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 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 6xHis-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni2+ 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 Ca2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca2+ 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.


CONCLUSIONS
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.
Tinghino
R
R
Department of Immunology, Istituto Superiore di Sanità, Rome, Italy.
Barletta
B
B
Palumbo
S
S
Afferni
C
C
Iacovacci
P
P
Mari
A
A
Di Felice
G
G
Pini
C
C
D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D003429:Cross Reactions; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D015723:Gene Library; D006801:Humans; D018653:Juniperus; D008969:Molecular Sequence Data; D011058:Pollen; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment
D000485:Allergens; D052179:Antigens, Plant; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; C113004:Jun o 2 allergen; D011994:Recombinant Proteins