Identification of Novel Short Ragweed Pollen Allergens Using Combined Transcriptomic and Immunoproteomic Approaches

Abstract

Background: Allergy to short ragweed (Ambrosia artemisiifolia) pollen is a serious and expanding health problem in North America and Europe. Whereas only 10 short ragweed pollen allergens are officially recorded, patterns of IgE reactivity observed in ragweed allergic patients suggest that other allergens contribute to allergenicity. The objective of the present study was to identify novel allergens following extensive characterization of the transcriptome and proteome of short ragweed pollen.

Methods: Following a Proteomics-Informed-by-Transcriptomics approach, a comprehensive transcriptomic data set was built up from RNA-seq analysis of short ragweed pollen. Mass spectrometry-based proteomic analyses and IgE reactivity profiling after high resolution 2D-gel electrophoresis were then combined to identify novel allergens.

Results: Short ragweed pollen transcripts were assembled after deep RNA sequencing and used to inform proteomic analyses, thus leading to the identification of 573 proteins in the short ragweed pollen. Patterns of IgE reactivity of individual sera from 22 allergic patients were assessed using an aqueous short ragweed pollen extract resolved over 2D-gels. Combined with information derived from the annotated pollen proteome, those analyses revealed the presence of multiple unreported IgE reactive proteins, including new Amb a 1 and Amb a 3 isoallergens as well as 7 novel candidate allergens reacting with IgEs from 20-70% of patients. The latter encompass members of the carbonic anhydrase, enolase, galactose oxidase, GDP dissociation inhibitor, pathogenesis related-17, polygalacturonase and UDP-glucose pyrophosphorylase families.

Conclusions: We extended the list of allergens identified in short ragweed pollen. These findings have implications for both diagnosis and allergen immunotherapy purposes.

Fig 1. Experimental workflow of short ragweed pollen transcriptome, proteome and allergome characterization.

Messenger RNAs from short ragweed pollen were analyzed by deep RNA sequencing using 454 sequencing technology. After de novo transcript assembly, a translated sequence database was generated and used to assign MS/MS spectra from proteomics experiments and perform homology searches. After obtaining a reference 2D-map of the short ragweed pollen proteome, IgE reactivity was analyzed with sera from 22 allergic patients.

Fig 2. Functional analysis of the short ragweed pollen proteome.

Proteome mining and classification into functional categories was performed using the KAAS server. The histogram denotes numbers of occurrences of KEGG Orthology (KO) annotations (abscissa) assembled in selected categories (ordinate). Among allergens, Amb a 1 and carbonic anhydrase were related to carbohydrate and energy metabolisms, respectively.

Fig 3. 2D-gel reference map of the short ragweed pollen proteome.

Proteins from an aqueous short ragweed pollen extract were separated by 2D-gel electrophoresis and stained with Sypro Ruby. Proteins spots were picked and analyzed by LC-MS/MS after trypsin digestion. Proteins were identified using the Transcriptome-Derived Proteome collection supplemented with missing known allergens. Numbers refer to spots analyzed by mass spectrometry. Identification details are provided in Table 1.

Fig 4. Mapping of IgE reactivity with short ragweed pollen proteins.

Water soluble short ragweed pollen proteins were resolved by 2D-gel electrophoresis, then probed by western blot with seric IgEs from 22 ragweed-allergic patients. Representative patterns of IgE reactivity obtained from 6 patients (a-f) are shown. Identification details are provided in Table 1.