) with the apricot homolog. Similarly to Hev b 5 and to Man e 5, the structural characterization indicated that ENEA is an intrinsically disordered protein. The immunological properties, investigated by dot blotting, the ABA system and the FABER test, showed that ENEA is recognized by specific IgE of allergic patients. In a selected population of 31 patients reporting allergic reactions to peach fruit and/or IgE positive to Hev b 5, 28 and 27 subjects resulted co-sensitized to rENEA and Hev b 5 in the ABA and ISAC test, respectively. In a random population of 3305 suspected allergic patients, analyzed with the FABER test, 17 of them were sensitized to rENEA and 10 of them were also positive to Hev b 5. In addition, both the natural molecule from peach and the recombinant protein of apricot partially inhibited the IgE binding to Hev b 5. In conclusion, a new peach and apricot IgE-binding protein, cross-reacting with the major latex allergen Hev b 5, has been identified. Its variable concentration in the fruit might explain some occasionally occurring allergic reactions. The apricot molecule has recently been registered by the WHO/IUIS Allergen Nomenclature Sub-Committee with the allergen name Pru ar 5. The recombinant form of apricot ENEA, now available, will contribute to allergy diagnosis.

Allergens in allergy diagnosis: a glimpse at emerging new concepts and methodologies.

Allergic diseases are important concern of public health. A reliable diagnosis is of utmost importance for the management of allergic patients both when immunotherapy is planned and when the treatment is essentially based on the avoidance of the allergy source. However, the available diagnostic systems sometimes fail to detect specific IgE antibodies thus impairing the correct diagnosis. The traditional test systems are generally based on the use of protein extracts derived from the allergenic sources whose composition is very variable and cannot be standardized. The development of a new methodology combining the so-called allergenic molecule-based diagnosis with the multiplex microarray technology and allowing the analysis of multiple purified allergens in a single test represents an important improvement in allergy diagnosis. In addition, the biochemical and immunological characterisation of individual allergens has provided new insights into the understanding of allergen-IgE recognition that could be exploited for further improvements of allergy diagnostic tests.

Allergenic lipid transfer proteins from plant-derived foods do not immunologically and clinically behave homogeneously: the kiwifruit LTP as a model.

BACKGROUND: Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet. OBJECTIVE: Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins. METHODS: Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out. RESULTS: Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients' diet. CONCLUSION: The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention.

Nanostructured silver-based surfaces: new emergent methodologies for an easy detection of analytes.

In this work, we describe how to realize a new sensing platform for an easy and fast detection of analytes. In particular, we utilized enhanced fluorescence emission on silver island films (SIFs) coupled to the total internal reflection fluorescence mode (TIRF) to develop a new assay format for the detection of target analytes. Here, as an example, we report on the detection of the toxic peptides present in gliadin (Gli). Our assay was performed as follows: (1) gliadin was first captured on surfaces coated with anti-Gli antibodies; (2) the surfaces were then incubated with fluorophore-labeled anti-Gli antibodies; (3) the signal from the fluorophore-labeled anti-Gli antibody bound to the antigen was detected by TIRF. The system was examined on glass surfaces and on SIFs. We observed a relevant enhancement of the signal from SIFs compared to the signal from the glass substrate not modified with a SIF. In addition, the estimated detection limit (EDL) of our methodology was 60 ng/mL (or lower). This limit is therefore lower than the clinical cut-off for Gli presence in food for celiac patients. The advantage of our method is a reduced number of testing steps, which allows for easy detection of residual toxic peptides in food labeled as gluten free. The proposed technology can be easily expanded to the determination of different target analytes.

Molecular strategies for protein stabilization: the case of a trehalose/maltose-binding protein from Thermus thermophilus.

The trehalose/maltose-binding protein (MalE1) is one component of trehalose and maltose uptake system in the thermophilic organism Thermus thermophilus. MalE1 is a monomeric 48 kDa protein predominantly organized in alpha-helix conformation with a minor content of beta-structure. In this work, we used Fourier-infrared spectroscopy and in silico methodologies for investigating the structural stability properties of MalE1. The protein was studied in the absence and in the presence of maltose as well as in the absence and in the presence of SDS at different p(2)H values (neutral p(2)H and at p(2)H 9.8). In the absence of SDS, the results pointed out a high thermostability of the MalE1 alpha-helices, maintained also at basic p(2)H values. However, the obtained data also showed that at high temperatures the MalE1 beta-sheets underwent to structural rearrangements that were totally reversible when the temperature was lowered. At room temperature, the addition of SDS to the protein solution slightly modified the MalE1 secondary structure content by decreasing the protein thermostability. The infrared data, corroborated by molecular dynamics simulation experiments performed on the structure of MalE1, indicated that the protein hydrophobic interactions have an important role in the MalE1 high thermostability. Finally, the results obtained on MalE1 are also discussed in comparison with the data on similar thermostable proteins already studied in our laboratories.

Mutant bovine odorant-binding protein: Temperature affects the protein stability and dynamics as revealed by infrared spectroscopy and molecular dynamics simulations.

Bovine odorant-binding protein (bOBP), a member of the lipocalin family, presents the so-called 3D "domain-swapped" protein structure. In fact, in solution, it appears as a dimer in which each monomer is composed by the classical lipocalin fold, with a central beta-barrel followed by a stretch of residues and the alpha-helix domain protruding out of the barrel and crossing the dimer interface. Recently, a deswapped mutant form of bOBP was obtained, in which a Gly residue was inserted after position 121 and the two residues in position 64 and 156 were replaced by Cys residues for restoring the disulfide bridge common to the lipocalin family. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the effect of temperature on the structural stability and conformational dynamics of the mutant bOBP. The spectroscopic and molecular simulation data pointed out that the hydrophobic regions of the protein matrix appear to be an important factor for the protein stability and integrity. In addition, it was also found that the mutant bOBP is significantly stabilized by the binding of the ligand, which may have an impact on the biological function of bOBP. The obtained results will allow for a better use of this protein as probe for the design of advanced protein-based biosensors for the detection of compounds used in the fabrication of explosive powders.

Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features.

Kissper is a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa). Its primary structure, elucidated by direct protein sequencing, is identical to the N-terminal region of kiwellin, a recently reported kiwi fruit allergenic protein, suggesting that kissper derives from the in vivo processing of kiwellin. The peptide does not show high sequence identity with any other polypeptide of known function. However, it displays a pattern of cysteines similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. A number of these proteins are also active on mammalian cells. Functional characterization of kissper showed pH-dependent and voltage-gated pore-forming activity, together with anion selectivity and channeling in model synthetic PLMs, made up of POPC and of DOPS:DOPE:POPC. A 2DNMR analysis indicates that in aqueous solution kissper has only short regions of regular secondary structure, without any evident similarity with other bioactive peptides. Comparative analysis of the structural and functional features suggests that kissper is a member of a new class of pore-forming peptides with potential effects on human health.

Wild-type and mutant bovine odorant-binding proteins to probe the role of the quaternary structure organization in the protein thermal stability.

The exploration of events taking place at different timescales and affecting the structural and dynamics properties of proteins, such as the interactions of proteins with ligands and the subunits association/ dissociation, must necessarily be performed by using different methodologies, each of which specialized to highlight the different phenomena that occur when proteins are exposed to chemical or physical stress. In this work, we investigated the structure and dynamics of the wild-type bovine odorant-binding protein (wt-bOBP), which is a domain-swapped dimeric protein, and the triple mutant deswapped monomeric form of the protein (m-bOBP) to shed light on the role of the quaternary and tertiary structural organization in the protein thermal stability. Difference infrared spectra, 2D-IR correlation spectroscopy and molecular dynamics simulations were used to probe the effect of heating on protein structure and dynamics in microsecond and nanoseconds temporal ranges, respectively. The obtained results show that there is a heating-induced transition toward a less structured state in m-bOBP, that it is detectable around 70-80 degrees C. On the contrary, in wt-bOBP this transition is almost negligible, and changes are detectable in the protein spectra in the range of temperature between 75 and 85 degrees C. A detailed 3D inspection of the structure of the two proteins that takes into the account the spectroscopic results indicates that (a) ion pairs and hydrophobic interactions appear to be the major determinants responsible for the protein stability and (b) the protein intersubunit interactions confer an increased resistance toward the thermal stress.

A strategic fluorescence labeling of D-galactose/D-glucose-binding protein from Escherichia coli helps to shed light on the protein structural stability and dynamics.

The D-glucose/D-galactose-binding protein (GGBP) of Escherichia coli serves as an initial component for both chemotaxis toward D-galactose and D-glucose and high-affinity active transport of the two sugars. GGBP is a monomer with a molecular weight of about 32 kDa that binds glucose with micromolar affinity. The sugar-binding site is located in the cleft between the two lobes of the bilobate protein. In this work, the local and global structural features of GGBP were investigated by a strategic fluorescence labeling procedure and spectroscopic methodologies. A mutant form of GGBP containing the amino acid substitution Met to Cys at position 182 was realized and fluorescently labeled to probe the effect of glucose binding on the local and overall structural organization of the protein. The labeling of the N-terminus with a fluorescence probe as well as the protein intrinsic fluorescence were also used to obtain a complete picture of the GGBP structure and dynamics. Our results showed that the binding of glucose to GGBP resulted in no stabilizing effect on the N-terminus portion of GGBP and in a moderate stabilization of the protein matrix in the vicinity of the ligand-binding site. On the contrary, it was observed that the binding of glucose has a strong stabilization effect on the C-terminal domain of the GGBP structure.