Abstract
The bovine immune system is known for its unusual traits relating to immunoglobulin and antiviral responses. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes that cause post-translational deimination, contributing to protein moonlighting in health and disease. PADs also regulate extracellular vesicle (EV) release, forming a critical part of cellular communication. As PAD-mediated mechanisms in bovine immunology and physiology remain to be investigated, this study profiled deimination signatures in serum and serum-EVs in Bos taurus. Bos EVs were poly-dispersed in a 70–500 nm size range and showed differences in deiminated protein cargo, compared with whole sera. Key immune, metabolic and gene regulatory proteins were identified to be post-translationally deiminated with some overlapping hits in sera and EVs (e.g., immunoglobulins), while some were unique to either serum or serum-EVs (e.g., histones). Protein–protein interaction network analysis of deiminated proteins revealed KEGG pathways common for serum and serum-EVs, including complement and coagulation cascades, viral infection (enveloped viruses), viral myocarditis, bacterial and parasitic infections, autoimmune disease, immunodeficiency intestinal IgA production, B-cell receptor signalling, natural killer cell mediated cytotoxicity, platelet activation and hematopoiesis, alongside metabolic pathways including ferroptosis, vitamin digestion and absorption, cholesterol metabolism and mineral absorption. KEGG pathways specific to EVs related to HIF-1 signalling, oestrogen signalling and biosynthesis of amino acids. KEGG pathways specific for serum only, related to Epstein–Barr virus infection, transcription mis-regulation in cancer, bladder cancer, Rap1 signalling pathway, calcium signalling pathway and ECM-receptor interaction. This indicates differences in physiological and pathological pathways for deiminated proteins in serum-EVs, compared with serum. Our findings may shed light on pathways underlying a number of pathological and anti-pathogenic (viral, bacterial, parasitic) pathways, with putative translatable value to human pathologies, zoonotic diseases and development of therapies for infections, including anti-viral therapies.
Highlights
Cattle are mammalian ruminants of the genus Bos, comprised of domesticated and wild cattle with five main extant species [1]
A particular research focus has been on their unusual ultralong CDR3H “cattlebodies”, which are being developed for immunotherapy, including against retroviral infections such as HIV [3,4,5]
Our findings suggest that protein deimination may play hitherto unidentified roles in the known unusual anti-pathogenic functions of cow serum, including via extracellular vesicle (EV)-transport, contributing to complement function in homeostatic processes
Summary
Cattle are mammalian ruminants of the genus Bos, comprised of domesticated and wild cattle with five main extant (living) species [1]. Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which cause an irreversible post-translational conversion of arginine to citrulline in target proteins. Deimination can cause the generation of neo-epitopes and affect gene regulation [6,7,8,9,10,11] This post-translational modification is most effective on beta-sheets and disordered proteins [7] and can facilitate protein moonlighting, where one polypeptide can exhibit multifaceted functions that are physiologically relevant. Roles for anti-viral responses via PAD-mediated neutrophil extracellular trap formation (NETosis) have been identified in cattle respiratory syncytial virus disease, via the detection of deiminated/citrullinated histone H3 [42].
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