Plant-expressed recombinant mountain cedar allergen Jun a 1 is allergenic and has limited pectate lyase activity.

BACKGROUND: Mountain cedar (Juniperus ashei) pollen commonly causes a winter time allergic rhinitis in the central USA. Jun a 1 is the dominant allergenic protein, but biologically active recombinant Jun a 1 has not been successfully expressed, despite numerous attempts with several expression systems. METHOD: Jun a 1 cDNA was inserted into a tobacco mosaic virus vector and transferred to Agrobacterium tumefaciens. Bacteria were syringe-inoculated into leaves of Nicotiana benthamiana (agroinoculation). The interstitial (apoplastic) fluid containing Jun a 1 was isolated. The recombinant protein was analyzed by SDS-PAGE, N-terminal sequencing and MALDI-TOF to confirm identity. Immunogenicity was examined with IgE from allergic patient's sera, mouse monoclonal anti-Jun a 1 antibodies, IgE-binding inhibition and by degranulation of RBL SX-38 cells sensitized with sera from allergic patients. Pectate lyase activity was assayed by capillary zone electrophoresis and mass spectrometry analysis. RESULTS: Recombinant Jun a 1 was recovered in good quantity (100 µg/g leaf material), was confirmed as Jun a 1, bound IgE from sera from cedar hypersensitive patients and inhibited IgE binding to native Jun a 1. Jun a 1 mutants were created and their pectate lyase activity quantified. For the first time, Jun a 1 pectate lyase activity was demonstrated, which may explain the necrosis seen on host plants, which was similar to that of control plants expressing banana pectate lyase. CONCLUSIONS: A means of producing recombinant Jun a 1 is now available for structure/function studies and potentially for diagnostic and therapeutic uses.

Structural basis for epitope sharing between group 1 allergens of cedar pollen.

The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development.

Crystal structure of Jun a 1, the major cedar pollen allergen from Juniperus ashei, reveals a parallel beta-helical core.

Pollen from cedar and cypress trees is a major cause of seasonal hypersensitivity in humans in several regions of the Northern Hemisphere. We report the first crystal structure of a cedar allergen, Jun a 1, from the pollen of the mountain cedar Juniperus ashei (Cupressaceae). The core of the structure consists primarily of a parallel beta-helix, which is nearly identical to that found in the pectin/pectate lyases from several plant pathogenic microorganisms. Four IgE epitopes mapped to the surface of the protein are accessible to the solvent. The conserved vWiDH sequence is covered by the first 30 residues of the N terminus. The potential reactive arginine, analogous to the pectin/pectate lyase reaction site, is accessible to the solvent, but the substrate binding groove is blocked by a histidine-aspartate salt bridge, a glutamine, and an alpha-helix, all of which are unique to Jun a 1. These observations suggest that steric hindrance in Jun a 1 precludes enzyme activity. The overall results suggest that it is the structure of Jun a 1 that makes it a potent allergen.

Simple synthesis of a weak nucleophilic base (4-ethyl-2,6-diisopropyl-3,5-dimethylpyridine) evidencing a double Janus group effect.

By analogy with 2,6-di-tert-butylpyridine and its 4-methyl-substituted derivatives, which are nonnucleophilic bases, 4-ethyl-2,6-diisopropyl-3,5-dimethylpyridine (4) is also such a base. The isopropyl groups (Janus-like groups) are forced by the neighboring methyl groups to turn their "tert-butyl-analogue face" toward the heteroatom, thereby protecting it sterically against electrophilic attack. The synthesis proceeds in two stages via the corresponding pyrylium salt 3 that is obtained by alkene diacylation. X-ray data for 4, its picrate, and the hexafluorophosphate of 3 confirm that the ground-state conformation agrees with the Janus effect prediction. The chemical behavior of 4 indicates that it is indeed a weak nucleophilic base, which is able to substitute the nonnucleophilic bases in organic syntheses. The compound 3 reacts at normal pressure with methylamine or ethylamine, forming N-alkylpyridinium salts. The cationic polymerization of isobutene in the presence of 4 was also investigated.

Crystallization and preliminary X-ray diffraction analysis of Jun a 1, the major allergen isolated from pollen of the mountain cedar Juniperus ashei.

Jun a 1, the major allergen of pollen from the mountain cedar Juniperus ashei, has been crystallized using the hanging-drop vapor-diffusion method at 277 K. The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 53.38, b = 113.48, c = 72.44 A, beta = 96.36 degrees and four molecules in the unit cell. A complete 2.5 A data set has been collected at 100 K with X-rays from a Cu Kalpha rotating-anode generator.