Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen

Background: A rapid method for the purification of the major 43-kDa allerge n, of Cupressus arizonica pollen, Cup a 1, was developed. Methods: The salient feature was a wash of the pollen in acidic buffer, fol lowed, by an extraction of the proteins and their purification by chromatog raphy. Immunoblotting, ELISA, and lectin binding were tested on both the cr ude extract and the purified Cup a 1. Biochemical analyses were performed t o assess the Cup a 1 isoelectric point, its partial amino-acid sequence, an d its glycan composition. Results: Immunochemical analysis of Cup a I confirmed that the allergenic r eactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergen ic proteins from the Cupressaceae and Taxodiaceae families displaying a sim ilar molecular mass. The purified protein shows one band with an isoelectri c point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cyp ress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mas s spectrometry indicated the presence of three N-linked oligosaccharide str uctures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide sub stituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF (3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF-/Gn (GF)XF- (with a Lewis(a) epitope on one arm) in the molar ratio 67:8:23. Conclusions: The rapid purification process of Cup a 1 allowed some fine st udies on its properties and structure, as well as the evaluation of its I-E reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-re activity among the Cupressaceae and Taxodiaceae families.