Examinando por Autor "San Segundo-Acosta, Pablo"

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Biophysical and biological impact on the structure and IgE-binding of the interaction of the olive pollen allergen Ole e 7 with lipids
(ELSEVIER, 2020) Oeo-Santos, Carmen; López-Rodríguez, Juan Carlos; García-Mouton, Cristina; San Segundo-Acosta, Pablo; Jurado, Aurora; Moreno-Aguilar, Carmen; García-Álvarez, Begoña; Pérez-Gil, Jesús; Villalba, Mayte; Barderas, Rodrigo; Cruz, Antonio
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 behavior of the allergen once inhaled at the airways.
Delineation of the Olive Pollen Proteome and Its Allergenome Unmasks Cyclophilin as a Relevant Cross-Reactive Allergen
(ACS Publications, 2019) San Segundo-Acosta, Pablo; Oeo-Santos, Carmen; Benedé, Sara; de Los Ríos, Vivian; Navas, Ana; Ruíz-León, Berta; Moreno-Aguilar, Carmen; Pastor-Vargas, Carlos; Jurado, Aurora; Villalba, Mayte; Barderas, Rodrigo
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.
Epitope Mapping of Allergenic Lipid Transfer Proteins. Methods in Molecular Biology
(Springer Nature. Humana Press, 2021-06) San Bartolomé, Clara; Oeo-Santos, Carmen; San Segundo-Acosta, Pablo; Muñoz-Cano, Rosa; Martínez-Botas, Javier; Bartra, Joan; Pascal, Mariona
En este capítulo de libro se describe el protocolo para producir microarrays de proteínas utilizando una biblioteca de péptidos solapantes correspondientes a las secuencias primarias de las proteínas alergénicas de transferencia lipídica (LTPs). Este nuevo enfoque aporta una nueva herramienta para obtener información en detalle sobre la respuesta IgE a los alérgenos. El mapeo de epítopos tiene el potencial de convertirse en una herramienta fundamental para el diagnóstico y pronóstico de la alergia alimentaria y de conducir a una mejor comprensión de la patogénesis. Este método supone un nuevo enfoque en el mapeo de epítopos de alérgenos alimentarios vegetales que tienen gran relevancia clínica en el área mediterránea dadas las potenciales reacciones de reactividad cruzada entre ellos, que dificultan el diagnostico y tratamiento de los pacientes.
High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components
(ELSEVIER, 2018) San Segundo-Acosta, Pablo; Garranzo-Asensio, María; Oeo-Santos, Carmen; Montero-Calle, Ana; Quiralte, Joaquín; Cuesta-Herranz, Javier; Villalba, Mayte; Barderas, Rodrigo
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.
Ole e 15 and its human counterpart -PPIA- chimeras reveal an heterogeneous IgE response in olive pollen allergic patients
(Nature, 2019) San Segundo-Acosta, Pablo; Oeo-Santos, Carmen; Navas, Ana; Jurado, Aurora; Villalba, Mayte; Barderas, Rodrigo
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.