Deiminated proteins and extracellular vesicles as novel biomarkers in pinnipeds: Grey seal (Halichoerus gryptus) and harbour seal (Phoca vitulina)

Abstract

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, leading to functional and structural changes in target proteins. Protein deimination can cause the generation of neo-epitopes, affect gene regulation and also allow for protein moonlighting and therefore facilitate multifaceted functions of the same protein. PADs are furthermore a key regulator of cellular release of extracellular vesicle (EVs), which are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and EVs were assessed in sera of two seal species, grey seal and harbour seal. We report a poly-dispersed population of serum-EVs, which were positive for phylogenetically conserved EV-specific markers and characterised by transmission electron microscopy. A number of deiminated proteins critical for immune and metabolic functions were identified in the seal sera and varied somewhat between the two species under study, while some targets were in common. EV profiles of the seal sera further revealed that key microRNAs for inflammation, immunity and hypoxia also vary between the two species. Protein deimination and EVs profiles may be useful biomarkers for assessing health status of sea mammals, which face environmental challenges, including opportunistic infection, pollution and shifting habitat due to global warming.

The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles—Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways

“…Naked mole-rat ( Heterocephelus glaber ) plasma shows similar EV size profiles as human plasma at 50–300 nm [ 38 ], as does rat ( Rattus norvegicus ) plasma at 50–250 nm [ 43 ]. In sea mammals, such as pinnipeds and cetaceans, serum-EVs were observed at 50–600 nm in seals [ 40 ], similar to as observed in halibut in the current study. In four species of whale, EV profiles were seen in the ranges of 50–500 (minke whale Balaenoptera acutorostrata ), 50–400 (fin whale Balaenoptera physalus ), 80–300 (humpback whale Megaptera novaeangliae ) and 90–300 nm (Cuvier’s beaked whale Ziphius cavirostris ), respectively, while orca serum-EVs ( Orcinus orca ; dolphin family) were reported at 30–500 nm [ 39 ].…”

“…While EV research has been an exponentially expanding field in the past decade in relation to human disease, less is known about EV communication in other taxa. The comparative field of EV research has recently been growing, including by studies from our group [ 14 , 16 , 19 , 22 , 23 , 24 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Therefore, there is currently great interest in expanding EV studies, also in relation to teleost fish and biomarker discovery for aquaculture [ 32 , 33 , 38 , 41 ].…”

Post-Translational Protein Deimination Signatures in Serum and Serum-Extracellular Vesicles of Bos taurus Reveal Immune, Anti-Pathogenic, Anti-Viral, Metabolic and Cancer-Related Pathways for Deimination

“…It must be noted that neither anti-human PAD1 nor PAD6 antibodies were tested against bovine sera in the current study. The anti-human antibodies have previously been shown to cross-react with PADs from diverse taxa, with PAD2, which is considered the phylogenetically most conserved isoform, predominantly cross-reacting best with other species, including bony and cartilaginous fish, birds, mole-rat, alligator, camelids, cetaceans and pinnipeds [15][16][17]92,93,[96][97][98][99].…”

“…Roles for selenoproteins in the regulation of the contraction and relaxation of bronchiolar smooth muscle have furthermore been identified [192]. Sepp1 is known to be glycosylated, and recently it has been identified as a deimination candidate in cetaceans, pinnipeds and alligators [93,96,97]. The contribution of deimination in the functional diversity and conserved functions of Sepp1 throughout phylogeny will remain to be further investigated.…”

“…Following blocking the membranes were incubated overnight at 4 • C on a shaking platform with the primary antibodies, which were diluted in TBS-T. For detection of deiminated/citrullinated proteins, the F95 pan-deimination antibody was used (MABN328, Merck, 1/1000). For detection of PAD proteins in bovine serum, the following anti-human PAD antibodies were used: anti-PAD2 (ab50257, Abcam, Cambridge, 1/1000); anti-PAD3 (ab50246, Abcam, 1/1000) and anti-PAD4 (ab50247, Abcam, 1/1000), and have previously been shown to cross-react with PAD homologues in a range of taxa [15][16][17]25,26,92,93,[96][97][98][99]. EV isolates were blotted against two EV-specific markers: CD63 (ab216130, 1/1000) and Flotillin-1 (Flot-1, ab41927, 1/2000), for characterisation of EVs.…”