Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Darnhofer, B.
Functional elucidation of the pollen proteome
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Medizinische Universität Graz; 2022. pp.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Birner-Grünberger Ruth
- Tomazic Peter Valentin
- Altmetrics:
- Abstract:
- Non-allergenic constituents in addition to the known pollen allergens can further drive the development of an immune response in allergic patients. The degree of allergenic potential of different pollen species can vary, probably due to the contribution of these unknown protein allergens. The aim of this study was to identify other potentially allergy promoting proteins of highly related allergenic tree species, birch, hazel and alder. A special focus was led on proteases. Serine and cysteine proteases can destroy tight junctions in the nasal epithelium. The pollen derived allergens can therefore easier enter the underlying tissue and provoke allergic reactions. A comprehensive and comparative proteomic screening of the pollen from three fagales tree species, birch, hazel and alder, was performed. To reach a maximum in protein coverage and recovery we employed different protein isolation and extraction techniques during sample preparation. As there were no publicly available plant protein databases, RNA-seq runs were used to construct protein databases. In addition to protein identification and quantitation, the activity of pollen proteases was also addressed. For this purpose, activity-based protein profiling (ABPP) was employed. In ABPP the basic principle is the covalent attachment of a small probe to the active site of the protein. We employed four different probes for the enrichment and characterization of serine and cysteine hydrolases. As a result, we report 2500 – 3000 identified proteins in each of the pollen species. All of the identified proteins were used for further annotation steps. We provide insight into different distribution of peptidases, peptidase inhibitors and additional allergenic proteins across the different species. Furthermore, we could discriminate between two different sample preparation techniques: the total proteome with harsh lysis and a second approach, where pollen was shaken in PBS, more likely to mimic the nasal environment. In addition, using the ABPP approach, we successfully enriched a number of different serine and cysteine hydrolases. In total 19, 23 and 16 active serine hydrolases could be enriched in alder, birch and hazel, respectively. Moreover, eight, 15 and 13 different active cysteine hydrolases were enriched in alder, birch and hazel. Functional enrichment analyses resulted in high species similarity, despite of their distinct protein profiles. To our knowledge, we provide the first insight into two important allergenic pollen types, hazel and alder, where no transcriptomics datasets were available. Our datasets can further be used as protein databases for further functional analysis. Moreover, we provide an overview of active serine and cysteine hydrolase profiles as a solid ground for further studies.