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Rapid isolation, characterization, and glycan analysis of Cup a 1, the major all_ergen of Arizona cypress ( Cupressus arizonica ) pollen
Author(s) -
Alisi C.,
Afferni C.,
Iacovacci P.,
Barletta B.,
Tinghino R.,
Butteroni C.,
Puggioni E. M. R.,
Wilson I. B. H.,
Federico R.,
Schininà M. E.,
Ariano R.,
Di Felice G.,
Pini C.
Publication year - 2001
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1034/j.1398-9995.2001.103125.x
Subject(s) - cypress , isoelectric point , oligosaccharide , chromatography , isoelectric focusing , chemistry , biochemistry , allergen , pollen , biology , botany , enzyme , allergy , immunology
Background: A rapid method for the purification of the major 43‐kDa all_ergen of Cupressus arizonica pollen, Cup a 1, was developed. Methods: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino‐acid sequence, and its glycan composition. Results: Immunochemical analysis of Cup a 1 confirmed that the all_ergenic reactivity is maintained after the purification process. Partial amino‐acid sequencing indicated a high degree of homology between Cup a 1 and all_ergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients all_ergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI‐TOF mass spectrometry indicated the presence of three N‐linked oligosaccharide structures: GnGnXF 3 (i.e., a horseradish peroxidase‐type oligosaccharide substituted with two nonreducing N ‐acetylglucosamine residues), GGnXF 3 /GnGXF 3 (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF 3 /Gn(GF)XF 3 (with a Lewis a epitope on one arm) in the molar ratio 67:8:23. Conclusions: The rapid purification process of Cup a 1 all_owed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino‐acid sequences and some complex glycan stuctures could explain the high degree of cross‐reactivity among the Cupressaceae and Taxodiaceae families.