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Molecular and Immunologic Characterization of a Highly Cross-Reactive Two EF-Hand Calcium-Binding Alder Pollen Allergen, Aln g 4: Structural Basis for Calcium-Modulated IgE Recognition¹

Brigitte Hayek^*, Luca Vangelista^§, Annalisa Pastore^¶, Wolfgang R. Sperr, Peter Valent, Susanne Vrtala^*, Verena Niederberger, Anna Twardosz^*, Dietrich Kraft^* and Rudolf Valenta^2,*

Departments of ^* General and Experimental Pathology, and Otorhinolaryngology, Division of Hematology and Hemostaseology, Department of Internal Medicine I, AKH, University of Vienna, Vienna, Austria; ^§ European Molecular Biology Laboratory, Heidelberg, Germany; and ^¶ Molecular Structure Division, National Institute for Medical Research, London, United Kingdom

Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coliand purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1–41 and 42–85 to patients’ IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.

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