Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: A novel calcium-binding allergen

• Published in: Journal of allergy and clinical immunology 1998-06, Vol.101 (6), p.772-777
• Main Authors: Tinghino, Raffaella, Barletta, Bianca, Palumbo, Sabrina, Afferni, Claudia, Iacovacci, Patrizia, Mari, Adriano, Di Felice, Gabriella, Pini, Carlo
• Format: Article
• Language: English
• Subjects: Allergens - genetics; Allergens - immunology; Allergic diseases; Amino Acid Sequence; Antigens, Plant; Biological and medical sciences; calcium-binding protein; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - immunology; cDNA cloning; Cloning, Molecular; Cross Reactions; cross-reactivity; Cupressaceae; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; Escherichia coli; Gene Library; Humans; IgE; Immunopathology; Juniperus; Juniperus oxycedrus; Medical sciences; Molecular Sequence Data; Pollen; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Respiratory and ent allergic diseases; Sequence Alignment
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/S0091-6749(98)70306-9

Background: Species belonging to the Cupressaceae family are a relevant 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 Cupressaceae 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 Cupressaceae. A recombinant 6×His-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni 2+ affinity chromatography. It was studied further by immunoblotting inhibition 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 absence of EGTA. Results: A cDNA coding for a newly identified Juniperus oxycedrus pollen allergen, rJun o 2, was isolated. The deduced amino acid sequence contained four typical Ca 2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca 2+ in the plaque assay led to a loss of IgE-binding capacity of rJun o 2. Immunoblotting inhibition revealed that J. oxycedrus, J. ashei, Cupressus arizonica, C. sempervirens , Parietaria judaica, Olea europaea, and Lolium perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations. Conclusion: rJun o 2 contains IgE-binding epitopes shared by taxonomically unrelated species, and therefore it can be regarded as a new panallergen. These findings could contribute to an explanation for the phenomenon of multiple positive test results in polysensitized patients and the potential symptom-eliciting role of allergenic sources previously not encountered. (J Allergy Clin Immunol 1998;101:772-7)