1 Scientific RepoRts | 6:32552 | DOI: 10.1038/srep32552 www.nature.com/scientificreports structural insights into the Ige mediated responses induced by the allergens Hev b 8 and Zea m 12 in their dimeric forms Israel Mares-Mejía 1 , siseth Martínez-Caballero 1 , Claudia Garay-Canales 2 , patricia Cano- sánchez 1 , Alfredo torres-Larios 3 , samuel Lara-González 4 , enrique ortega 2 & Adela Rodríguez-Romero 1 Oligomerization of allergens plays an important role in IgE-mediated reactions, as efective crosslinking of Ige- FcεRI complexes on the cell membrane is dependent on the number of exposed B-cell epitopes in a single allergen molecule or on the occurrence of identical epitopes in a symmetrical arrangement. Few studies have attempted to experimentally demonstrate the connection between allergen dimerization and the ability to trigger allergic reactions. Here we studied plant allergenic proflins rHev b 8 (rubber tree) and rZea m 12 (maize) because they represent an important example of cross-reactivity in the latex-pollen-food syndrome. Both allergens in their monomeric and dimeric states were isolated and characterized by exclusion chromatography and mass spectrometry and were used in immunological in vitro experiments. Their crystal structures were solved, and for Hev b 8 a disulfde-linked homodimer was found. Comparing the structures we established that the longest loop is relevant for recognition by Ige antibodies, whereas the conserved regions are important for cross-reactivity. We produced a novel monoclonal murine IgE (mAb 2F5), specifc for rHev b 8, which was useful to provide evidence that proflin dimerization considerably increases the IgE-mediated degranulation in rat basophilic leukemia cells. It has been recognized that the study of protein oligomerization is relevant from several perspectives, since this phe- nomenon can regulate the function of the protein or can create higher-order structures 1 . An important implication of protein oligomerization has been acknowledged in type I hypersensitivity reactions, where the efect of allergen dimer- ization and their multivalent characteristics promotes its recognition by specifc IgE antibodies bound to high afnity FcεRI receptors on the surface of mast cells and basophils 2 . Tis interaction triggers the cross-linking of FcεRI on the efector cell membranes, and a concomitant activation of biochemical pathways leads to degranulation and the release of various mediators such as histamine and lipids, which cause infammatory reactions 3 . Oligomerization phenomena and its consequences have been explained for some allergens such as Ara h 1(cupin; vicillin-type protein) 4 , Ara h 3 (cupin; legumin-type protein) 5 , Bos d 5 (β-lactoglobulin) 6 , Bet v 1 (pathogenesis-related protein) 7,8 , Alt a 1 (β-barrel protein) 9 , Bla g 2 (aspartic proteinase from the cockroach) 10 , and Per a 3 (hemocyanin from the American cockroach) 11 . Proflin is an ubiquitous cytoskeletal protein that interacts with several molecules, such as actin, proline-rich ligands, and phosphoinositides to perform several cellular functions. For the human proflin isoforms PFN1 and PFN2, oligomerization has been implicated in their regulation and binding to G-actin, where PFN1 acts as a tetramer 12 . It has also been reported that dimers of these human isoforms bind peptides derived from the vasodilator-stimulated phosphoprotein, thus afecting the regulation of G-actin polymerization 13 . Tese reports provide evidence that the oligomerization of proflin confers diferent properties by afecting its binding to 1 instituto de Química, Universidad nacional Autónoma de México, circuito exterior, cd. Universitaria, coyoacán, Ciudad de México, 04510. 2 instituto de investigaciones Biomédicas, Universidad nacional Autónoma de México, Circuito Escolar, Cd. Universitaria, Coyoacán, Ciudad de México, 04510. 3 instituto de fisiología celular, Universidad Nacional Autónoma de México, Circuito Exterior, Cd. Universitaria, Coyoacán, Ciudad de México, 04510. 4 instituto Potosino de Investigación Científca y Tecnológica, Camino a la Presa San José 2055, Col. Lomas 4a. Sección, San Luis Potosí, México, 78216. Correspondence and requests for materials should be addressed to A.R.-R. (email: adela@ unam.mx) Received: 23 March 2016 Accepted: 09 August 2016 Published: 02 September 2016 opeN