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 allergen 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 allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic 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 allergic 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 Lewisa epitope on one arm) in the molar ratio 67:8:23.


CONCLUSIONS
The rapid purification process of Cup a 1 allowed 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.
Alisi
C
C
Department of Immunology, Istituto Superiore di Sanità, Rome, Italy.
Afferni
C
C
Iacovacci
P
P
Barletta
B
B
Tinghino
R
R
Butteroni
C
C
Puggioni
E M
EM
Wilson
I B
IB
Federico
R
R
Schininà
M E
ME
Ariano
R
R
Di Felice
G
G
Pini
C
C
D000485:Allergens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D052179:Antigens, Plant; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007525:Isoelectric Focusing; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011058:Pollen / Q000276:immunology; D011134:Polysaccharides / Q000032:analysis; D020539:Sequence Analysis, Protein; D014197:Trees
D000485:Allergens; D052179:Antigens, Plant; C419643:Cup a 1 protein, Cupressus arizonica; D010940:Plant Proteins; D011134:Polysaccharides; D007073:Immunoglobulin E