MOLECULAR CHARACTERIZATION OF A CROSS-REACTIVE JUNIPERUS OXYCEDRUS POLLEN ALLERGEN, JUN O 2 - A NOVEL CALCIUM-BINDING ALLERGEN

Background: Species belonging to the Cupressaceae family are a relevan t source of allergens that are present in a wide number of countries. Objective:We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus, a species belonging to the Cupress aceae family. Methods: Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector. IgE screening of the Library was performed with a pool of sera from subjects allergic to C upressaceae. A recombinant 6 x His-tagged Juniperus oxycedrus allergen , Jun o 2, was expressed in Escherichia coli and purified by Ni2+ affi nity chromatography. It was studied further by immunoblotting inhibiti on with pollen extracts from other Cupressaceae, Oleaceae, Urticaceae, and Graminaceae. The role of protein-bound calcium on the allergen's IgE-binding capacity was tested in a plaque assay in the presence or a bsence of EGTA. Results: A cDNA coding for a newly identified Juniperu s oxycedrus pollen allergen, dun o 2, was isolated. The deduced amino acid sequence contained four typical Ca2+ binding sites and showed a s ignificant sequence similarity to calmodulins. Depletion of Ca2+ in th e plaque assay led to a loss of IgE-binding capacity of dun o 2. Immun oblotting inhibition revealed that J. oxycedrus, J. ashei, Cupressus a rizonica, C. sempervirens, Parietaria judaica, Olea europaea, and Loli um perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations. Conclusion: rJun o 2 contains I gE-binding epitopes shared by taxonomically unrelated species, and the refore it can be regarded as a new panallergen. These findings could c ontribute to an explanation for the phenomenon of multiple positive te st results in polysensitized patients and the potential symptom-elicit ing role of allergenic sources previously not encountered.