Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier
Figure 2
SDS-PAGE and immunoblotting analyses of in-lab preparations of pollen surface (coat), cytoplasmic proteins and commercial defatted pollen from Bermuda grass, Timothy grass, and Johnson grass.
Raw un-defatted pollens were extracted with cyclohexane. Cyclohexane-soluble proteins (pollen coat) were separated, and the remaining cyclohexane-defatted pollen was used for isolation of the cytoplasmic fraction. MW markers, surface pollen coat and cytoplasmic proteins, and commercial defatted Bermuda grass pollen (Greer) were resolved by (A) SDS-PAGE and stained with Coomassie blue (left panel) or transferred to nitrocellulose membranes, probed with human pooled allergic sera, and detected with anti-human IgE antibodies (right panel) as described in Materials and Methods. Three numbered protein bands, 1, 2, and 3, from the Bermuda pollen surface, denoted IgE-reactive proteins identified in the immunoblot, were sequenced and identified via MALDI-TOF-MS and peptide sequencing as endoxylanase (EXY), major allergen Cyn d 1, and cysteine protease (CP). The BGP coat proteins were resolved by SDS-PAGE. Proteins identified as IgE-reactive CP and EXY were cut from the gel and electro-eluted for high purification of each protein (B). The pollen surfaces of (C) Timothy and (D) Johnson grass pollens also contain IgE-reactive CP. Three protein bands from the Timothy pollen surface, indicated by stars, were sequenced and identified as major allergen Phl p 4, EXY, and CP. A CP was identified from the Johnson grass surface. The data shown are taken from one representative experiment repeated three times. (E) Frequency and identity of IgE-reactivity of Bermuda pollen surface proteins. Proteins were analyzed by Western blotting for the presence of IgE and tested with pooled (p) and individual (a–g) pollen-allergic patients’ sera. Each lane corresponds to an allergic patient serum. The 23 kDa band was identified as CP. The data shown are taken from 1 representative experiment repeated three times.