Supplementary Material for: 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.