Abstract
Introduction
Grass pollens are major outdoor sources of aeroallergens worldwide. Compared with timothy (
Phleum pratense
), other common allergenic grasses have far fewer officially recognized pollen allergens, even though most, if not all, timothy allergens are expected to have homologous counterparts in related species. This study aimed to expand the pollen allergomes of sweet vernal-grass (
Anthoxanthum odoratum
), cocksfoot (
Dactylis glomerata
), rye-grass (
Lolium perenne
), and meadow-grass (
Poa pratensis
) to improve the molecular characterization of allergens products, particularly those used for
in vivo
allergy diagnosis.
Methods
Grass pollen transcriptomes were sequenced.
De novo
assembly was followed by coding DNA sequence prediction, translation, and annotation. Each novel allergen homologous to one of the timothy pollen allergens Phl p 1–7 and 11–13 was cloned and expressed in
E. coli
. IgE reactivity of recombinant proteins was assessed by immunoblotting. Two-dimensional gel electrophoresis of pollen extracts was performed, and IgE-binding spots were identified by immunoblotting, excised, and analyzed by mass spectrometry using a database derived from the extended transcriptomes. Two-dimensional western blots were performed on grass pollen extracts and IgE-reactive spots were analyzed by mass spectrometry using the transcriptome-derived database. Mass spectrometry-based allergen characterization of products used for
in vivo
diagnosis of allergy by skin prick testing was performed using the extended allergomic database, in comparison with the official database.
Results
Homologous counterparts of all timothy pollen allergens from groups 1–7 and 11–13 were found in all four other grass species studied. Varying degrees of sequence identity and similarity were observed, depending on the species and allergen group considered. Of the allergens that were successfully cloned and expressed, IgE reactivity was confirmed for nearly all recombinant protein. Additionally, novel allergens unrelated to the allergen groups studied were also identified. The enriched allergome information substantially enhanced the molecular resolution achieved in the characterization of diagnostic allergen products.
Discussion
In-depth proteomics informed by transcriptomics enabled a major expansion of the pollen allergomes of the grass species studied. This extended allergomic knowledge, in turn, substantially improved the allergen characterization of
in vivo
diagnostic allergen products, confirming the added value of comprehensive grass pollen allergomes.