Proteomic profiling of the weed feverfew, a neglected pollen allergen source

Abstract

Feverfew (Parthenium hysterophorus), an invasive weed from the Asteraceae family, has been reported as allergen source. Despite its relevance, knowledge of allergens is restricted to a partial sequence of a hydroxyproline-rich glycoprotein. We aimed to obtain the entire sequence for recombinant production and characterize feverfew pollen using proteomics and immunological assays. Par h 1, a defensin-proline fusion allergen was obtained by cDNA cloning and recombinantly produced in E. coli. Using two complementary proteomic strategies, a total of 258 proteins were identified in feverfew pollen among those 47 proteins belonging to allergenic families. Feverfew sensitized patients' sera from India revealed IgE reactivity with a pectate lyase, PR-1 protein and thioredoxin in immonoblot. In ELISA, recombinant Par h 1 was recognized by 60 and 40% of Austrian and Indian sera, respectively. Inhibition assays demonstrated the presence of IgE cross-reactive Par h 1, pectate lyase, lipid-transfer protein, profilin and polcalcin in feverfew pollen. This study reveals significant data on the allergenic composition of feverfew pollen and makes recombinant Par h 1 available for cross-reactivity studies. Feverfew might become a global player in weed pollen allergy and inclusion of standardized extracts in routine allergy diagnosis is suggested in exposed populations.

Figure 1

Sequence analysis, purification and secondary structure elements of recombinant Par h 1. (a) Sequence alignment of defensin-like domains obtained with the multiple sequence alignment program Clustal Omega (b), comparison of proline-rich regions. Art v 1 from Artemisia vulgaris (Q84ZX5), Amb a 4 from Ambrosia artemisiifolia (D4IHC0) and SF18 from Helianthus annuus (P22357) were included. Conserved cysteine residues boxed in grey, conserved residues of defensin-like proteins in bold, proline clusters underlined. The symbols shown in the alignment correspond with identical amino acids (*), conserved substitutions (:), and semi-conserved substitutions (.). (c) Purification process of recombinant Par h 1: BE, bacterial extract; ASP, ammonium sulfate precipitation; HIC1 and HIC2, hydrophobic interaction chromatography and SEC, size exclusion chromatography. (d) Circular dichroism spectra of purified Par h 1. The unprocessed gel is shown in the Related Manuscript File.

Figure 2

Proteomics of feverfew pollen reveals the presence of allergenic protein families. (a) Summarized workflow and results of the proteomic analyses. (b) Two-dimensional gel electrophoresis of feverfew pollen extract. After the isoelectric focusing, proteins were separated by SDS-PAGE and the spots were visualized by Coomassie staining. All excised spots subjected to mass analysis are circled and spots containing allergenic molecules are indicated in red circles. Detailed information of identified allergenic protein families is shown in Table 1. The unprocessed gel is shown in the Related Manuscript File.

Figure 3

Immunoblot analysis of feverfew pollen extract (E) and purified recombinant Par h 1 (P). Extract and Par h 1 were loaded on reducing SDS-PAGE and stained with Coomassie. Immunoblot analysis was performed with a serum pool of Austrian weed pollen allergic patients (n = 15) and Indian feverfew pollen sensitized patients (n = 25). The unprocessed gels and immunoblots are shown in the Related Manuscript File. NHS, non-atopic human sera (n = 2) and BC, buffer control.

Figure 4

Feverfew extract and recombinant Par h 1 are recognized by patients’ sera in ELISA. IgE reactivity to immobilized feverfew pollen extract and purified recombinant Par h 1 was assessed with individual patients’ sera from Austria (a,b) and India (c,d). NHS, non-atopic human sera (n = 3). The dotted line indicates the response threshold calculated as 5*SD of the buffer control signal.

Figure 5

Feverfew pollen inhibits IgE reactivity to allergens from mugwort and ragweed. Purified weed pollen allergens were immobilized on ELISA plates. A pool of patients’ sera (n = 6) was pre-incubated with different concentrations of (a), mugwort (self-inhibition control) and feverfew pollen extract or (b), ragweed (self-inhibition control) and feverfew pollen extract. The sensitization profile to the single allergens of the patients’ pool is shown in the Supplementary Table S2.