How α-Helical Motifs Form Functionally Diverse Lipid-Binding Compartments.

Lipids are produced site-specifically in cells and then distributed nonrandomly among membranes via vesicular and nonvesicular trafficking mechanisms. The latter involves soluble amphitropic proteins extracting specific lipids from source membranes to function as molecular solubilizers that envelope their insoluble cargo before transporting it to destination sites. Lipid-binding and lipid transfer structural motifs range from multi-ß-strand barrels, to ß-sheet cups and baskets covered by α-helical lids, to multi-α-helical bundles and layers. Here, we focus on how α-helical proteins use amphipathic helical layering and bundling to form modular lipid-binding compartments and discuss the functional consequences. Preformed compartments generally rely on intramolecular disulfide bridging to maintain conformation (e.g., albumins, nonspecific lipid transfer proteins, saposins, nematode polyprotein allergens/antigens). Insights into nonpreformed hydrophobic compartments that expand and adapt to accommodate a lipid occupant are few and provided mostly by the three-layer, α-helical ligand-binding domain of nuclear receptors. The simple but elegant and nearly ubiquitous two-layer, α-helical glycolipid transfer protein (GLTP)-fold now further advances understanding.

Basophil Activation Test Utility as a Diagnostic Tool in LTP Allergy.

Plant-food allergy is an increasing problem, with nonspecific lipid transfer proteins (nsLTPs) triggering mild/severe reactions. Pru p 3 is the major sensitizer in LTP food allergy (FA). However, in vivo and in vitro diagnosis is hampered by the need for differentiating between asymptomatic sensitization and allergy with clinical relevance. The basophil activation test (BAT) is an ex vivo method able to identify specific IgE related to the allergic response. Thus, we aimed to establish the value of BAT in a precise diagnosis of LTP-allergic patients. Ninety-two individuals with peach allergy sensitized to LTP, Pru p 3, were finally included, and 40.2% of them had symptoms to peanut (n = 37). In addition, 16 healthy subjects were recruited. BAT was performed with Pru p 3 and Ara h 9 (peanut LTP) at seven ten-fold concentrations, and was evaluated by flow cytometry, measuring the percentage of CD63 (%CD63+) and CD203c (%CD203chigh) cells, basophil allergen threshold sensitivity (CD-Sens), and area under the dose−response curve (AUC). Significant changes in BAT parameters (%CD63+ and %CD203chigh) were found between the controls and patients. However, comparisons for %CD63+, %CD203chigh, AUC, and CD-Sens showed similar levels among patients with different symptoms. An optimal cut-off was established from ROC curves, showing a significant positive percentage of BAT in patients compared to controls and great values of sensitivity (>87.5%) and specificity (>85%). In addition, BAT showed differences in LTP-allergic patients tolerant to peanut using its corresponding LTP, Ara h 9. BAT can be used as a potential diagnostic tool for identifying LTP allergy and for differentiating peanut tolerance, although neither reactivity nor sensitivity can distinguish the severity of the clinical symptoms.

Lysine crotonylation of DgTIL1 at K72 modulates cold tolerance by enhancing DgnsLTP stability in chrysanthemum.

Lysine crotonylation of proteins is a recently identified post-translational modification (PTM) in plants. However, the function of lysine-crotonylated proteins in response to abiotic stress in plants has not been reported. In this study, we identified a temperature-induced lipocalin-1-like gene (DgTIL1) from chrysanthemum and showed that it was notably induced in response to cold stress. Overexpression of DgTIL1 enhanced cold tolerance in transgenic chrysanthemum. Ubiquitin membrane yeast two-hybrid (MYTH) system and bimolecular fluorescence complementation (BIFC) assays showed that DgTIL1 interacts with a nonspecific lipid transfer protein (DgnsLTP), which can promote peroxidase (POD) gene expression and POD activity to reduce the accumulation of reactive oxygen species (ROS) and improve resistance to cold stress in DgnsLTP transgenic chrysanthemum. In addition, we found that DgTIL1 was lysine crotonylated at K72 in response to low temperature in chrysanthemum. Moreover, lysine crotonylation of DgTIL1 prevented DgnsLTP protein degradation in tobacco and chrysanthemum. Inhibition of DgnsLTP degradation by lysine crotonylation of DgTIL1 further enhanced POD expression and POD activity, reduced the accumulation of ROS under cold stress in DgTIL1 transgenic chrysanthemum, thus promoting the cold resistance of chrysanthemum.

Crystal structure of nonspecific lipid transfer protein from Solanum melongena.

Lipids are considered to protect protein allergens from proteolysis and are generally seen to exist in a bound form. One of the well-known plant protein families with bound lipids is non-specific lipid transfer proteins (nsLTPs). Structure-function relationships in the case of the members of non-specific lipid transfer protein family are not clearly understood. As part of exploring the seed proteome, we have analyzed the proteome of a member of Solanaceae family, Solanum melongena (eggplant) and a non-specific lipid transfer protein from S. melongena, SM80.2 was purified, crystallized and the structure was determined at 1.87 Å resolution. Overall, the tertiary structure is a cluster of α-helices forming an internal hydrophobic cavity. Absence of conserved Tyr79, known to govern the plasticity of hydrophobic cavity, and formation of hydrogen bond between Asn79 and Asn36 further reduced the pocket size. Structural analysis of SM80.2 thus gives insight about a new hydrogen bond mediated mechanism followed in closure of the binding pocket. Extra electron densities observed at two different places on the protein surface and not in the cavity could provide interesting physiological relevance. In light of allergenic properties, probably overlapping of epitopic region and ligand binding on surface could be a main reason. This work shows first crystal structure of A-like nsLTP with a close binding pocket and extra density on the surface suggesting a plausible intermediate state during transfer.

Lipid transfer protein sensitization: reactivity profiles and clinical risk assessment in an Italian cohort.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) represent a major cause of systemic food allergic reactions in the Mediterranean area. This study investigate hierarchical patterns and cluster relationships of IgE sensitization to different nsLTPs, and the relationship to clinical allergy in a large Italian cohort. METHODS: A total of 568 nsLTP-positive subjects after IgE ImmunoCAP-ISAC microarray analysis with Ara h 9, Art v 3, Cor a 8, Jug r 3, Pla a 3, Pru p 3 and Tri a 14 allergens were studied. IgE inhibition experiments were carried out with mugwort and plane tree pollen extracts. RESULTS: Eighty-two per cent of nsLTP-positive participants (94% if <6 years old) were Pru p 3(pos) , and 71% were Jug r 3(pos) . Participants who reacted to >5 nsLTPs reported a higher incidence of food-induced systemic reactions. Only Art v 3 and Pla a 3 (mugwort and plane tree nsLTPs, respectively) were associated with respiratory symptoms, and a correlation was observed between sensitization to pollen and plant food nsLTPs, particularly between Pla a 3 and tree nut/peanut nsLTPs. Co-sensitization to Par j 2 and PR-10 or profilin pan-allergens was associated with a lower prior prevalence of severe food-induced reactions. In inhibition assays, plane and mugwort pollen extracts inhibited 50-100% of IgE binding to food nsLTPs in microarrays. CONCLUSIONS: Testing IgE reactivity to a panel of nsLTP allergens unveils important associations between nsLTP sensitization profiles and clinical presentation and allows the identification of novel cluster patterns indicating likely cross-reactivities and highlighting potential allergens for nsLTP immunotherapy.

Ara h 2 and Ara h 6 sensitization predicts peanut allergy in Mediterranean pediatric patients.

BACKGROUND: Peanut allergy (PA) management was improved by the introduction of molecular allergology, but guidelines for Mediterranean patients are lacking. We aimed at evaluating peanut component-resolved diagnosis as a diagnostic and prognostic tool in children from Southern France. METHODS: In 181 pediatric patients, PA diagnosis was founded on medical history, skin prick testing, serum-specific IgE to Arachis hypogea extract and components, Pru p 4, and plant carbohydrates, and oral food challenge. Allergen microarray was also performed in 68 of these patients. RESULTS: In peanut-allergic children (n = 117), IgE to Ara h 6 were most prevalent (64%), followed by Ara h 2 (63%), Ara h 1 (60%), and Ara h 9 (52%). Ara h 6 was the best predictor of PA. The second best predictor was the ratio of Ara h 2 IgE to peanut IgE (cutoff 0.113). Persistent childhood PA was associated with complex molecular profiles. Comparison of singleplex and microarray results showed poor concordance for Ara h 2 and Ara h 9. CONCLUSION: Ara h 6 and Ara h 2 are the best predictors of PA at diagnosis in Mediterranean pediatric patients. Ara h 1, Ara h 8, and molecular complexity are associated with PA persistence.