Aplicaciones de la proteómica en la alergia: identificación y caracterización de alérgenos de relevancia clínica en la cuenca meditarránea

La alergia respiratoria, que afecta a un 25‐30% de la población, es una de las principales causas de hipersensibilidad tipo I. Aunque la etiología de la alergia es compleja, la influencia tanto de factores genéticos como ambientales parece ser crucial en el desarrollo de esta enfermedad. La respuesta alérgica, que cursa con un predominio de células T colaboradoras tipo 2 (Th2) y la producción de anticuerpos IgE, se produce como consecuencia de una reacción inflamatoria frente a antígenos de diferentes fuentes biológicas. Se pueden distinguir dos fases de la enfermedad, una fase de sensibilización en la que se producen células de memoria, y otra efectora, en la que, tras una reexposición al alérgeno, tiene lugar la liberación de mediadores de la alergia por parte de células específicas, como basófilos y mastocitos, que provocan la aparición de los síntomas. Existen una gran variedad de pólenes implicados en el desarrollo de la respuesta alérgica, constituyendo el polen de gramíneas la principal causa de alergia en la cuenca mediterránea. La expansión de la desertificación en todo el mundo ha provocado un aumento de la presencia de plantas adaptadas a condiciones extremas de salinidad y sequía que ha provocado que la sensibilización a su polen haya aumentado. Entre ellas, Salsola kali actúa como un importante agente inductor de la alergia en la Península Ibérica debido a su papel como especie invasora de ciertos cultivos, incluyendo el olivo. Por otro lado, el polen del olivo, segunda causa de polinosis en la cuenca mediterránea, es el principal inductor de alergia en Andalucía debido a su intenso cultivo..

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.We thank the excellent technical support of Sara Abián Saz. This work was supported by grants cofounded by Fondo Europeo de Desarrollo Regional (FEDER): SAF2014-53209-R to M.V. and R.B. and SAF2017-86483-R to M.V. from the Ministerio de Economía y Competitividad and by the Thematic Networks and Co-operative Research Centres: RIRAAF Network RD12/0013/0015; and ARADyAL (RD16/0006/0014) from the Instituto de Salud Carlos III (ISCIII). A.N. and A.J. acknowledge PI-01119-2016 from the Consejería de Salud (Junta de Andalucía) and the Alergosur Foundation. R.B. also acknowledges the financial support of the PI17CIII/00045 grant from the AES-ISCIII program. The FPU predoctoral contract to P.S.S.-A. is supported by the Spanish Ministerio de Educación, Cultura y Deporte. C.O.-S. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.S

A relevant IgE-reactive 28 kDa protein identified from Salsola kali pollen extract by proteomics is a natural degradation product of an integral 47 kDa polygalaturonase

[EN] A highly prevalent IgE-binding protein band of 28 kDa 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 39,554.2 Da. 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 28 kDa band together with an allergenic band of about 47 kDa in the pollen extract. Therefore, the 28 kDa was assigned as a natural degradation product of the 47 kDa 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 47 kDa 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/m(3) 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.This work was supported by grants SAF2011-26716 and SAF2014-53209-R from the Ministerio de Economia y Competitividad and RIRAAF Network RD12/0013/0015 from the ISCIII. R.B. was a fellow of the Ramon y Cajal program of the Ministerio de Economia y Competitividad (Spain). C.O-S. is supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejeria de Education, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.Mas-García, S.; Oeo-Santos, C.; Cuesta-Herranz, J.; Díaz-Perales, A.; Colás, C.; Fernández, J.; Barber, D.... (2017). A relevant IgE-reactive 28 kDa protein identified from Salsola kali pollen extract by proteomics is a natural degradation product of an integral 47 kDa polygalaturonase. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1865(8):1067-1076. https://doi.org/10.1016/j.bbapap.2017.05.007S106710761865

New insights into the sensitization to nonspecific lipid transfer proteins from pollen and food: New role of allergen Ole e 7.

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. 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). 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. 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.S

Delineation of the olive pollen proteome and its allergenome unmasks cyclophilin as a relevant cross-reactive allergen

37 p.-5 fig.-1 tab.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.This work was supported by grants cofounded by Fondo Europeo de Desarrollo Regional (FEDER): SAF2014-53209-R to M.V. and R.B. and SAF2017-86483-R to M.V. from theMinisterio de Economı́a y Competitividad and by the Thematic Networks and Co-operative Research Centres:RIRAAF Network RD12/0013/0015; and ARADyAL(RD16/0006/0013 and RD16/0006/0014) from the Instituto de Salud Carlos III (ISCIII). B.R.-L., A.J., A.N, and C.M.acknowledge PI-01119-2016 from the Consejerı́a de Salud(Junta de Andalucı́a) and the Alergosur Foundation. R.B. also acknowledges the financial support of the PI17CIII/00045grant from the AES-ISCIII program.Peer reviewe

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

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