Epitope Mapping of Allergenic Lipid Transfer Proteins.

Food allergy is becoming a great problem in industrialized countries. Thus, there is the need for a robust understanding of all aspects characterizing IgE response to allergens. The epitope mapping of B-cell epitopes has the potential to become a fundamental tool for food allergy diagnosis and prognosis and to lead to a better understanding of the pathogenesis. Using this approach, we have worked on epitope mapping of the most important plant food allergens identified in the Mediterranean area. The final aim of this study is to define the immune response regarding B epitopes and its clinical relevance in LTP allergy. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of allergenic lipid transfer proteins.

The key to the allergenicity of lipid transfer protein (LTP) ligands: A structural characterization.

Plant lipid transfer proteins are a large family that can be found in all land plants. They have a hydrophobic cavity that allows them to harbor lipids and facilitates their traffic between membranes. However, in humans, this plant protein family is responsible for the main food allergies in the Mediterranean area. Nevertheless, not only the protein itself but also its ligand is relevant for allergic sensitization. The main aim of the present work is to analyse the natural ligands carried by four allergenic LTPs (Tri a 14, Art v 3, Par j 2, and Ole e 7), compared with the previously identified ligand of Pru p 3 (CPT-PHS ligand), and clarify their role within the immunological reactions. Results showed that the ligands of the LTPs studied shared a chemical identity, in which the presence of a polar head was essential to the protein-ligand binding. This ligand was transported through a skin cellular model, and phosphorylated phytosphingosine could be detected as result of cell metabolism. Since sphingosine kinase 1 was overexpressed in keratinocytes incubated with the LTP-ligand complex, this enzyme might be responsible for the phosphorylation of the phytosphingosine fraction of the CPT-PHS ligand. This way, phytosphingosine-1-phosphate could be mimicking the role of the human inflammatory mediator sphingosine-1-phosphate, explaining why LTPs are associated with more severe allergic responses. In conclusion, this work contributes to the understanding of the chemical nature and behavior of lipid ligands carried by allergens, which would help to gain insight into their role during allergic sensitization.

Biophysical and biological impact on the structure and IgE-binding of the interaction of the olive pollen allergen Ole e 7 with lipids.

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways.

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.

Ole e 15 and its human counterpart -PPIA- chimeras reveal an heterogeneous IgE response in olive pollen allergic patients.

Olive pollen is a major cause of immunoglobulin E (IgE)-mediated allergy in Mediterranean countries. It is expected to become a worldwide leading allergenic source because olive cultivation is increasing in many countries. Ole e 15 belongs to the cyclophilin pan-allergen family, which includes highly cross-reactive allergens from non-related plant, animal and mold species. Here, the amino acid differences between Ole e 15 and its weak cross-reactive human homolog PPIA were grafted onto Ole e 15 to assess the contribution of specific surface areas to the IgE-binding. Eight Ole e 15-PPIA chimeras were produced in E. coli, purified and tested with 20 sera from Ole e 15-sensitized patients with olive pollen allergy by ELISA experiments. The contribution of linear epitopes was analyzed using twelve overlapping peptides spanning the entire Ole e 15 sequence. All the patients displayed a diverse reduction of the IgE-reactivity to the chimeras, revealing a highly polyclonal and patient-specific response to Ole e 15. IgE-epitopes are distributed across the entire Ole e 15 surface. Two main surface areas containing relevant conformational epitopes have been characterized. This is the first study to identify important IgE-binding regions on the surface of an allergenic cyclophilin.

Delineation of the Olive Pollen Proteome and Its Allergenome Unmasks Cyclophilin as a Relevant Cross-Reactive Allergen.

Olive pollen is a major allergenic source worldwide due to its extensive cultivation. We have combined available genomics data with a comprehensive proteomics approach to get the annotated olive tree (Olea europaea L.) pollen proteome and define its complex allergenome. A total of 1907 proteins were identified by LC-MS/MS using predicted protein sequences from its genome. Most proteins (60%) were predicted to possess catalytic activity and be involved in metabolic processes. In total, 203 proteins belonging to 47 allergen families were found in olive pollen. A peptidyl-prolyl cis-trans isomerase, cyclophilin, produced in Escherichia coli, was found as a new olive pollen allergen (Ole e 15). Most Ole e 15-sensitized patients were children (63%) and showed strong IgE recognition to the allergen. Ole e 15 shared high sequence identity with other plant, animal, and fungal cyclophilins and presented high IgE cross-reactivity with pollen, plant food, and animal extracts.

A Hypoallergenic Polygalacturonase Isoform from Olive Pollen Is Implicated in Pollen-Pollen Cross-Reactivity.

BACKGROUND: Cross-reactivity reactions between allergenic polygalacturonases (PGs) from different biological sources, especially foods and pollens from the Oleaceae family, have been described using Salsola kali PG (Sal k 6). No PG from olive pollen has been characterized to date, hampering further knowledge about cross-reactions through PGs. OBJECTIVES: The aim of this work was to determine the potential allergenicity of the PG from olive pollen and clarify its role in cross-reactivity. METHODS: A cDNA-encoding olive pollen PG sequence was subcloned into the pET41b vector and used to transform BL21(DE3) Escherichia coli cells to produce a His-tag fusion recombinant protein. The allergenic properties of olive pollen PG were determined by immunoblotting and ELISA in comparison to Sal k 6. The cross-reactivity potential of the protein with other pollen sources was analyzed by inhibition immunoassays. RESULTS: The existence of other isoforms of Ole e 14 with different allergenicity was confirmed by proteomics and a meta-analysis of the recently reported olive genome. Sal k 6 showed a higher IgE recognition than Ole e 14 regardless of patient sensitization, suggesting the existence of more allergenic Ole e 14 isoforms in olive pollen. IgG and IgE inhibition assays supported the existence of cross-reactions between them and with other PGs from Oleaceae and Poaceae plant families. CONCLUSIONS: A new allergen from olive pollen, Ole e 14, has been identified, produced as a recombinant isoform, and structurally and immunologically characterized. Its role in cross-reactivity has been confirmed and, due to its smaller IgE binding capacity, it could have an important role for therapeutic purposes.

A recombinant isoform of the Ole e 7 olive pollen allergen assembled by de novo mass spectrometry retains the allergenic ability of the natural allergen.

The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software. Thirteen out of the 457 de novo sequenced peptides obtained allowed assembling its full-length amino acid sequence. Then, Ole e 7-encoding cDNA was synthesized and cloned in pPICZαA vector for its expression in Pichia pastoris yeast. The analyses by Circular Dichroism, and WB, ELISA and cell-based tests using sera and blood from olive pollen-sensitized patients showed that rOle e 7 mostly retained the structural, allergenic and antigenic properties of the natural allergen. In summary, rOle e 7 allergen assembled by de novo peptide sequencing by MS behaved immunologically similar to the natural allergen scarcely isolated from pollen. SIGNIFICANCE: Olive pollen is an important cause of allergy. The non-specific lipid binding protein Ole e 7 is a major allergen with a high incidence and a phenotype associated to severe clinical symptoms. Despite its relevance, its cloning and recombinant expression has been unable by classical techniques. Here, we have inferred the primary amino acid sequence of Ole e 7 by mass-spectrometry. We separated Ole e 7 isolated from pollen by 2DE. After in-gel digestion with trypsin and a direct analysis by nLC-MS/MS in an LTQ-Orbitrap Velos, we got the complete de novo sequenced peptides repertoire that allowed the assembling of the primary sequence of Ole e 7. After its protein expression, purification to homogeneity, and structural and immunological characterization using sera from olive pollen allergic patients and cell-based assays, we observed that the recombinant allergen retained the antigenic and allergenic properties of the natural allergen. Collectively, we show that the recombinant protein assembled by proteomics would be suitable for a better in vitro diagnosis of olive pollen allergic patients.

High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components.

Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning. The screening of the phage microarrays with individual sera from allergic patients enabled the identification of 10 and 9 IgE-reactive unique amino acid sequences from olive pollen and mustard seeds, respectively. Five immunoreactive amino acid sequences displayed on phages were selected for their expression as His6-GST tag fusion proteins and validation. After immunological characterization, we assessed the IgE-reactivity of the constructs. Our results show that protein microarrays printed with T7 phages displaying peptides from allergenic sources might be used to identify allergenic components -peptides, proteins or mimotopes- through their screening with specific IgE antibodies from allergic patients.

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.