Variable expression of pathogenesis-related protein allergen in mountain cedar (Juniperus ashei) pollen

Abstract

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.

FIGURE 1

HPLC chromatograms of the ammonium sulfate fraction of mountain cedar pollen extracts. The chromatograms of five different lots of mountain cedar pollen are shown. The pollens were harvested in (a) 1992 Arkansas, (b) 1993 Oklahoma, (c) 1993 Arizona, (d) 1995 Oklahoma, and (e) 1998 Texas. The relative percentage of the Jun a 3 peak (A), to the Jun a 1 peak (B) is indicated to the right of each chromatogram. f, Whole pollen was analyzed by SDS-PAGE, transferred to nitrocellulose, and stained with colloidal gold. Lane 1, 1992 Arkansas; lane 2, 1993 Oklahoma; lane 3, 1995 Oklahoma; lane 4, 1998 Texas. The Jun a 3 content as a percent of total protein and relative to Jun a 1, as determined by densitometry, are shown below the each lane.

FIGURE 2

The 1993 (lanes 1, 2, and –9) and 1995 (lanes 3 and 4) Oklahoma pollen was analyzed further. HPLC peak A (lanes 1, , and 5) and peak B (lanes 2, , and 6) were examined by SDS-PAGE and Coomassie staining (lanes 1–4), and immunoblotting (anti-Cry j 1; lanes 5 and 6). Both peaks stained with Coomassie blue but only peak B bound anti-Cry j 1 Ab in immunoblotting. Recombinant Jun a 3 (lanes 7–9) stained with Comassie (lane 7), anti-Juna3 (lane 8), and anti-IgE from patients’ sera (lane 9).

FIGURE 3

Homology analysis. a, Nucleotide and amino acid sequence of Jun a 3 is shown with its N-terminal amino acid sequences of intact protein and tryptic peptides (underlined). b, The amino acid sequence of Jun a 3 is compared with other PR proteins. a29581, α-amylase/trypsin inhibitor (21); js0646, 22K antifungal protein in maize (22); s34794, osmotin in common tobacco (20); jh0231, thaumatin-like protein E2 (19); p50694, Pru a 2 from cherries (24). The potential N-glycosylation site is double underlined.

FIGURE 4

ELISA for cedar allergen-specific IgE and total IgE content of sera from Japanese cedar-hypersensitive patients. A, Allergen-specific IgE levels directed against Jun a 3 in sera from cedar-hypersensitive patients and normal donors were examined by ELISA. MC, Mountain cedar-hypersensitive patients; JC, Japanese cedar-hypersensitive patients. The mean plus 2 SD of the IgE binding to each allergen for a group of 12 normal subjects was regarded as the negative cut off. B, The correlation of IgE reactivity to Jun a 3 with the total IgE levels are shown. The total serum IgE level for the Jun a 3-positive group (●) was 1481.73 ± 2277.18 IU/ml, while that for the Jun a 3-negative group (○) was 1000.10 ± 1154.23 IU/ml.