Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier

Abstract

BackgroundThe pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored.Methodology/Principal FindingsUsing a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity.Conclusions/SignificanceUsing an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is warranted and could potentially lead to the development of improved diagnostic and therapeutic tools.

Grass pollen allergens globally: the contribution of subtropical grasses to burden of allergic respiratory diseases

“…Sequences for Cyn d, 15, Cyn d 2, and Cyn d 23 are listed in the Allergome database (http://www.allergome.org), and names of Cyn d 5, Cyn d 6, Cyn d 11, and Cyn d 13 are on the same site, but no evidence of their protein expression in mature pollen, IgE reactivity with patient sera, or other supporting data on their allergenic status has been published. Two new classes of allergens discovered within the lipid soluble fraction of the pollen coat; exoxylanase, and cysteine protease, of C. dactylon showed IgE reactivity in five and six of eight patients, respectively, who had high IgE levels to C. dactylon .…”

“…Bashir et al. observed an IgE‐reactive homologue of the newly described cysteine protease by immunoblotting a lipid soluble coat protein fraction of S. halepense GP (Sor h CP).…”

The Pollen Coat Proteome: At the Cutting Edge of Plant Reproduction

“…After apoptosis, this enzyme is released with the tapetum remnants to the anther loculus and deposited on the pollen exine surface [70]. An endoxylanase of ~30 kDa was also identified in the pollen coat of Bermuda ( Cynodon dactylon ) and Timothy ( Phleum pratense ) grass [36]. Both proteins share a high level of identity with maize and rice xylanase enzymes and were undetectable in the cytosolic fraction of the cyclohexane-defatted pollen.…”

“…These data suggest that Zm PCP protease synthesis is regulated at the transcriptional level. Maize Cys protease homologs have recently been identified in the pollen coat eluates of Bermuda, Timothy and Johnson ( Sorghum halepense ) grass [36]. …”

“…At the ultrastructural level, Zea m 1d was mainly localized in the tectum and the foot layer of the exine (Table S2), although gold labeling was also occasionally observed in the pollen coat [40]. A novel Cyn d 1 form (β-expansin) was also immunodetected in the pollen coat fraction of the Bermuda grass [36]. …”