D2.395 - Characterization of Juniperus oxycedrus pollen extract and identification of putative allergens

The pollen of Cupressaceae species contains the allergenic proteins responsible for cypress allergies. High loads of pollen are released during the pollinosis season, and it can potentially travel long distances, increasing the risk of sensitization and trigger allergic symptoms. Juniperus oxycedrus belongs to the cypress family, extensively present in the Mediterranean basin, but its role as risk factor for allergic sensitization has been scarcely studied. Two allergens from this species, Jun o 1 (pectate lyase) and Jun o 4 (polcalcin) have been reported in the official allergen database. However, the complete allergenic composition of J. oxycedrus pollen remains unknown.

Objective: To characterize the protein and allergenic profile of J. oxycedrus pollen extract and identify putative allergens.

Allergenic extracts were prepared from J. oxycedrus pollen by extraction in PBS. Purified native (n) Jun o 1 was obtained by chromatography. 108 allergic individuals residing in three localities of Spain (Albacete, Seville and Malaga) were skin prick tested (SPT), and sera was obtained on behalf of "Precision allergic rhinitis: Multicentric MOLecular mapping of the allergic EXPOsome" study by SEAIC Rhinology and Allergic Conjunctivitis Committee. Specific IgE determination was studied by ELISA, western blot and competition assays. For 2D-electrophoresis, pollen extract previously precipitated with ammonium sulphate was used. Total protein profile and IgE reactive spots in the 2D gels were detected by Coomassie stain and western blot. Characterization of the IgE reactive spots was performed by mass spectrometry (MS).

32 out of 108 allergic individuals (29.63%) had positive SPT to J. oxycedrus pollen extract, and the sera from 22 (68.75%) and 27 (84.38%) of them were positive to J. oxycedrus extract and nJun o 1, by ELISA. In competition assays, nJun o 1 accounted for the majority of IgE reactivity in J. oxycedrus extract (96.39% of IgE binding inhibition). Western blot of the 2D gel revealed 34 IgE-reactive spots. 14 were further characterized by MS: 8 spots corresponded to isoforms or variants of Jun o 1 (Uniprot: Q93X51). 6 additional IgE reactive spots are indicated in decreasing order of intensity of reactivity:  calreticulin (2 spots), identified as an allergen in Penicillium chrysogenum; enolase (2 spots), represents an allergen in several sources; aldose 1-epimerase (1 spot); aconitate hydratase (1 spot); unknown proteins (20 spots).

The allergenic profile of J. oxycedrus pollen is complex and goes beyond Jun o 1 and Jun o 4, including putative allergens such as calreticulin and enolase.