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Article

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

by
Michael F. Criscitiello
1,2,
Igor Kraev
3 and
Sigrun Lange
4,*
1
Comparative Immunogenetics Laboratory, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA
2
Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, Texas A&M University, College Station, TX 77843, USA
3
Electron Microscopy Suite, Faculty of Science, Technology, Engineering and Mathematics, Open University, Milton Keynes MK7 6AA, UK
4
Tissue Architecture and Regeneration Research Group, School of Life Sciences, University of Westminster, London W1W 6XH, UK
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(8), 2861; https://doi.org/10.3390/ijms21082861
Submission received: 6 April 2020 / Revised: 15 April 2020 / Accepted: 16 April 2020 / Published: 19 April 2020
(This article belongs to the Special Issue Extracellular Vesicles in Phylogeny)
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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.

Graphical Abstract

1. Introduction

Cattle are mammalian ruminants of the genus Bos, comprised of domesticated and wild cattle with five main extant (living) species [1]. The lifespan of Bos is 18-25 years in the wild and as cattle are valuable livestock that form an important part of food security, bovine research is important for livestock management. Furthermore cows fall under a group of long-lived mammals that display considerable cancer resistance [2]. With considerably long life spans and unusual immunological characteristics cows may hold information for molecular pathways underlying such physiological traits. The bovine immune system has received considerable attention in the medical field due to its unique immunoglobulin traits, including exceptional ability to reach recessed viral epitopes on enveloped viruses. Therefore, 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].
Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which cause an irreversible post-translational conversion of arginine to citrulline in target proteins. This modification causes structural, and sometimes functional, changes of target cytoskeletal, cytoplasmic, mitochondrial and nuclear proteins, including loss or gain of function or denaturation. Deimination can furthermore 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 also facilitate protein moonlighting, where one polypeptide can exhibit multifaceted functions that are physiologically relevant. As this is an evolutionarily acquired phenomenon, moonlighting facilitated by post-translational changes, such as deimination, may contribute to protein’s diverse and conserved functions throughout phylogeny [12,13]. PADs are identified throughout phylogeny from bacteria to mammals. In mammals, five tissue specific PAD isozymes with deimination activity are described, three in chicken and alligator, one in bony and cartilaginous fish [6,14,15,16,17], and PAD homologues (arginine deiminases, ADI) in parasites [18], fungi [19] and bacteria [20,21]. While in Bos taurus five PAD isozymes have been reported: PAD1 (NP_001094742.1), PAD2 (NP_001098922.1), PAD3 (XP_010800991.1), PAD4 (NP_001179102.1) and PAD6 (XP_002685843.1), few studies, besides assessment of cattle PAD ability to deiminate human myelin basic protein [22] and inhibitory effects of paclitaxel on PAD activity in bovine brain extract [23], have hitherto been carried out on PAD protein function, or on putative physiological relevance for PAD-mediated post-translational deimination in cattle.
PADs play important roles in a range of pathologies, including chronic, autoimmune and neurodegenerative diseases, as well as in cancer [9,10,11,24]. PADs also play roles in hypoxia and CNS regeneration [25,26,27,28,29], and PAD-mediated mechanisms have been related to ageing [30,31]. Importantly, PADs have also been implicated in infection, including sepsis, endotoxemia [32,33,34,35,36,37,38,39], in antibiotic resistance [21] and other anti-pathogenic responses, including anti-viral ones [37,40,41]. Roles for anti-viral responses via PAD-mediated neutrophil extracellular trap formation (NETosis) have furthermore been identified in cattle respiratory syncytial virus disease, via the detection of deiminated/citrullinated histone H3 [42]. Roles for PADs in tissue remodeling and immunity have also recently been described [15,16,43]. PADs have furthermore been identified as phylogenetically conserved key regulators of cellular extracellular vesicle (EV) release [21,44,45,46]. EV-mediated cellular communication is a phylogenetically conserved phenomenon [47], with EVs transferring cargo proteins and genetic material characteristic of the cells of origin [11,48,49,50,51]. As EV cargo is comprised of a large range of proteins, enzymes and genetic material, and as EVs can easily be isolated from a range of body fluids, including serum and plasma, EV signatures can be useful biomarkers [52,53].
While work on EVs has largely focused on human pathologies [53], EVs are gaining increasing interest also in veterinary medicine, including in cattle [47,54,55,56,57]. Studies on EVs in cattle have been in relation to host-pathogen interactions [58,59,60,61], including in bovine respiratory disease [62,63] and in response to infection [64,65], as well as for export of viral proteins [66,67,68]. EV research in cattle has furthermore been in relation to the estrous cycle, fertility and reproduction [56,69,70,71,72,73,74], as well as for roles during embryonic development [75,76,77,78]. Bovine milk EVs have been assessed for biological activities [79,80,81,82,83], for the application of milk EVs as safe drug delivery vehicles [84,85,86], as well as assessment of viral transfer via milk to calves [87]. Bovine milk EVs have also been investigated as an anti-inflammatory treatment in autoimmune disease [88,89] and in necrotizing enterocolitis [90]. Cattle EVs have furthermore been investigated in relation to neurological disease, including bovine spongiform encephalopathy (BSE) [54].
A recent body of comparative studies with respect to EVs and EV cargo has been performed in a range of taxa throughout the phylogenetic tree, including with a particular emphasis on deiminated protein cargo by our group [17,91,92,93,94,95,96,97,98,99] Hitherto though, no such evaluation of post-translational protein cargo in cattle serum or serum-EVs has been carried out and therefore warrants further exploration. In the current study, post-translationally deiminated protein signatures were assessed in serum and serum-EVs of Bos taurus. We report for the first time post-translational deimination of key immune, metabolic and nuclear proteins in cattle, and identify differences in KEGG pathways enriched for deiminated proteins in serum-EVs compared to whole serum. Our findings provide novel insight into the unusual immunological traits of cattle, including a new angle on their unusual anti-viral activity. Our findings further current understanding of protein moonlighting via deimination in physiological and immunological pathways, underlying anti-pathogenic responses throughout phylogeny.

2. Results

2.1. Characterisation of Bovine Serum-EVs

Bos taurus serum-EVs were assessed by nanoparticle tracking analysis (NTA) for particle numbers and size distribution using the NanoSight NS300 system, revealing a poly-dispersed population of EVs in the size range of 70–500 nm (Figure 1A). Further characterization was performed by western blotting using the EV-specific markers CD63 and Flot-1 (Figure 1B), which showed positive at protein band sizes corresponding to what is observed for these EV markers in other taxa, including in human [17,46,91,92,93,96,97,98,99], and by transmission electron microscopy (TEM), confirming typical EV morphology (Figure 1C). Some variation was observed between the three individuals with respect to EV yield, ranging from 1.72 × 1010 to 2.46 × 1010 per mL, and modal EV size, which fell in the range of 146–165 nm.

2.2. PAD Protein Homologues and Deiminated Proteins in Bovine Serum and Serum-EVs

For assessment of total deiminated proteins present in serum, F95-positive proteins were detected by western blotting in the size range of mainly 50–150 kDa (Figure 2A). Following immunoprecipitation for F95-enrichment of deiminated proteins in serum and serum-EVs, silverstaining revealed F95-enriched protein bands between 15–150 kDa in serum and in EVs protein bands were observed mainly in the size range of 50–150 kDa (Figure 2B). For assessment of bovine PAD protein homologues, anti-human PAD-isozyme specific antibodies were used for western blotting, identifying a positive protein band at 70–75 kDa mainly cross-reacting with anti-human PAD4 antibody in bovine serum (Figure 2C). A neighbor-joining phylogeny tree was furthermore constructed for bovine PADs (PAD1, NP_001094742.1; PAD2, NP_001098922.1; PAD3, XP_010800991.1; PAD4, NP_001179102.1 and PAD6, XP_002685843.1) compared with human PADs (PAD1, NP_037490.2; PAD2, NP_031391.2; PAD3, NP_057317.2; PAD4, NP_036519.2 and PAD6, NP_997304.3), using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), showing that the individual bovine and human PAD isoforms clustered together (Figure 2D).

2.3. LC-MS/MS Analysis of Deiminated Proteins in Bovine Serum and Serum-EVs

Protein identification of deiminated proteins in bovine serum and serum-EVs was carried out using F95-enrichment and LC-MS/MS analysis, searching for species-specific protein hits using the Bos taurus protein database. In serum, 118 species-specific deiminated protein hits were identified and 31 were specific to total serum only (Table 1; see detailed analysis for all hits in Supplementary Table S1). In serum-EVs, 179 species-specific deiminated protein hits were identified and 90 were specific to EVs only (Table 2; see detailed analysis for all hits in Supplementary Table S2).
The Venn diagram in Figure 3 represents common and specific deimination hits in serum and serum-EVs. Overall 87 deiminated protein hits were common to serum and serum-EVs, while 31 hits were identified to be deiminated in serum only and 90 hits were identified to be deiminated in EVs only (Figure 3A). Following KEGG pathway analysis for these deiminated protein hits, a number of common KEGG pathways enriched in deiminated proteins were identified in serum and serum EVs, while differences were observed in KEGG pathways relating to infection, immunity, disease and metabolism, that were specific to serum or serum-EVs only, respectively (Figure 3B).

2.4. Protein–Protein Interaction Network Identification of Deiminated Proteins in Bovine Serum and Serum-EVs

For the prediction of protein–protein interaction networks of the deimination candidate proteins, the protein ID lists for serum and serum-EVs respectively, were submitted to STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis (https://string-db.org/) and analyzed for KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (Figure 3B and Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8). Protein interaction networks were based on known and predicted interactions and represent all deiminated proteins identified in serum (Figure 4), all deiminated proteins identified in EVs (Figure 5) as well as deiminated proteins identified in serum only (Figure 6) or in EVs only (Figure 7), or common deimination candidates in EVs and serum (Figure 8). The PPI enrichment p-value for all deiminated proteins identified in bovine serum (based on protein identifier sequences) was found to be p < 1.0 × 10−16 and for all deiminated proteins identified in the serum-derived EVs, the PPI enrichment p-value was also found to be p < 1.0 × 10−16 (Figure 4 and Figure 5). For deiminated proteins identified in serum only (but not EVs) the PPI enrichment p-value was also p < 1.0 × 10−16 (Figure 6), while for deiminated proteins identified specifically in EVs, the PPI enrichment p-value was p = 4.83 × 10−8 (Figure 7). For common proteins found deiminated in serum and in serum-EVs, the PPI enrichment p-value was p < 1.0 × 10−16 (Figure 8). This indicates that in all cases, the identified protein networks have significantly more interactions than what would be expected for a random set of proteins of similar size, based on information drawn from the genome.
The KEEG pathways related to all deiminated proteins identified as deiminated in whole serum are shown in Figure 4A–E. For KEGG pathways relating to infection these belonged to: “Complement and coagulation cascades”, “S. aureus infection”, “systemic lupus erythematous”, “prion diseases”, “pertussis”, “trypanosomiasis”, “amoebiasis”, “leishmaniosis”, “tuberculosis”, “Epstein–Barr virus infection” (Figure 4B); For KEGG pathways relating to immunity these related to: “phagosome”, “primary immunodeficiency”, “FC-gamma R-mediated phagocytosis”, “intestinal immune network for IgA production”, “allograft rejection”, “Fc epsilon RI signalling pathway”, “autoimmune thyroid disease”, “B-cell receptor signalling pathway”, “rheumatoid arthritis”, “ferroptosis” (Figure 4C); For KEGG pathways relating to cancer and disease these related to: “transcription misregulation in cancer”, “bladder cancer”, “viral myocarditis”, “Rap1 signalling pathway”, “natural killer cell mediated cytotoxicity”, “measles”, “dilated cardiomyopathy”, “asthma” (Figure 4D); For KEGG metabolic pathways these related to: “phospholipidase D signalling pathway”; “ECM-receptor interaction”; “vitamin digestion and absorpition”, “calcium signalling pathway”, “fat digestion and absorption”, “NF-kappa B signalling pathway”, “cholesterol catabolism”, “hematopoietic cell lineage”, “PI3K-Akt signalling pathway” and “platelet activation”(Figure 4E).
The KEEG pathways related to deiminated proteins identified as deiminated in whole serum only are shown in Figure 5A,B. These were: “complement and coagulation cascades”, “phagosome”, “Rap1 signalling pathway” and “bladder cancer”.
The KEEG pathways related to all deiminated proteins identified in EVs are shown in Figure 6A–C. For KEGG pathways relating to infection these belonged to: “complement and coagulation cascades”, “S. aureus infection”, “systemic lupus erythematous”, “pertussis”, “prion diseases”, “amoebiasis”, “trypanosomiasis”, “leishmaniosis” (Figure 6B); Other KEGG pathways identified were: “oestrogen signalling pathway”, “phagosome” “vitamin digestion and absorption”, “asthma”, “primary immunodeficiency”, “Fc gamma R-mediated phagocytosis”, “ferroptosis”, “ fat digestion and absorption”, “cholesterol metabolism”, “HIF-1 signalling pathway”.
The KEEG pathways related to deiminated proteins identified as deiminated in EVs only are shown in Figure 7A,B. These were: “oestrogen signalling pathway”, “S. aureus infection”, “complement and coagulation cascades”, “biosynthesis of amino acids”.
Deiminated proteins that were common to serum and serum-EVs were furthermore analyzed for KEGG pathways (Figure 8A–E). Pathways identified for infection were: “complement and coagulation cascades”, “S. aureus infection”, “systemic lupus erythematous”, “pertussis”, “prion diseases”, “amoebiasis”, “trypanosomiasis”, “leishmaniosis”, “tuberculosis”, “malaria” (Figure 8B); KEGG pathways for immunity (and diseases) were: “Fc gamma R-mediated phagocytosis”, “primary immunodeficiency”, “Fc epsilon RI signalling pathway”, “intestinal immune network for IgA production”, “autoimmune thyroid disease”, “allograft rejection”, B-cell receptor signalling pathway”, “phagosome”, ”viral myocarditis”, rheumatoid arthritis (Figure 8C); KEGG pathways for cancer and disease were: “natural killer cell mediated cytotoxicity”, “dilated cardiomyopathy (DCM)”, “viral myocarditis”, “asthma”, “measles” (Figure 8D); KEGG metabolic pathways for common deiminated proteins in serum and serum-EVs were: “Vitamin digestion and absorption”, “ferroptosis”, “fat digestion and absorption”, “cholesterol metabolism”, “mineral absorption”, “phospholipase D signalling pathway”, PI3K-Akt signalling pathway”, NF-kappa B signalling pathway”, “hematopoietic cell lineage” and “platelet activation” (Figure 8E).

3. Discussion

The current study is the first to profile deiminated protein signatures in serum and serum-EVs of cattle, using Bos taurus as a model species. F95-enrichment for deiminated proteins from serum and serum-EVs revealed a range of immunological, metabolic and gene regulatory proteins as candidates for this post-translational modification, therefore indicating hitherto under-recognized modes for protein-moonlighting of these proteins in bovine immunity and physiology. Few studies have hitherto assessed roles for PADs and deimination in cattle [22,23,42], while a range of studies have been carried out on EVs in relation to cattle immunity, fertility and development [54,57,60,61,63,65,68,74,78,83,85,88,90]. Export of post-translationally modified proteins, such as deiminated proteins in the current study, has not been assessed before in cattle serum.
PAD proteins were assessed in bovine serum via cross-reaction to antibodies raised against human PAD2, PAD3 and PAD4 isozymes, with the strongest cross-reaction found with anti-human PAD4 at a predicted size of 70–75 kDa for PAD proteins. Protein sequence alignment of cow and human PADs showed that the individual isozymes clustered together between the two species, therefore the lack of cross-reaction with anti-human PAD2 and PAD3 antibodies may possibly be explained by differences in folding of the PADs, but will remain to be further investigated. 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].
A number of bovine species-specific deiminated protein candidates were identified in both serum and serum-EVs, using F95-enrichment in tandem with LC-MS/MS analysis. This analysis revealed some key metabolic and immune related proteins, with 87 characterised common deiminated proteins in serum and EVs, while 31 characterised deiminated protein hits were specific for serum and 90 characterised deiminated protein hits were specific for serum-EVs. Overall, the hit for deiminated proteins was higher in the serum-EVs, with a total hit of 179, compared to 118 in serum. Upon assessment of protein–protein interaction networks using STRING analysis, the PPI enrichment p-value for all deiminated proteins identified in bovine serum and serum-EVs, as well as for deiminated proteins identified either in serum or EVs only, indicated that the identified protein networks have significantly more interactions than expected for a random set of proteins of similar size, drawn from the genome, and that the proteins are at least partially biologically connected, as a group (Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8). When assessing KEGG pathways for deiminated proteins relating to infection and immunity, diseases and metabolism, a number of pathways overlapped between serum and serum-EVs, while KEGG pathways only found in 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 misregulation in cancer, bladder cancer, Rap1 signalling pathway, calcium signalling pathway, ECM-receptor interaction. This indicates differences in cell-communication via export of deiminated proteins in serum-EVs (Figure 3B).
KEGG pathways in common with both serum and serum-EVs related to a number of immunological pathways including the complement and coagulation cascade, bacterial infection, parasitic infection and importantly viral infections, as well as viral myocarditis. The role for deimination of immunoglobulins may be of considerable importance, particularly in relation to enveloped viruses as identified in the current study via deiminated KEGG pathways highlighted for Epstein–Barr virus in cow serum. Deimination in this KEGG pathway has also recently been identified in aggressive glioblastoma cells [100], and the relationship between viral infection cancers still remains to be fully understood [101,102]. In the cow, a link between viral infections and cancer has also been made [103,104,105]. Furthermore, oncolytic viruses from cow, such as bovine herpesvirus type 1, have been considered for application as a human broad spectrum cancer therapeutic [106]. The cow is known for its unusual immune responses and is a model organism for studying neutralizing antibodies against viral infections, including HIV [3,4,5]. Understanding of immune responses against enveloped viruses is of pivotal importance as indeed coronaviruses (CoVs) including severe acute respiratory syndrome (SARS) and COVID-19, also fall under enveloped viruses. CoVs are enveloped, positive-stranded RNA viruses with a nucleocapsid, and the structure of the envelope has a crucial role in virus pathogenicity as it promotes viral assembly and release [107]. While a range of studies has focused on modelling the envelope proteins, post-translational deimination has not yet been investigated in relation to SARS and CoVs, while it has been previously identified to play roles in rhinovirus infection [41]. Therefore, the roles for post-translational modifications via deimination may play some roles in structural interactions with the virus, particularly as cow immunoglobulins were here identified as deimination candidates, both in serum as well as in serum-EVs, and cow antibodies with an extended knob structure formed from the third complementarity determining region of the heavy chain are known to bind bovine and human pathogens and be capable of remarkably broad viral neutralization [3,4,5,108]. Post-translational deimination of cow Ig’s, which are reported in the current study for the first time, have yet to be further explored. Roles for PADs in anti-viral responses have previously been identified via the generation of NETosis [37], which can be PAD-mediated and is also a recognized mechanism in anti-pathogenic functions in cattle [109]. Deimination has also been identified to modulate chemokines in anti-HIV responses [40] and higher levels of anti-cyclic citrullinated peptide antibodies have been found sera of HIV patients [110]. Higher amounts of cyclic citrullinated peptides in sera have indeed been related to infectious diseases including a number of viral, bacterial and parasitic infections [111]. The ability of PAD homologues in bacteria, for example bacterial arginine deiminase in Mycoplasma arginini, a Gram-negative bacterium, has furthermore been shown to act as an effective anti-viral agent against HIV [112]. Interestingly, a recent comparison of transcriptomics data between healthy and SARS-CoV-2 infected patients’ lung biopsies (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615032), reveals a 6-fold downregulation of PAD4 mRNA level, assessing 17 SARS-CoV-2 versus 106 healthy individuals. This highlights importance for PADs in virus host-pathogen interactions, including in COVID-19. Only PAD4 was reported in this COVID-19 patient cohort. Importantly, as PAD4 is involved in gene regulation and also considered one key-driver of NETosis [37], reduced PAD4 levels, observed in these COVID-19 patients, may contribute to less defences against viral infection due to gene regulatory changes, changes in deimination of immune related proteins, or impaired NETosis in these individuals. Whether the virus manipulates PAD4 expression, as an immune evasion mechanism, or whether individuals with lower PAD4 are more prone to SARS-CoV-2 infection, remains to be further investigated. Myocarditis is furthermore one of the hallmarks of COVID-19 [113] and roles for deimination/citrullination have previously been identified in a murine model of coxsackievirus B3-induced viral myocarditis [114]. As viral myocarditis was here identified as a KEGG pathway for deiminated proteins in cow sera and serum-EVs, to what extent PADs and deimination could be involved in viral induced myocarditis, including in relation to COVID-19, may be of considerable interest and remains to be further investigated.
The complement and coagulation cascades were identified as a common KEGG immune pathway enriched in deiminated proteins in cow serum and serum-EVs. The complement system bridges innate and adaptive immunity, has roles in clearing apoptotic and necrotic cells and is in the front line for clearing invading pathogens [115,116,117,118,119]. Dysregulation of the complement system is furthermore associated with a range of pathologies [120]. In the current study a range of proteins from the complement cascade were identified as deiminated in cow serum and serum EVs, with complement components identified both in serum and serum-EVs including C1q, C3, C4A, C5a, C7, C8, C9 factor B, Factor H, C4-binding protein; while complement components found deiminated only in whole serum were C6, clusterin, and collectin-43 (CL43). C3 is the central component of the complement cascade and has furthermore been implicated in tissue remodeling during teleost ontogeny [121,122,123,124] as well as in regeneration [125]. A range of complement proteins have recently been identified by our group to be deiminated in a range of taxa throughout the phylogenetic tree including teleost and cartilaginous fish, camelids, cetaceans, pinnipeds, camelids and peglagic seabirds [16,17,91,92,93,94,95,96,98,99]. The bovine complement system [126] has been widely studied, including in immune defences against bovine anaplasmosis [127], in relation to heat-stress in dairy cows [128], susceptibility to bacterial infection relating to copper-deficiency [129]. Roles for modulation of complement regulation in relation to poxviral and pestiviral infections have been identified [130,131,132], including immune evasion by bovine herpesvirus 1 (BHV-1) [133], while and roles for antibody-dependent complement-mediated killing of mycoplasma have recently been revealed [134]. The finding of deiminated complement components highlight hitherto understudied roles for post-translational deimination in the known diversity of complement function throughout phylogeny [135,136,137,138,139]. Furthermore, immune evasion of bacteria (Gingivalis) by deimination of the host’s C5a has previously been identified [20] and indeed C5a was here identified as a deimination candidate in cow serum. Our findings suggest that protein deimination may play hitherto unidentified roles in the known unusual anti-pathogenic functions of cow serum, including via EV-transport, also contributing to complement function in homeostatic processes.
Alpha-2-macroglobulin was found to be deiminated in cow serum and serum-EVs. It forms part of the innate immune system and clears active proteases from tissue fluids [140]. In cow plasma-EVs, alpha-2-macroglobulin has been identified [55], but the current study is the first report of deiminated bovine alpha-2-macroglobulin. Alpha-2-macroglobulin is related to the other thioester-containing proteins, complement proteins C3, C4 and C5, and is a phylogenetically conserved protein from arthropods to mammals [115,141,142]. The deimination of alpha-2-macroglobulin has recently been identified by our group in teleost fish, camelid, alligator and birds [15,92,93,99]. In the cow, alpha-2-macroglobulin has been widely studied and has amongst other binding affinities to TGF-beta and a range of cytokines [143] While alpha-2-macroglobulin is a known glycoprotein, less is known about deimination and such post-translational modification may facilitate protein moonlighting contribute both to its conserved immunological, as well as multifaceted functions in a range of taxa.
Serotransferrin was identified as a major deimination candidate in the current study both in cow serum and serum-EVs. It is an iron-binding protein with multifaceted functions in development and immunity [144,145]. Serotransferrin is found both in plasma and mucosal tissue and has important roles in anti-pathogenic responses across phylogeny, including in cattle, by withdrawing iron from pathogens including bacteria, fungi and viruses [146,147,148,149]. Furthermore, some viruses, including zoonitic ones, have been identified to utilize human transferrin receptor for viral entry and infection [150,151]. In chronic disease, for example lung diseases, the dysregulation of iron homeostasis can facilitate viral respiratory infections and secondary bacterial infections [152]. Deimination of serotransferrin has previously been identified by our group in teleost serum and mucosa [15,16]. Therefore, its deimination identified here in cow indicates that serotransferrin is deiminated in several taxa and this may be of considerable importance in its immune responses to a range of pathogens throughout phylogeny, as well as with respect to a range of human diseases and infections.
Intestinal immune network for IgA production was identified as deiminated for proteins common in serum and serum-EVs. The gut associated lymphoid system in the intestinal mucosa produces the highest level of antibody-secreting (IgA) plasma cells in the body [153,154]. It has been established that IgA cells play roles beyond antibody production, including having monocytic potential and antimicrobial properties via rapid secretion of cytokines as well as acting as regulators of inflammation [153]. IgA production in the gut mucosa plays major roles in maintaining the integrity of the mucosa and is therefore imperative for the host’s homeostasis and survival [154]. IgA is also produced in the BALT of the respiratory mucosal system [154] and is therefore important in defences against respiratory infections, including viral infections and this has also been identified in cattle bovine respiratory syncytial virus [155]. Deimination has been related to mucosal immunity in teleost fish, including in response to bacterial immune challenge and in EVs mediated mucosal immunity [15,16]. Roles for deimination in the regulation of intestinal immune networks for IgA production identified here may be of considerable interest and remain to be further investigated.
Various immunoglobulin (Ig) proteins and Ig superfamily members were identified here to be deiminated in cow serum and serum-EVs, confirming that Ig’s can be exported via EVs. Ig’s identified both in serum and serum-EVs were immunoglobulin lambda-like and immunoglobulin heavy chain constant mu. Ig’s identified in addition as deiminated in serum only, were the immunoglobulin J (joining) chain responsible for dimeric IgA and pentameric IgM and the polymeric immunoglobulin receptor (pIgR) that facilitates IgA’s translocation from the lamina propria through intestinal epithelia to the lumen. This is the first report of deiminated Ig’s in bovine serum and serum-EVs. Previous studies have identified that cow plasma EVs contain immunoglobulin J chain [55], but this did not come up as a deimination candidate in EVs, only in whole serum in the current study, and therefore may not be exported in EVs in the deiminated form. We have previously confirmed post-translational deimination if Ig’s in several other taxa, including shark, camelid, alligator and birds [17,92,93,99], as well as in teleost fish [15,16], and furthermore reported EV-mediated transport of Ig’s in shark and camelid [17,92]. Ig’s are key molecules in adaptive immunity and studied in diverse taxa. Post-translational deimination of Ig’s and roles in Ig functions remain to be further investigated but have been related to the IgG Fc region in bronchiectasis and RA [156]. Current understanding of Ig diversity throughout the phylogenetic tree is still incomplete [157,158,159,160,161,162]. Therefore, our identification of deimination of Ig’s in diverse taxa, including in bovine Ig’s in the current study, highlights a novel phylogenetically conserved concept of the diversification of Ig’s via this post-translational change. Our reported findings may furthermore shed some light on the unusual immune responses in bovine sera, relating to their immunoglobulins.
Serpins (serine proteases) were identified both in serum and serum-EVs, with both common candidates in serum and EVs as well as some specific serpin targets deiminated in serum or EVs. Serpins have multifaceted roles, ranging from protease inhibition to transport of hormones as well as regulation of chromatin organization [163]. Anti-viral roles for serine proteases have been identified in the airway, and serine proteases have been identified as drug targets for respiratory diseases, including virus infections [164,165]. Bovine serpins are cross-class inhibitors and shown to be strongly active against trypsin, as well as regulating caspases, therefore playing important roles in control of apoptosis in mammals [166]. In cow, serine proteases are for example involved in defences against Babesia bovis, a tick-borne and major apicomplexan pathogen in the cattle industry worldwide [167]. Serpins are identified in a range of taxa and for example serpin based peptides from alligator have been assessed as antimicrobials against multi-drug resistant pathogens [168]. Interestingly, serpins have been identified to be deiminated in alligator, and furthermore in humans, where deimination has been related to the modulation of protease activity and downstream effects on serpin-regulated pathways in rheumatoid arthritis [169]. As deimination of serpin pathways seems therefore a conserved phenomenon in a range of taxa, including in cattle as observed here, such post-translational regulation via deimination may contribute to its various functions, relating to immunity and anti-pathogenic defences, as well as also autoimmune diseases, and this remains to be further investigated.
Fat digestion and absorption was identified as a KEGG pathway in both cow serum and serum-EVs alongside adiponectin as a specific target. The adipocytokine signalling pathway has key regulatory roles in metabolism and glucose regulation [170,171,172,173] and is linked to a range of pathologies, including type II diabetes and insulin resistance [174], to myopathies [175] and cancer [176]. Adiponectin has also been linked to longevity [177] and to regenerative functions [178]. In the cow, adiponectin is a known glycoprotein and has been assessed in bovine tissues as well as body fluids [179] and amongst other been identified as a pro-survival signal in ER stress in the mammary gland [180]. Adiponectin has recently been identified as a deimination candidate in several taxa with unusual metabolism, including camelids, the naked mole-rat, orca and alligator [92,93,96,98]. A range of apolipoproteins was furthermore identified to be deiminated in the current study in cow serum and serum-EVs. In cattle, apolipoprotein B-100, apolipoprotein A-I and apolipoprotein C-III have previously been identified as biomarkers for fatty liver and related diseases [181]. In other taxa, apolipoproteins have also been found to display antimicrobial activity against a range of pathogenic bacteria [182,183,184,185] as well as against viral (HIV) replication [186]. Various apolipoproteins have recently been identified as deimination protein candidates by our group in several taxa [15,16,92,93,99]. The deimination of apolipoproteins therefore needs further investigation in relation to their multifaceted phylogenic immune and metabolic functions.
Selenoprotein P (Sepp1) was identified to be deiminated in whole serum and serum-EVs. It is a plasma glycoprotein, mainly secreted from liver but also other tissues and contains most of the selenium in mammalian plasma [187,188,189]. In the cow roles for selenoproteins in mammary gland physiology and in milk formation have been identified [190] and roles for responses to oxidative stress in bovine arterial endothelial cells have also been suggested [191]. It has antioxidant properties [188] and serves in homeostasis and distribution of selenium [189]. 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.
KEGG pathways for ECM-receptor interactions were identified to be enriched in deiminated proteins in whole cow serum only. ECM-receptor interactions play both direct and indirect roles in controlling multifaceted cellular activities, such as cell differentiation, migration and proliferation as well as cell adhesion and apoptosis [193]. ECM-receptor interaction KEGG pathways have been identified in cancer [193] and enrichment for this pathway has been reported mesenchymal stem cell EVs [194]. Regulation of ECM-receptor interactions via post-translational deimination has recently been identified by our group via enrichment of deiminated proteins in KEGG pathways for ECM-receptor interactions in the fin whale, a long-lived cancer-resistant animal [96], in the alligator, an animal with unusual antibacterial and anti-viral responses [93], in the wandering albatross (Diomedea exulans), also an unusually long-lived bird for an avian species [99], as well as in aggressive brain cancer cells [100]. In the cow, ECM-receptor interactions are amongst other related to depot-specific adipogenesis [195] and found to be over-represented in specific cattle breeds [196]. ECM-receptor interaction pathway has furthermore been identified in Holstein cattle in relation to adaptive immune responses following viral vaccination [197] and in intra-mammary infection with Streptococcus agalactiae [198]. Therefore, deimination in this pathway may be of significant importance in anti-bacterial and anti-viral responses. Deimination in ECM-receptor interaction pathway is here described for the first time in cow and may play roles in the multifaceted functions of this pathway.
KEGG pathways for calcium signalling pathway were here identified to be enriched in deiminated proteins in whole bovine serum only. Calcium is a key modulator in a range of immunological, metabolic and developmental functions [199,200,201,202]. Calcium signalling is linked to a wide range of physiological and pathological processes [203,204,205], is a key driver for post-translational deimination [206,207,208] and plays an important role in the regulation of EVs [11,48]. In cattle, calcium is linked to many immunological pathways including inflammatory diseases [209], virus replication [210], parasitic infection [211], wound healing [212], reproduction [213,214] and adrenal function [215]. Interestingly, calcium regulation has been shown to differ between Holstein and Jersey cows, including via serotonergic stimulation of the calcium pathway [216]. The identification in the current study of enrichment in deiminated proteins in the calcium signalling pathway in cow serum indicates a regulatory function in multiple key pathways of physiological and pathological processes via this post-translational modification.
RAP1 (Ras-related protein 1) signalling pathway was identified as enriched in deiminated proteins in whole bovine serum. It belongs to a superfamily of small GTPases, with pleiotrophic regulatory functions in cellular processes, such as nuclear transport, cell cycle progression, vesicle trafficking, cell adhesion and cytoskeletal rearrangement [217]. Rap1 is an inflammatory regulator, is highly expressed in platelets and is a key molecule for platelet activation and adhesion in injury [218,219]. In mammals, Rap1 is a phylogenetically conserved telomere-interacting protein and promotes endothelial barrier function and angiogenesis [220,221], as well as being involved in cancer cell invasion and metastasis [222]. Rap1 also has roles in oxidative stress and metabolism, and besides cancer it is linked to metabolic and cardiac disorders, including cardiomyopathy [217] as well as Kabuki syndrome (characterised by congenital anomalities and developmental delay) [223]. Rap1 has also been linked to bone growth, including in bovine chondrocyte models [224]. Rap1 has been identified to undergo post-translational modifications by phosphorylation, geranylgeranylation or guanine nucleotide exchange factors [225], but deimination in this pathway has not been identified before, to our knowledge. To what extent deimination contributes to the multifaceted functions of Rap1 signalling in health and disease may therefore be of considerable interest.
Transcription misregulation in cancer and bladder cancer KEGG pathways were identified as enriched in deiminated proteins in bovine serum. Cows belong to a group of long-lived mammals that display cancer resistance and are considered a translationally valuable model for human cancers [2], including for breast cancer as they have been reported to be resistant to mammary cancer [226]. Cattle do generate bladder tumors and these have been studied in relation to various molecular mechanisms as well as in relation to viral infections, including papilloma and leukemia viruses, also in a comparative context to human cancer [103,227,228,229]. Bovine leukemia virus has furthermore been linked to telomerase upregulation and tumor induction in cattle [230]. PADs and deimination have been widely studied in a range of cancers, including via epigenetic regulation [9,10,11,100,231,232,233,234] but this is the first identification of post-translational regulation of such pathways via deimination in cattle.
HIF-1 (hypoxia inducible factor) signalling KEGG pathways were enriched in deiminated proteins in serum-EVs only. HIF-1 regulates oxygen homeostasis and is the key factor mediating the mammalian hypoxic response [235]. HIF-1 has roles in inflammation, cancer, angiogenesis and cardiovascular disease [236,237]. Furthermore, roles for HIF have been identified in bacterial infections [235] and a link between pathogenic agents and cancer has been identified, as viruses, bacteria and parasites may deregulate HIF-1 signalling associated cancer cells [238]. In cows, HIF-1 has for example been found to play roles in immune responses against Theileria annulata, a protozoan parasite of major economically importance for the cattle industry [239]. HIF-1 has also been identified to be important in angiogenesis and the maintenance of capillary structures for final follicle maturation in the cow ovary [240]. Furthermore, HIF-1 has recently been identified as a genomic marker differing between cattle breeds, including highland-adapted cattle [241]. Interestingly, enrichment in deiminated proteins in KEGG HIF-1 signalling pathways has previously been identified in EVs only, in some species with unusual immune and metabolic function including the naked mole-rat and deep-diving whales, both of which are animal models of hypoxia-tolerance and cancer-resistance [96,98]. Furthermore, deimination of HIF-1 regulation were recently identified in aggressive brain cancer (glioblastoma) cells [100]. To what extent deimination regulates HIF-1 signalling in relation to cancer, inflammation and infectious disease, including in cattle, remains to be further investigated.
KEGG pathways for biosynthesis of amino acids were identified as deiminated in serum-EVs only. This may be of considerable interest for comparative metabolic studies, particularly as amino acid assessment for mammalian metabolism and for research into ageing and disease has received some attention [242]. In dairy cows, these pathways are studies as they are of importance for understanding of optimization of diets and increased efficiency of microbial protein synthesis [243]. Enrichment for deiminated proteins in this KEGG pathway has previously been identified in alligator serum-EVs [93] as well as in cetacean sera [96] by our group, while mechanisms for deimination in this pathway remain to be elucidated.
The presence of deiminated histones H2A, H2B, H3 and H4 was identified in serum-EVs only, but not in whole bovine serum. Interestingly, a similar EV-mediated export of deiminated histones, as observed in cow serum-EVs in the current study, was recently identified in the naked mole-rat, an animal with unusual immunological and metabolic traits [98], as well as in alligator, also an animal with unusual antimicrobial, including anti-viral responses [93]. Deiminated histone H3 is commonly used as an indicator for neutrophil extracellular trap formation (NETosis) [31,244], which has been related to PAD4 and implicated in anti-viral responses in cattle respiratory syncytial virus disease [42], in response to parasitic infections in cattle [245,246,247] as well as in response to certain antibiotics [248]. Interestingly, in crocodilians, extracellular histones H2A and H4 have been identified to act as inhibitors of viral (HIV) infection in vitro [249], although roles for post-translational deimination were not assessed. Anti-microbial effects for histones have furthermore been observed in teleost fish mucosal immunity for H2A [250] as well as for deiminated histone H3 [15]. Histone H3 deimination has furthermore been linked to inflammatory and hypoxic responses during CNS regeneration in avian and murine models [25,26]. Histone deimination is also a known epigenetic mechanism in cancer [11,231,233]. The multifaceted functions of histones in immunity, including via post-translational regulation, such as deimination identified here, remains to be further investigated in cattle, including in relation to possible roles in anti-viral and other anti-pathogenic or disease related responses.
The present study highlights novel aspects of protein moonlighting in immunity and metabolism of cattle, via post-translational deimination, including via EV-mediated transport. Our findings indicate differences in physiological and pathological pathways for deiminated proteins in serum-EVs, compared with whole serum, and 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. While these data fall short of directing immediate clinical intervention for human disease, they do mandate further study of deimination in future work of broader scope and pre-clinical focus. For example, deimination will need further exploration as an additional diversification mechanism of ultralong CDR3 “cattlebodies” that have shown remarkable reach for broad viral neutralization. Also, importance for PADs in host-pathogen interactions, including in viral infections and in relation to COVID-19, may be of considerable importance and needs to be further investigated.

4. Materials and Methods

4.1. Serum Sampling from Cow

Blood was collected from the jugular vein of three healthy 5 months old Holstein breed steer and serum was prepared following clotting of blood for 2 h at room temperature (RT), by centrifugation for five minutes at 200× g. Sample collection was conducted under Texas A&M Institutional Animal Care and Use Protocol # 2015-078. Serum was aliquoted and kept at –80 °C until used. These samples were excess from a distinct study protocol and thus no animals needed to be purchased or euthanized for the study described here, consistent with the ethical principle of replacement, reduction and refinement.

4.2. Isolation of Extracellular Vesicles and Nanoparticle Tracking Analysis (NTA)

Serum-EVs were isolated from serum of individual animals (n = 3), using sequential centrifugation and ultracentrifugation in accordance to previously established protocols [17,46,98] and according to the recommendations of MISEV2018 (the minimal information for studies of extracellular vesicles 2018; [251]). For each individual EV preparation, 100 μL of bovine serum were diluted 1:5 in Dulbecco’s PBS (DPBS, ultrafiltered using a 0.22 μm filter, before use) and then centrifuged at 4000× g for 30 min at 4 °C, to ensure the removal of aggregates and apoptotic bodies. Thereafter the supernatants were collected and centrifuged further, using ultracentrifugation at 100,000× g for 1 h at 4 °C. The EV-enriched pellets were resuspended in 1 mL DPBS and ultracentrifuged again at 100,000× g for 1 h at 4 °C. The resulting washed EV pellets were then resuspended in 100 µL DPBS and frozen at –80 °C until further use. For EV size distribution profiles and EV quantification, NTA analysis was carried out using the NanoSight NS300 system (Malvern Panalytical, Malvern, UK), which analyses particle size based on Brownian motion. The EV samples were diluted 1/100 in DPBS (10 μL of EV preparation diluted in 990 μL of DPBS) and applied to the NanoSight using a syringe pump to ensure continuous flow of the sample. For each sample, five 60 second videos were recorded, keeping the number of particles per frame in-between 40 to 60. Replicate histograms were generated from the videos, using the NanoSight software 3.0 (Malvern), representing mean and confidence intervals of the 5 recordings for each sample.

4.3. Transmission Electron Microscopy (TEM)

A pool of EVs, isolated from serum of the three individual animals as described above, was used for morphological analysis using TEM. Following isolation, the EVs were resuspended in 100 mM sodium cacodylate buffer (pH 7.4) and a drop (~3–5 μL) of the suspension was placed onto a grid with previously glow discharged carbon support film. After the suspension had partly dried, the EVs were fixed by placing the grid onto a drop of a fixative solution (2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0)) for 1 min at room temperature and washed afterwards by touching the grid to the surface of three drops of distilled water. Excess water was removed by touching the grid to a filter paper. Next, the EVs were stained with 2% aqueous Uranyl Acetate (Sigma-Aldrich) for 1 min, the excess stain was removed by touching the grid edge to a filter paper and the grid was let to dry. Imaging of EVs was performed using a JEOL JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV at a magnification of 30,000× to 60,000×. Digital images were recorded using an AMT XR60 CCD camera (Deben UK Limited, Bury Saint Edmunds, UK).

4.4. Isolation of Deiminated Proteins in Serum and EVs Using F95-enrichment

Immunoprecipitation and isolation of deiminated proteins in serum and serum-derived EVs was carried out using the Catch and Release®v2.0 immunoprecipitation kit (Merck, UK) in conjunction with the F95 pan-deimination antibody (MABN328, Merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination [252]. Bovine serum pools of the three individual animals (3 × 25 μL) were used for F95-enrichment from whole serum, while for EVs, total protein was first extracted from the pool of EVs derived from 3 animals (EV pellets derived from 100 μL serum per animal), using RIPA+ buffer (Sigma, UK). Following application of RIPA+ buffer, the EVs were incubated on ice for 2 h followed by centrifugation at 16,000× g for 30 min to collect the protein containing supernatant. Thereafter, immunoprecipitation (F95-enrichment) was carried out overnight on a rotating platform at 4 °C. The F95 bound proteins were eluted using denaturing elution buffer (Merck), according to the manufacturer’s instructions (Merck) and diluted 1:1 in Laemmli sample buffer. The F95-enriched eluates from whole serum and serum -EVs were then analyzed by SDS-PAGE, followed by silver staining, Western blotting or LC-MS/MS.

4.5. Western Blotting Analysis

Bovine serum and serum-EVs were diluted 1:1 in denaturing 2× Laemmli sample buffer (containing 5% beta-mercaptoethanol, BioRad, Kidlington, UK) and boiled for 5 min at 100 °C. Proteins were separated by SDS-PAGE using 4%-20% gradient TGX gels (BioRad UK). Western blotting was carried out using the Trans-Blot® SD semi-dry transfer cell (BioRad, UK); even transfer was assessed by staining the membranes with PonceauS (Sigma, UK). Blocking was performed for 1 h at room temperature using 5% bovine serum albumin (BSA; Sigma, UK), in Tris buffered saline (TBS) containing 0.1% Tween20 (BioRad, UK; TBS-T). 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. After primary antibody incubation the membranes were washed for 3 × 10 min in TBS-T at RT and incubated for 1 h, at RT with HRP-conjugated secondary antibodies (anti-rabbit IgG (BioRad) or anti-mouse IgM (BioRad) respectively, diluted 1/3000 in TBS-T). The membranes were then washed in TBS-T for 5 × 10 min and positive proteins bands were visualized digitally, using enhanced chemiluminescence (ECL, GE Healthcare, Amersham Pl, Little Chalfont, UK) and the UVP BioDoc-ITTM System (Thermo Fischer Scientific, Hemel Hempstead, UK).

4.6. Silver Staining

F95-enriched protein eluates from bovine serum and serum-EVs were silver stained following SDS-PAGE (using 4–20% gradient TGX gels, BioRad, UK) under reducing conditions, using the BioRad Silver Stain Plus Kit (1610449, BioRad, UK), according to the manufacturer’s instructions (BioRad).

4.7. Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Analysis of Deiminated Protein Candidates

F95-enriched eluates from bovine serum and serum-EVs respectively, were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS), according to previously described methods [93,99]. For LC-MS/MS analysis, the F95-enriched eluates were run 0.5 cm into a 12% TGX gel (BioRad, UK), the band cut out, trypsin digested and subjected to proteomic analysis on a Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scientific Inc, Waltham, MA, USA.) system and a QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific Inc, Waltham, MA, USA) according to previously described methods [93,99]. In brief, peptides were separated by reverse-phase chromatography and a Thermo Scientific reverse-phase nano Easy-spray column (Thermo Scientific PepMap C18, 2 µm particle size, 100A pore size, 75 µm i.d. × 50 cm length). First, a pre-column (Thermo Scientific PepMap 100 C18, 5 µm particle size, 100A pore size, 300 µm i.d. × 5 mm length) was used for loading the peptides from the Ultimate 3000 autosampler, in the presence of 0.1% formic acid for 3 min (flow rate 10 µL/min). Therafter, peptides were eluted from the pre-column onto the analytical column (solvent A = water + 0.1% formic acid; solvent B = 80% acetonitrile, 20% water + 0.1% formic acid). A linear gradient of 2–40% B was applied for 30 min. An easy-Spray source (Thermo Fischer Scientific Inc., Waltham, MA, USA) as used to spray the LC eluant into the mass spectrometer. The m/z values of the eluting ions were measured using an Orbitrap mass analyzer. The resolution was set at 70,000 and scanning was performed at m/z 380–1500. Fragment ions were automatically isolated and generated based on data dependent scans (top 20) in the HCD collision cell by higher energy collisional dissociation (HCD, NCE:25%). The resulting fragment ions were measured using the Orbitrap analyzer, set at a resolution of 17500. Ions that were singly charged, or with unassigned charge states, were excluded from selection for MS/MS. Furthermore, a dynamic exclusion window of 20 seconds was applied. The Protein Discoverer (version 2.1., Thermo Scientific) was used to process the data post-run and the MS/MS data was thereafter converted to mgf files. For identification of deiminated protein hits, the files were then submitted to the Mascot search algorithm (Matrix Science, London, UK) and searched against the UniProt Bos taurus_ 20170607 database (24148 sequences; 40594 residues) and Bos_primigenius primigenius_20191209 database (54 sequences; 14783 residues), as well as a common contaminant sequences (123 sequences; 40594 residues). Fragment and peptide mass tolerances and were set at 0.1 Da and 20 ppm, respectively. The peptide cut-off score was set at 31 and significance at p < 0.05 (carried out by Cambridge Proteomics, Cambridge, UK).

4.8. Protein–Protein Interaction Network Analysis

For the identification and prediction of putative protein–protein interaction networks for deiminated proteins identified in bovine serum and serum-EVs, STRING analysis (Search Tool for the Retrieval of Interacting Genes/Proteins; https://string-db.org/) was used. Protein networks were built based on the protein IDs and using the function of “search multiple proteins” in STRING, choosing “Bos taurus” for the species database. For analysis, “medium conficence” and “basic settings” were selected and the colour lines connecting nodes indicated the following evidence-based interactions for the network edges: “known interactions”, which are based on curated databases or experimentally determined interactions; “predicted interactions”, which are based on protein homology, co-expression, gene fusion, gene neighborhood, gene co-occurrence, or established by text mining.

4.9. Phylogenetic Comparison of Bos PADs with Human PADs

A neighbor-joining phylogeny tree was constructed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) based on sequence alignment of amino acid sequences for bovine PADs 1-6 (PAD1, NP_001094742.1; PAD2, NP_001098922.1; PAD3, XP_010800991.1; PAD4, NP_001179102.1 and PAD6, XP_002685843.1) compared with human PADs 1-6 (PAD1, NP_037490.2; PAD2, NP_031391.2; PAD3, NP_057317.2; PAD4, NP_036519.2 and PAD6, NP_997304.3).

4.10. Statistical Analysis

The histograms and graphs were prepared using the Nanosight 3.0 software (Malvern Panalytical) and GraphPad Prism version 7 (GraphPad Software, San Diego, CA, USA). NTA curves represent mean and standard error of mean (SEM), indicated by confidence intervals. STRING analysis (https://string-db.org/) was used for prediction of protein–protein interaction networks. Significance was considered as p ≤ 0.05.

5. Conclusions

In the current study we report for the first time deimination profiles of serum and serum-EVs of Bos taurus. Post-translational deimination of key immune, metabolic and nuclear proteins was identified and these related to KEGG pathways for a number of immunological pathways with relevance to viral, bacterial and parasitic infections, to chronic and autoimmune diseases, as well as a range of metabolic pathways. Our findings highlight novel aspects of protein moonlighting in immunity and metabolism of cattle, via post-translational deimination, including via EV-mediated transport.
While these data fall short of directing immediate clinical intervention for human disease, they do mandate further study of deimination as an additional diversification mechanism of ultralong CDR3 “cattlebodies” that have shown remarkable reach for broad viral neutralization. This study limitation of this comparative biochemical basic science must be addressed in future work of broader scope and pre-clinical focus. Furthermore, transcriptomics data revealing downregulation in PAD4 mRNA levels in SARS-CoV-2 infected patient’ lung biopsies, compared to healthy controls (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA615032), indicates importance for PADs in host-virus interactions in COVID-19 and will need to be further explored.
EVs research in comparative immunology and veterinary sciences is a rapidly growing field and while EVs have been previously assessed in cattle, this study is the first to characterise deiminated protein signatures in bovine serum and serum-EVs. The identification of post-translational deimination and EV-mediated communication in bovine immunity and physiology, revealed here, contributes to current understanding of protein moonlighting functions and EV-mediated communication in cattle, providing novel insight into their unusual immune systems and physiological traits. Our findings may shed light on pathways underlying a number of pathological and anti-pathogenic, including anti-viral, pathways, with putative translatable value to human pathologies, zoonotic diseases and development of therapies for infections, including anti-viral therapies.

Supplementary Materials

The following are available online at https://www.mdpi.com/1422-0067/21/8/2861/s1, Table S1. Full LC-MS/MS analysis of F95-enriched proteins in bovine serum. Supplementary Table S2. Full LC-MS/MS analysis of F95-enriched proteins in bovine serum-EVs.

Author Contributions

Conceptualization, S.L.; methodology, I.K. and S.L.; validation, M.F.C. and S.L.; formal analysis, S.L.; investigation, M.F.C., and S.L.; resources, M.F.C., I.K. and S.L.; data curation, S.L.; writing—original draft preparation, S.L.; writing—review and editing, M.F.C. and S.L.; visualization, I.K., and S.L.; supervision, S.L.; project administration, S.L.; funding acquisition, M.F.C. and S.L. All authors have read and agreed to the published version of the manuscript.

Funding

The study was funded in part by a University of Westminster start-up grant to SL and U.S. National Science Foundation grant IOS 1656870 to M.F.C.

Acknowledgments

The authors would like to thank Yagnesh Umrania and Michael Deery at the Cambridge Centre for Proteomics for the LC–MS/MS analysis. Thanks are due to The Guy Foundation for funding the purchase of equipment utilized in this work.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

BSABovine serum albumin
CD63CD63 antigen; granulophysin; lysosomal-associated membrane protein 3
CoVCoronavirus
COVID-19Coronavirus disease 2019
ECLEnhanced chemiluminescence
ECMExtracellular matrix
EVsExtracellular vesicles
F95Pan-deimination/citrullination antibody
FBSFetal bovine serum
Flot-1Flotillin-1
HIFHypoxia inducible factor
IgImmunoglobulin
KEGG Kyoto encyclopedia of genes and genomes
LC-MS/MSLiquid chromatography mass spectrometry
NETosisNeutrophil extracellular trap formation
NTANanoparticle tracking analysis
PADPeptidylarginine deiminase
RAP1Ras-related protein 1
SARSSevere acute respiratory syndrome
SDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresis
TBSTris buffered saline
TEMTransmission electron microscopy
WBWestern blotting

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Figure 1. Extracellular vesicle profiling in bovine serum. (A) Nanoparticle tracking analysis shows a size distribution of plasma-EVs from Bos taurus in the size range of 70 to 500 nm, with main peaks at approximately 120–240 nm. (B) Transmission electron microscopy (TEM) analysis of bovine serum-derived EVs shows typical EV morphology; scale bar is 50 nm in all figures. (C) Western blotting analysis confirms that bovine EVs are positive for the phylogenetically conserved EV-specific markers CD63 and Flot-1, showing positive at expected molecular weight size corresponding to what is observed in other taxa (kDa = kilodaltons).
Figure 1. Extracellular vesicle profiling in bovine serum. (A) Nanoparticle tracking analysis shows a size distribution of plasma-EVs from Bos taurus in the size range of 70 to 500 nm, with main peaks at approximately 120–240 nm. (B) Transmission electron microscopy (TEM) analysis of bovine serum-derived EVs shows typical EV morphology; scale bar is 50 nm in all figures. (C) Western blotting analysis confirms that bovine EVs are positive for the phylogenetically conserved EV-specific markers CD63 and Flot-1, showing positive at expected molecular weight size corresponding to what is observed in other taxa (kDa = kilodaltons).
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Figure 2. Deiminated proteins and peptidylarginine deiminases (PADs) in bovine serum. (A) Total deiminated proteins were identified in bovine serum using the pan-deimination specific F95 antibody. (B) F95-enriched IP fraction from bovine serum and serum-extracellular vesicles (EVs), shown by silver-staining. (C) Immunodetection of PAD homologues in bovine sera by western blotting, using anti-human PAD2, PAD3 and PAD4 antibodies. (D) A neighbor-joining phylogeny tree for bovine and human PAD isozymes.
Figure 2. Deiminated proteins and peptidylarginine deiminases (PADs) in bovine serum. (A) Total deiminated proteins were identified in bovine serum using the pan-deimination specific F95 antibody. (B) F95-enriched IP fraction from bovine serum and serum-extracellular vesicles (EVs), shown by silver-staining. (C) Immunodetection of PAD homologues in bovine sera by western blotting, using anti-human PAD2, PAD3 and PAD4 antibodies. (D) A neighbor-joining phylogeny tree for bovine and human PAD isozymes.
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Figure 3. Deiminated proteins identified in bovine serum and serum-EVs. (A) Species specific hits identified for deiminated proteins in bovine serum (Table 1) and serum-EVs (Table 2) respectively, as well as number of overlapping hits are presented. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways identified to be enriched in deiminated proteins in bovine serum and serum-EVs respectively, as well as number of overlapping KEGG pathways, are presented. For specific KEGG pathways presented in the Venn diagram, see the protein–protein interaction networks in Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8.
Figure 3. Deiminated proteins identified in bovine serum and serum-EVs. (A) Species specific hits identified for deiminated proteins in bovine serum (Table 1) and serum-EVs (Table 2) respectively, as well as number of overlapping hits are presented. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways identified to be enriched in deiminated proteins in bovine serum and serum-EVs respectively, as well as number of overlapping KEGG pathways, are presented. For specific KEGG pathways presented in the Venn diagram, see the protein–protein interaction networks in Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8.
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Figure 4. Protein–protein interaction networks show all deiminated proteins which were identified in bovine serum. Protein–protein interactions were reconstructed based on both known and predicted interactions in serum of Bos taurus, using STRING analysis. (A) Query proteins are indicated by the coloured nodes represent and first shell of interactors. (B) KEGG pathways relating to the identified proteins and reported in STRING and relating to infection are highlighted (see colour code included in the figure). (C). KEGG pathways relating to the identified proteins and reported in STRING are highlighted for immunity (see colour code included in the figure). (D). KEGG pathways relating to cancer and disease for deiminated proteins identified are highlighted (see colour code included in the figure). (E). KEEG pathways relating to metabolism for deiminated proteins identified are highlighted (see the colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
Figure 4. Protein–protein interaction networks show all deiminated proteins which were identified in bovine serum. Protein–protein interactions were reconstructed based on both known and predicted interactions in serum of Bos taurus, using STRING analysis. (A) Query proteins are indicated by the coloured nodes represent and first shell of interactors. (B) KEGG pathways relating to the identified proteins and reported in STRING and relating to infection are highlighted (see colour code included in the figure). (C). KEGG pathways relating to the identified proteins and reported in STRING are highlighted for immunity (see colour code included in the figure). (D). KEGG pathways relating to cancer and disease for deiminated proteins identified are highlighted (see colour code included in the figure). (E). KEEG pathways relating to metabolism for deiminated proteins identified are highlighted (see the colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
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Figure 5. Protein–protein interaction networks of deiminated protein candidates identified in bovine serum only (not identified in EVs). Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEGG pathways reported in STRING and related to the identified proteins are highlighted (see the colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
Figure 5. Protein–protein interaction networks of deiminated protein candidates identified in bovine serum only (not identified in EVs). Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEGG pathways reported in STRING and related to the identified proteins are highlighted (see the colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
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Figure 6. Protein–protein interaction networks of all deiminated proteins identified in serum-EVs of Bos taurus. Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEGG pathways reported in STRING and relating to infection are highlighted (see colour code included in the figure). (C) KEGG pathways relating to the identified proteins and reported in STRING are highlighted for other pathways (see colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
Figure 6. Protein–protein interaction networks of all deiminated proteins identified in serum-EVs of Bos taurus. Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEGG pathways reported in STRING and relating to infection are highlighted (see colour code included in the figure). (C) KEGG pathways relating to the identified proteins and reported in STRING are highlighted for other pathways (see colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
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Figure 7. Protein–protein interaction networks of deiminated protein candidates identified in bovine serum-EVs only (not identified in total serum). Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEEG pathways relating to the identified deiminated proteins and reported in STRING are highlighted (see colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
Figure 7. Protein–protein interaction networks of deiminated protein candidates identified in bovine serum-EVs only (not identified in total serum). Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEEG pathways relating to the identified deiminated proteins and reported in STRING are highlighted (see colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
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Figure 8. Protein–protein interaction networks for common deiminated protein candidates identified in bovine serum and serum-EVs (excluding serum-specific or EV specific candidates). Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEGG pathways for to the identified deiminated proteins and reported in STRING and relating to infection are highlighted (see colour code included in the figure). (C) KEGG pathways relating to the identified proteins and reported in STRING are highlighted for immunity (see colour code included in the figure). (D) KEGG pathways relating to cancer and disease for deiminated proteins identified are highlighted for (see colour code included in the figure). (E) KEEG pathways relating to metabolism for deiminated proteins identified are highlighted (see colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
Figure 8. Protein–protein interaction networks for common deiminated protein candidates identified in bovine serum and serum-EVs (excluding serum-specific or EV specific candidates). Protein–protein interactions were reconstructed based on both known and predicted interactions by STRING analysis. (A) Query proteins are indicated by the coloured nodes and represent the first shell of interactors. (B) KEGG pathways for to the identified deiminated proteins and reported in STRING and relating to infection are highlighted (see colour code included in the figure). (C) KEGG pathways relating to the identified proteins and reported in STRING are highlighted for immunity (see colour code included in the figure). (D) KEGG pathways relating to cancer and disease for deiminated proteins identified are highlighted for (see colour code included in the figure). (E) KEEG pathways relating to metabolism for deiminated proteins identified are highlighted (see colour code included in the figure). The coloured lines highlight which protein interactions are identified through known interactions (this refers to curated databases, experimentally determined), through predicted interactions (this refers to gene neighborhood, gene fusion, gene co-occurrence) or through co-expression, text mining or protein homology (the colour key for connective lines is included in the figure).
Ijms 21 02861 g008aIjms 21 02861 g008b
Table
Table 1. Deiminated proteins in bovine serum (Bos taurus), as identified by F95-enrichment and LC-MS/MS analysis. Deiminated proteins from serum were isolated by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analysed by LC-MS/MS and peak list files submitted to Mascot. Bos taurus species-specific peptide sequence hits are listed, showing number of sequences for protein hits and total score. Blue highlighted rows indicate protein hits identified in whole sera only. Uncharacterised proteins based on LC-MS/MS search were further confirmed in STRING, based on the protein symbol, and protein identification from STRING, where retrieved, is shown in brackets ([]). For further full LC-MS/MS analysis of all protein hits see Supplementary Table S1.
Table 1. Deiminated proteins in bovine serum (Bos taurus), as identified by F95-enrichment and LC-MS/MS analysis. Deiminated proteins from serum were isolated by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analysed by LC-MS/MS and peak list files submitted to Mascot. Bos taurus species-specific peptide sequence hits are listed, showing number of sequences for protein hits and total score. Blue highlighted rows indicate protein hits identified in whole sera only. Uncharacterised proteins based on LC-MS/MS search were further confirmed in STRING, based on the protein symbol, and protein identification from STRING, where retrieved, is shown in brackets ([]). For further full LC-MS/MS analysis of all protein hits see Supplementary Table S1.
Protein NameSymbolSequencesTotal Score (p < 0.05) 1
SerotransferrinG3X6N3765712
SerotransferrinQ29443765711
Complement factor HQ28085775123
Alpha-2-macroglobulinQ7SIH1775079
Serum albuminA0A140T897593944
Uncharacterised protein [complement factor H]F1MC45543745
Complement C3Q2UVX4683483
Uncharacterised protein [ceruloplasmin]F1N076442712
Embryo-specific fibronectin 1B8Y9S9372263
HemopexinQ3SZV7302019
Uncharacterised protein [complement C4A]E1BH06341997
C4b-binding protein alpha chainQ28065331841
Kininogen-1A0A140T8C8231505
Kininogen-1P01044221449
Histidine-rich glycoproteinF1MKS5261432
Uncharacterised protein (uncharacterised)F1MJK3261291
Kininogen-2P01045191271
Uncharacterised protein (uncharacterised)G3X7F3161259
Uncharacterised protein (uncharacterised)G3N0V0191186
Apolipoprotein A-I preproproteinV6F9A2191122
Uncharacterised protein (uncharacterised)F1MVK117953
SERPIND1 proteinA6QPP216865
Uncharacterised protein (uncharacterised)F1MZ9613837
Uncharacterised protein [Bos taurus immunoglobulin lambda-like polypeptide 1 (IGLL1)]F1MLW710815
Uncharacterised protein [Bos taurus immunoglobulin lambda-like polypeptide 1 (IGLL1)]F1MCF810796
Uncharacterised protein [Immunoglobulin heavy constant mu]G5E5T512784
Uncharacterised protein [Immunoglobulin heavy constant mu]G5E51310732
Uncharacterised protein (uncharacterised)G3N0S912648
Histidine-rich glycoproteinP3343312623
Alpha-2-antiplasminP2880011531
Uncharacterised protein (uncharacterised)F1MLW85434
Hemoglobin subunit betaP020706422
Vitamin D-binding proteinF1N5M29420
Alpha-1-antiproteinaseP349558418
AdiponectinQ3Y5Z36411
Uncharacterised protein (uncharacterised)F1MW797403
ECM1 proteinA5PJT78395
Complement C1q subcomponent subunit BQ2KIV98390
Plasma kallikreinQ2KJ638385
Inter-alpha-trypsin inhibitor heavy chain H4F1MMD76367
C1QC proteinQ1RMH55347
Uncharacterised protein [Serpin A3-5; Serine protease inhibitor]G8JKW76338
Apolipoprotein A-IVF1N3Q77338
Inter-alpha-trypsin inhibitor heavy chain H2F1MNW46338
Selenoprotein PP499076323
PlasminogenE1B7266302
Complement C1q subcomponent subunit AQ5E9E35300
Uncharacterised protein [Bos taurus insulin-like growth factor binding protein, acid labile subunit (IGFALS)]F1MJZ46277
Thrombospondin-1F1N3A17273
Serpin A3-3G3N1U45271
Protein HP-20 homologQ2KIT05269
Uncharacterised protein (uncharacterised)G5E6044234
Inter-alpha-trypsin inhibitor heavy chain H1Q0VCM54234
Uncharacterised protein (uncharacterised)E1B8055232
Uncharacterised protein (uncharacterised)G3MXD93209
Uncharacterised protein [Bos taurus immunoglobulin lambda-like polypeptide 1 (IGLL1)]G3N2D73204
Keratin, type II cytoskeletal 7Q29S214199
Keratin, type I cytoskeletal 14F1MC114199
Uncharacterised protein [Heparan sulfate proteoglycan 2]F1MER74195
Alpha-2-HS-glycoproteincRAPR1|FETUA_BOVIN4190
Uncharacterised protein (uncharacterised)G3N1H52187
Serpin A3-8A0A0A0MP893184
Complement factor BP811874183
Protein AMBPF1MMK95181
Serpin A3-2A2I7M94180
Antithrombin-IIIF1MSZ65176
Paraoxonase 1Q2KIW14174
GelsolinF1MJH13171
Uncharacterised protein (uncharacterised)G3N3Q32168
Complement C5a anaphylatoxinF1MY853166
Uncharacterised protein (uncharacterised)G5E5H23162
Serpin A3-7A0A0A0MP924160
C1QTNF3 proteinA7MB823160
Uncharacterised protein [Bos taurus serotransferrin-like]E1BI824157
Uncharacterised protein [Apolipoprotein B]E1BNR04150
Primary amine oxidase, liver isozymeQ294373137
Coagulation factor XIF1MUT42127
Hepatocyte growth factor-like proteinE1BDW72121
Fibrinogen beta chainF1MAV03108
Immunoglobulin J chainQ3SYR82107
Uncharacterised protein (uncharacterised)G3MXB52100
Uncharacterised protein [CD5 molecule-like]F1N514294
Uncharacterised protein (uncharacterised)G3MXG6294
Complement component C9Q3MHN2287
Complement component C6F1MM86386
Uncharacterised protein (uncharacterised)G5E5V1286
Hemoglobin subunit alphaP01966285
Uncharacterised protein (uncharacterised)G3N028275
Fibrinogen alpha chainA5PJE3174
VitronectinQ3ZBS7273
Alpha-1B-glycoproteinQ2KJF1166
Coagulation factor XIIF1MTT3165
Uncharacterised protein (uncharacterised)F1MSF0263
ProthrombinP00735161
Uncharacterised protein [Nidogen 1]F1MWN3157
Protein HP-25 homolog 2Q2KIU3157
ClusterinF1MWI1155
Contactin-1F1MVI0154
Polymeric immunoglobulin receptorF1MR22251
Uncharacterised protein [Sperm associated antigen 9]F1MZ69251
Uncharacterised protein [Ankyrin repeat domain 11]E1BAT5251
Beta-2-glycoprotein 1A0A140T843150
Carboxypeptidase N catalytic chainQ2KJ83149
Uncharacterised protein [complement component 8, alpha polypeptide (C8A)]F1MX87148
Fibrinogen gamma-B chainF1MGU7148
Uncharacterised protein [ADAM metallopeptidase with thrombospondin type 1 motif, 13]F1MVP0147
Uncharacterised protein [Talin 1]F1MDH3146
Complement C1s subcomponentQ0VCX1146
Uncharacterised protein [CL43—Collectin-43]F1MFY6145
Complement component C7F1N045144
CLEC11A proteinA5D7L1143
Uncharacterised protein [KCTD12—Potassium channel tetramerization domain containing 12]G3N3D4143
Uncharacterised protein [BRIP1—BRCA1 interacting protein C-terminal helicase 1]E1BNG9139
Cadherin-1F1N619139
Ubiquitin carboxyl-terminal hydrolase isozyme L5Q9XSJ0138
CPN2 proteinA6QP30137
Acyl-coenzyme A thioesterase THEM4A1A4L1137
Uncharacterised protein [SLAMF9—SLAM family member 9]E1BNF9136
Uncharacterised protein [MED12; Mediator complex subunit 12]F1MZ95135
TransthyretinO46375133
Voltage-dependent L-type calcium channel subunit beta-3Q9MZL3133
1 Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores > 31 indicated identity or extensive homology (p < 0.05). Protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits.
Table
Table 2. Deiminated proteins in serum-EVs of cow (Bos taurus) as identified by F95-enrichment. Deiminated proteins from serum-EVs were isolated by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analysed by LC-MS/MS and peak list files submitted to Mascot. Bos taurus species-specific peptide sequence hits are listed, showing number of sequences for protein hits and total score. Rows highlighted in pink indicate protein hits identified in serum-EVs only. Uncharacterised proteins based on LC-MS/MS search were further confirmed in STRING, based on the protein symbol, and protein identification from STRING, where retrieved, is shown in brackets ([]).For further full LC-MS/MS analysis of all protein hits see Supplementary Table S2.
Table 2. Deiminated proteins in serum-EVs of cow (Bos taurus) as identified by F95-enrichment. Deiminated proteins from serum-EVs were isolated by immunoprecipitation using the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analysed by LC-MS/MS and peak list files submitted to Mascot. Bos taurus species-specific peptide sequence hits are listed, showing number of sequences for protein hits and total score. Rows highlighted in pink indicate protein hits identified in serum-EVs only. Uncharacterised proteins based on LC-MS/MS search were further confirmed in STRING, based on the protein symbol, and protein identification from STRING, where retrieved, is shown in brackets ([]).For further full LC-MS/MS analysis of all protein hits see Supplementary Table S2.
Protein NameSymbolSequencesTotal Score (p < 0.05) 1
SerotransferrinG3X6N3754958
SerotransferrinQ29443744941
Complement factor HQ28085854700
Alpha-2-macroglobulinQ7SIH1774633
Serum albuminA0A140T897764604
Complement C3Q2UVX4834258
Uncharacterised protein (CFH—Complement factor H)F1MC45613469
FibronectinG5E5A9562492
Uncharacterised protein (Ceruloplasmin)F1N076371994
Uncharacterised protein (uncharacterised)F1MJK3381870
Uncharacterised protein (C4A—Complement C4)E1BH06351840
C4b-binding protein alpha chainQ28065361761
Keratin, type II cytoskeletal 5M0QVZ6351750
HemopexinQ3SZV7301594
Keratin, type I cytoskeletal 14F1MC11291592
Uncharacterised protein (uncharacterised)G3N0V0251542
Histidine-rich glycoproteinF1MKS5261407
Uncharacterised protein (keratin 33A (KRT33A))F1MXG6221311
KRT33A proteinA5PJJ1221306
Keratin 31Q148I8221291
Uncharacterised protein (uncharacterised)G3X7F3201288
Keratin, type II cuticular Hb1Q148H4251225
Uncharacterised protein (keratin 34 (KRT34))F1MSA6211209
Kininogen-1A0A140T8C8211188
Kininogen-1P01044211164
Uncharacterised protein (Desmoplakin)E1BKT9271156
Uncharacterised protein (keratin 86 (KRT86))E1B898221104
Apolipoprotein A-I preproproteinV6F9A2221101
Uncharacterised protein (uncharacterised)F1MVK1201066
Uncharacterised protein (KRT6A—Keratin, type II cytoskeletal 59 kDa, component IV)M0QVY0211049
Kininogen-2P01045191042
Uncharacterised protein (KRT3—Keratin, type II cytoskeletal 68 kDa, component IB)G3MXL3211034
Junction plakoglobinQ8SPJ121987
Keratin, type II cytoskeletalQ08D9119971
Uncharacterised protein (Immunoglobulin heavy constant mu)G5E5T516938
Keratin, type I cytoskeletal 17A0A140T86717890
Uncharacterised protein (IGLL1—immunoglobulin lambda-like polypeptide 1)F1MCF812886
Uncharacterised protein (uncharacterised)F1MH4013872
Uncharacterised protein (uncharacterised)F1MZ9614863
Keratin 10 (Epidermolytic hyperkeratosis; keratosis palmaris et plantaris)A6QNZ715854
Uncharacterised protein (uncharacterised)F1MLW710849
Complement C4P0103016826
Uncharacterised protein (Immunoglobulin heavy constant mu)G5E51315810
KRT15 proteinQ17QL713734
Uncharacterised protein (uncharacterised)G3N0S914631
Keratin, type I cytoskeletal 19P0872812625
KRT4 proteinA4IFP212623
SERPIND1 proteinA6QPP212606
Uncharacterised protein (uncharacterised)F1MLW87588
Histidine-rich glycoproteinP334339566
Inter-alpha-trypsin inhibitor heavy chain H2F1MNW412554
Inter-alpha-trypsin inhibitor heavy chain H4F1MMD710521
Vitamin D-binding proteinF1N5M212517
Antithrombin-IIIF1MSZ611492
PlasminogenP0686814485
Apolipoprotein A-IVQ32PJ211475
Serpin A3-3G3N1U49442
Complement factor BP811879429
C1QC proteinQ1RMH58428
Complement C1q subcomponent subunit BQ2KIV97413
Uncharacterised protein (serotransferrin-like)E1BI828411
Uncharacterised protein (KRT16—Keratin 16)G3X7W89407
Complement C1q subcomponent subunit AQ5E9E37401
Serpin A3-4A2I7N09383
Alpha-1B-glycoproteinQ2KJF17381
Uncharacterised protein (uncharacterised)G3N3Q35367
Uncharacterised protein (Serpin A3-5)G8JKW77366
Keratin, type II cytoskeletal 78A6QNX57356
Primary amine oxidase, liver isozymeQ294378352
Serpin A3-2A2I7M97333
Uncharacterised protein (uncharacterised)F1MW796330
Uncharacterised protein (uncharacterised)G3N1H55325
Uncharacterised protein (KRT77—Keratin 77)G3MYU26310
Uncharacterised protein (uncharacterised)E1B8057309
Uncharacterised protein (uncharacterised)G3MXG64305
Uncharacterised protein (uncharacterised)G3MXD95300
Uncharacterised protein (uncharacterised)G3N2P65292
Uncharacterised protein (insulin-like growth factor binding protein, acid labile subunit (IGFALS))F1MJZ45286
AdiponectinQ3Y5Z35282
Alpha-2-antiplasminP288007271
Protein AMBPF1MMK96268
VitronectinQ3ZBS75264
Uncharacterised protein (uncharacterised)G5E6045254
Desmoglein-1F1MIW87252
Fibrinogen gamma-B chainF1MGU75252
Uncharacterised protein (uncharacterised)G3MY715251
Annexin A2P042725244
Fibrinogen alpha chainA5PJE35243
Uncharacterised protein (uncharacterised)G3MWT13241
Glyceraldehyde-3-phosphate dehydrogenaseP100966238
Serpin A3-7A0A0A0MP925218
Plakophilin-1Q281614216
Inter-alpha-trypsin inhibitor heavy chain H1Q0VCM55213
Hemoglobin subunit betaP020704212
Alpha-1-antiproteinaseP349554209
Keratin, type IIA0JND24198
Actin, cytoplasmic 1F1MRD05197
Keratin, type I cytoskeletal 28Q148H64194
Thrombospondin-1F1N3A16192
Keratin, type I cytoskeletal 20F1MPK14191
ECM1 proteinA5PJT75188
Uncharacterised protein (Bos taurus immunoglobulin lambda-like polypeptide 1 (IGLL1))G3N2D73186
Selenoprotein PP499075184
Uncharacterised protein (uncharacterised)A0A0A0MPA04176
GelsolinF1MJH14175
LactotransferrinP246274169
Fibrinogen beta chainF1MAV05162
Uncharacterised protein (uncharacterised)G5E5H23156
Protein HP-20 homologQ2KIT03151
Uncharacterised protein (TGM1—Transglutaminase 1)F1MBB72143
Actin, gamma-enteric smooth muscleF1MKC44143
Uncharacterised protein (CD5L—CD5 molecule-like)F1N5144131
Complement component C7F1N0454128
Uncharacterised protein (APOB—Apolipoprotein B)E1BNR04119
Arginase-1Q2KJ643119
Alpha-S1-caseinCASA1_BOVIN 2115
Uncharacterised protein (uncharacterised)G3N0282114
Alpha-2-HS-glycoproteincRAPR1|FETUA_BOVIN2106
Paraoxonase 1Q2KIW13106
Beta-2-glycoprotein 1A0A140T843297
Uncharacterised protein (uncharacterised)G5E5V1289
Uncharacterised protein (Histone H2B family)E1B7N8288
Complement component 1, r subcomponentA5D9E9383
Histone H2AA0A0A0MP90280
Uncharacterised protein (SLK-STE20-like kinase)G3X696380
ProthrombinP00735279
Peroxiredoxin-2Q9BGI3379
Uncharacterised protein (uncharacterised)G3MZE0169
Uncharacterised protein (complement component 8, alpha polypeptide (C8A))F1MX87169
Uncharacterised protein (C1orf68—Chromosome 1 open reading frame 68)G3N3D3268
Complement C5a anaphylatoxinF1MY85263
Uncharacterised protein (complement factor I (CFI))F1N4M7161
Uncharacterised protein (armadillo repeat gene deleted in velocardiofacial syndrome (ARVCF))E1BPV1259
Uncharacterised protein (NEK10-Uncharacterised protein; NIMA-related kinase 10)E1BHZ1258
Keratin associated protein 13-1A1A4M9257
Uncharacterised protein (uncharacterised)F1MSF0257
Histone H4E1B9M9156
Gamma-glutamylcyclotransferaseQ32LE4155
Uncharacterised protein (JAKMIP2—Janus kinase and microtubule interacting protein 2)G5E551254
TOX high mobility group box family member 4Q0P5K4153
Uncharacterised protein (TBC1D32—TBC1 domain family, member 32)F1N7V1251
Proteasome subunit alpha type-6Q2YDE4150
Uncharacterised protein (KIAA1009 ortholog)F1MZ01250
Uncharacterised protein (MYO5B—Myosin VB)F1MMQ6250
Uncharacterised protein (Phospholipid phosphatase related 4)F1MJ26249
Uncharacterised protein (MGA—MGA, MAX dimerization protein)E1BKB7249
Uncharacterised protein (GOLGB1—Golgin B1)E1BKZ5247
Histone H3E1BGN3146
Uncharacterised protein (CROCC—Ciliary rootlet coiled-coil, rootletin)E1BBS9346
Protein FAM149B1A0JNF3245
Alpha-enolaseF1MB08245
Uncharacterised protein (UBR4—Ubiquitin protein ligase E3 component n-recognin 4)E1BHT5345
Complement component C9Q3MHN2144
Uncharacterised protein (DOCK8—Bos taurus dedicator of cytokinesis 8 (DOCK8))E1BNA6244
Uncharacterised protein (KPRP—Keratinocyte proline-rich protein)E1BLN6144
Uncharacterised protein (CCDC14—Coiled-coil domain containing 14)F1MS02243
Uncharacterised protein (Strawberry notch homolog 1(Drosophila))E1BMP8342
DNA-directed RNA polymerase subunit betaA5PJW8142
Uncharacterised protein (CRYGN-Crystallin, gamma N)E1BDQ1142
Flotillin-1Q08DN8141
Uncharacterised protein (SMG6—Uncharacterised protein; Bos taurus smg-6 homolog, nonsense mediated mRNA decay factor (C. elegans) (SMG6))E1BFK4241
C1QTNF3 proteinA7MB82141
Uncharacterised protein (Protocadherin gamma subfamily B, 1)F1MCA2140
Uncharacterised protein (Death-associated protein kinase 1)F1MRL0139
Acyl-coenzyme A thioesterase THEM4A1A4L1137
Uncharacterised protein (Centrosomal protein 104kDa)E1BND2137
C8G proteinA8YXZ2136
Uncharacterised protein (SERPINB12—Serpin family B member 12)E1BDF5136
Uncharacterised protein (SLAMF9 SLAM family member 9)E1BNF9136
Complement C1s subcomponentQ0VCX1136
Uncharacterised protein (methyltransferase like 3 (METTL3))F1MX80135
Uncharacterised protein (SPAG9—Sperm associated antigen 9)F1MZ69135
AdenosylhomocysteinaseQ3MHL4135
Uncharacterised protein (HIVEP3—Human immunodeficiency virus type I enhancer binding protein 3)F1MBK6134
Coagulation factor VF1N0I3133
Uncharacterised protein (VPREB1—Uncharacterised protein)F1N160133
Uncharacterised protein (SERPING1)E1BMJ0133
PPARD proteinA4IFL4 32
Uncharacterised proteinF1MZ93132
Uncharacterised protein (NCBP1—Nuclear cap binding protein subunit 1)E1BMM0132
TransthyretinO46375132
Uncharacterised protein (Cryptochrome-1)F1MXB2132
Tubulin alpha chainF1MNF8131
1 Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores > 31 indicated identity or extensive homology (p < 0.05). Protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits.

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Criscitiello, M.F.; Kraev, I.; Lange, S. 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. Int. J. Mol. Sci. 2020, 21, 2861. https://doi.org/10.3390/ijms21082861

AMA Style

Criscitiello MF, Kraev I, Lange S. 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. International Journal of Molecular Sciences. 2020; 21(8):2861. https://doi.org/10.3390/ijms21082861

Chicago/Turabian Style

Criscitiello, Michael F., Igor Kraev, and Sigrun Lange. 2020. "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" International Journal of Molecular Sciences 21, no. 8: 2861. https://doi.org/10.3390/ijms21082861

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