Rapid isolation, characterization, and glycan analysis of Cup a 1, the major all_ergen of Arizona cypress (Cupressus arizonica) pollen (original) (raw)
Related papers
2001
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 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 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.
Molecular cloning and characterization of Cup a 4, a new allergen from Cupressus arizonica
Biochemical and Biophysical Research Communications, 2010
Sensitization to Cupressaceae pollen has become one of the most important causes of pollinosis in Western countries during winter and early spring. However, the characterization of the extracts, the allergens involved and the cross-reactivity with other pollen sources still remain poorly studied; in the case of Cupressus arizonica only two allergens have been described so far. A new allergen from C. arizonica pollen, Cup a 4, was cloned and expressed in Escherichia coli as an N-terminally His-tag recombinant protein that was characterized biochemically, immunologically and by circular dichroism spectroscopy. The new allergen has high sequence identity with Prickly Juniper allergen Jun o 4 and contains four EF-hand domains. The recombinant protein has structural similarities with other calcium binding allergens such as Ole e 3, Ole e 8 and Phl p 7. Cup a 4 is expressed in mature pollen grains and shares antigenic properties with the recombinant form. Sera from 9.6% C. arizonica allergic patients contain specific IgE antibodies against recombinant Cup a 4.
Clinical <html_ent glyph="@amp;" ascii="&"/> Experimental Allergy, 1999
Background A reduction of IgE immunoreactivity after periodate-treatment has been previously reported for various glycoprotein allergens. Objective The aim of this study was to investigate the role of glycan moiety of a C. arizonica extract in the binding of patients' IgE and to identify the carbohydrates possibly involved. Methods The reactivity of IgE with C. arizonica extract, before and after periodatetreatment, was evaluated by immunoblotting and ELISA inhibition. The specificity of carbohydrate-reactive IgE was evaluated by ELISA using unrelated glycoproteins with known sugar composition and structure, such as pineapple bromelain, honeybee venom phospholipase A 2 , and ovalbumin, before and after periodate treatment. Results When periodate-treated C. arizonica extract was probed after SDS-PAGE and immunoblotting with patients' IgE, no reactivity could be detected. Furthermore, a very poor inhibitory activity of the periodate-treated C. arizonica extract as compared with the untreated sample could be observed in the ELISA inhibition experiments performed using C. arizonica extract as antigen. When phospholipase A 2 and bromelain were used as antigens in ELISA, they were recognized by patients' IgE, whereas ovalbumin was negative. Treatment of phospholipase A 2 and bromelain with periodate completely abolishes the IgE reactivity. Conclusion A large portion of the IgE reactivity of Cupressaceae-allergic subjects appears to be associated with sugar moieties of C. arizonica extract which appear to be shared by bromelain and phospholipase A 2 , thus suggesting that the IgE of patients reacting with such epitopes probably react with b 1 → 2 xylose, a 1 → 3 fucose and/or a 1 → 6 fucose.
Turkish Journal of Biochemistry, 2018
Objective Cupressaceae pollen has commonly been reported to be an important aeroallergen and causal factor of spring, autumn and winter pollinosis in many countries. The aim of this study was to compare of the structure and allergenic protein content of Cupressus arizonica Greene., Cupressus sempervirens L. and Juniperus oxycedrus L. pollen in detail and contribute to Cupressaceae pollen allergen diagnosis and therapy studies in Turkey. Methods The pollen structure were examined by LM and SEM. Pollen protein content was investigated by Bradford protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), respectively. Pectate lyase (PL) enzyme activities were compared. Immunoblotting was carried out by using extracts of the three taxa pollen collected from Turkey. Results All three taxa was found very similar in terms of pollen morphology however, intine thickness was pro...
Journal of Allergy and Clinical Immunology, 1994
Species of the Cupressaceae family are an important cause of respiratory allergies in countries with a Mediterranean climate. An allergenic extract from Cupressus arizonica pollen was prepared with two extraction steps followed by ammonium sulfate precipitation, giving a protein yieM of about 3%. Cupressus arizonica pollen extract was also characterized by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by IgE and IgG immunoblotting and lectin blotting. IgE reactivity was restricted to six componemts, whereas IgG binding showed a more complex pattern. A 43 kd component, predominant both in its intensity and frequency of recognition by human IgE antibodies, was identified as the major allergen of C. arizonica~ Four of the six IgE-binding components, including the major allergen, seem to be glycoproteins, as confirmed by the lectin blotting analysis. The extract produced inhouse was used to set up an immunoenzymatic test to evaluate the specific IgE binding in a panel of sera from 33 immunotherapy-free subjects who were monosensitized to cypress pollen. The percent of positivity obtained was much higher than that reported in the literature for commercial immunoassays. (J ALLERGY CLIN IMMUNOL 1994;94:547-55.)
Annals of Allergy, Asthma & Immunology, 2006
Background-The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe. Objective-To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens. Methods-Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3. Results-Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen. Conclusion-Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.
Allergy, 2017
Since IgE glyco-epitopes, also referred to as cross-reactive carbohydrate determinants (CCDs), can share significant structural homologies between different plants, they are prone to extensive cross-reactivity among allergen pollen extracts. Here, cypress pollen allergens, especially a polygalacturonase (PG), were further characterized using double one dimensional electrophoresis (D1-DE). The presence of specific IgE directed against CCDs was investigated by bromelain IgE inhibition and concanavalin A binding assays using sera of cypress pollen sensitized patients. Our results showed that IgE reactivity to CCDs in Cupressus sempervirens pollen extracts is mainly related to bromelain-type epitopes of a newly-identified cypress PG. This glycoprotein has been further characterized through an immunoproteomic approach and officially indexed as Cup s 2 by the WHO/IUIS allergen nomenclature. Cup s 2 could thus be associated with the increased prevalence of IgE reactivity to cypress pollen ...
Journal of Biological …, 2005
Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As some allergic patients´ sera react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry and 800 MHz 1 H and 13 C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short β1,6-galactan core which is substituted by a variable number (5-28) of αarabinofuranose residues which form branched side chains with 5-, 2,5-, 3,5-and 2,3,5 substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans and we suggest to name it type III arabinogalactan.
Allergies
Pectate-lyase allergens, the group 1 of allergens from Cupressaceae pollen, consist of glycoproteins exhibiting an extremely well-conserved three-dimensional structure and sequential IgE-binding epitopes. Up to 10 IgE-binding epitopic regions were identified on the molecular surface, which essentially cluster at both extremities of the long, curved β-prism-shaped allergens. Most of these IgE-binding epitopes possess very similar conformations that provide insight into the IgE-binding cross-reactivity and cross-allergenicity commonly observed among Cupressaceae pollen allergens. Some of these epitopic regions coincide with putative N-glycosylation sites that most probably consist of glycotopes or cross-reactive carbohydrate determinants, recognized by the corresponding IgE antibodies from allergic patients. Pectate-lyase allergens of Cupressaceae pollen offer a nice example of structurally conserved allergens that are widely distributed in closely-related plants (Chamæcyparis, Crypto...
Allergies
The present work was aimed at identifying the IgE-binding epitopic regions on the surface of the Cup s 3 allergen from the common cypress Cupressus sempervirens, that are possibly involved in the IgE-binding cross-reactivity reported between Cupressaceae species. Three main IgE-binding epitopic regions were mapped on the molecular surface of Cup s 3, the PR-5 thaumatin-like allergen of common cypress Cupressus sempervirens. They correspond to exposed areas containing either electropositive (R, K) or electronegative (D, E) residues. A coalescence occurs between epitopes #1 and #2, that creates an extended IgE-binding regions on the surface of the allergen. Epitope #3 contains a putative N-glycosylation site which is actually glycosylated and could therefore comprise a glycotope. However, most of the allergenic potency of Cup s 3 depends on non-glycosylated epitopic peptides. The corresponding regions of thaumatin-like allergens from other closely related Cupressaceae (Cryptomeria, Ju...