Protein Deimination and Extracellular Vesicle Profiles in Antarctic Seabirds

Pelagic seabirds are amongst the most threatened of all avian groups. They face a range of immunological challenges which seem destined to increase due to environmental changes in their breeding and foraging habitats, affecting prey resources and exposure to pollution and pathogens. Therefore, the identification of biomarkers for the assessment of their health status is of considerable importance. Peptidylarginine deiminases (PADs) post-translationally convert arginine into citrulline in target proteins in an irreversible manner. PAD-mediated deimination can cause structural and functional changes in target proteins, allowing for protein moonlighting in physiological and pathophysiological processes. PADs furthermore contribute to the release of extracellular vesicles (EVs), which play important roles in cellular communication. In the present study, post-translationally deiminated protein and EV profiles of plasma were assessed in eight seabird species from the Antarctic, representing two avian orders: Procellariiformes (albatrosses and petrels) and Charadriiformes (waders, auks, gulls and skuas). We report some differences between the species assessed, with the narrowest EV profiles of 50-200 nm in the northern giant petrel Macronectes halli, and the highest abundance of larger 250-500 nm EVs in the brown skua Stercorarius antarcticus. The seabird EVs were positive for phylogenetically conserved EV markers and showed characteristic EV morphology. Post-translational deimination was identified in a range of key plasma proteins critical for immune response and metabolic pathways in three of the bird species under study; the wandering albatross Diomedea exulans, south polar skua Stercorarius maccormicki and northern giant petrel. Some differences in Gene Ontology (GO) biological and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins were observed between these three species. This indicates that target proteins for deimination may differ, potentially contributing to a range of physiological functions relating to metabolism and immune response, as well as to key defence mechanisms. PAD protein homologues were identified in the seabird plasma by Western blotting via cross-reaction with human PAD antibodies, at an expected 75 kDa size. This is the first study to profile EVs and to identify deiminated proteins as putative novel plasma biomarkers in Antarctic seabirds. These biomarkers may be further refined to become useful indicators of physiological and immunological status in seabirds-many of which are globally threatened.