Post-Translational Deimination of Immunological and Metabolic Protein Markers in Plasma and Extracellular Vesicles of Naked Mole-Rat (Heterocephalus glaber)

Naked mole-rats are long-lived animals that show unusual resistance to hypoxia, cancer and ageing. Protein deimination is an irreversible post-translational modification caused by the peptidylarginine deiminase (PAD) family of enzymes, which convert arginine into citrulline in target proteins. Protein deimination can cause structural and functional protein changes, facilitating protein moonlighting, but also leading to neo-epitope generation and effects on gene regulation. Furthermore, PADs have been found to regulate cellular release of extracellular vesicles (EVs), which are lipid-vesicles released from cells as part of cellular communication. EVs carry protein and genetic cargo and are indicative biomarkers that can be isolated from most body fluids. This study was aimed at profiling deiminated proteins in plasma and EVs of naked mole-rat. Key immune and metabolic proteins were identified to be post-translationally deiminated, with 65 proteins specific for plasma, while 42 proteins were identified to be deiminated in EVs only. Using protein-protein interaction network analysis, deiminated plasma proteins were found to belong to KEEG (Kyoto Encyclopedia of Genes and Genomes) pathways of immunity, infection, cholesterol and drug metabolism, while deiminated proteins in EVs were also linked to KEEG pathways of HIF-1 signalling and glycolysis. The mole-rat EV profiles showed a poly-dispersed population of 50–300 nm, similar to observations of human plasma. Furthermore, the EVs were assessed for three key microRNAs involved in cancer, inflammation and hypoxia. The identification of post-translational deimination of critical immunological and metabolic markers contributes to the current understanding of protein moonlighting functions, via post-translational changes, in the longevity and cancer resistance of naked mole-rats.

. Peptidylarginine deiminases (PADs) and deiminated proteins in naked mole-rat plasma and plasma-extracellular vesicles (EVs). (A) PAD positive bands were identified at the expected size of approximately 70-75 kDa using the human PAD2, PAD3 and PAD4 specific antibodies in naked mole-rat plasma. (B) Total deiminated proteins were identified in naked mole-rat plasma (n = 4) using the F95 pan-deimination specific antibody. (C) Total deiminated proteins were identified in naked mole-rat plasma-EVs using the F95 pan-deimination specific antibody (EV pools from plasma of 4 individuals are shown, respectively). (D) The F95-enriched IP fraction from mole-rat plasma (from a pool of 5 individual mole-rat plasma; F95_IP) is shown. The molecular weight marker is indicated next to each blot.

Deiminated Protein Profiles of Naked Mole-Rat Plasma and Plasma-Derived EVs
Total deiminated proteins were detected by Western blotting with the pan-deimination F95 antibody in mole-rat plasma and plasma-derived EVs, revealing a range of proteins mainly between 50-150 kDa ( Figure 1B). The mono-specific F95 antibody was used in this study for the identification of deiminated proteins, as it has been developed against a deca-citrullinated peptide and is predicted to react with all deiminated/citrullinated proteins based on 100% sequence homology (MABN328 Merck), and it has been used to identify deiminated proteins in human and animals from diverse taxa [11,12,16,18,19,21,22,31]. Deiminated proteins in mole-rat were also detected in the plasma-derived EVs, mainly in the size range of 20-100 kDa ( Figure 1C). Deiminated protein candidates in plasma and EVs were further identified by F95 enrichment (see F95 enriched fraction from plasma assessed by Western blotting, Figure 1D) and LC-MS/MS analysis (Tables 1 and 2;  Supplementary Tables S1 and S2). In plasma, 112 species-specific protein hits were identified (Table  1 and Supplementary Table S1) while in EVs, 80 protein hits were identified (Table 2 and  Supplementary Table S2). Overall, 48 proteins overlapped between plasma and plasma-derived EVs, while 65 proteins were specific for whole plasma only and 42 proteins for EVs only (Figure 2). The protein lists for deiminated proteins identified in naked mole-rat plasma and plasma-EVs were submitted to STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis (https://string-db.org/) to predict putative protein-protein interaction networks (Figures 3 and 4). Table 1. Deiminated proteins identified by F95 enrichment in total plasma of naked mole-rat (Heterocephalus glaber). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95 enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Species-specific peptide sequence hits scoring with H. glaber are

Deiminated Protein Profiles of Naked Mole-Rat Plasma and Plasma-Derived EVs
Total deiminated proteins were detected by Western blotting with the pan-deimination F95 antibody in mole-rat plasma and plasma-derived EVs, revealing a range of proteins mainly between 50-150 kDa ( Figure 1B). The mono-specific F95 antibody was used in this study for the identification of deiminated proteins, as it has been developed against a deca-citrullinated peptide and is predicted to react with all deiminated/citrullinated proteins based on 100% sequence homology (MABN328 Merck), and it has been used to identify deiminated proteins in human and animals from diverse taxa [11,12,16,18,19,21,22,31]. Deiminated proteins in mole-rat were also detected in the plasma-derived EVs, mainly in the size range of 20-100 kDa ( Figure 1C). Deiminated protein candidates in plasma and EVs were further identified by F95 enrichment (see F95 enriched fraction from plasma assessed by Western blotting, Figure 1D) and LC-MS/MS analysis (Tables 1 and 2; Supplementary Tables S1  and S2). In plasma, 112 species-specific protein hits were identified (Table 1 and Supplementary  Table S1) while in EVs, 80 protein hits were identified (Table 2 and Supplementary Table S2). Overall, 48 proteins overlapped between plasma and plasma-derived EVs, while 65 proteins were specific for whole plasma only and 42 proteins for EVs only (Figure 2). The protein lists for deiminated proteins identified in naked mole-rat plasma and plasma-EVs were submitted to STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis (https://string-db.org/) to predict putative protein-protein interaction networks (Figures 3 and 4).  G5BNV2_HETGA  1167  Antithrombin-III  G5ARS6_HETGA  1093  Coagulation factor V  G5CB46_HETGA  1092  Complement C5  G5AXS5_HETGA  1002  Hemoglobin subunit beta  G5BS33_HETGA  941  Transcobalamin-2 isoform 1  G5AVP0_HETGA  913  Hemopexin  G5BBR0_HETGA  899  Coagulation factor XIII B chain  G5BM72_HETGA  821  Protein AMBP  G5B1Y4_HETGA  817  N-acetylmuramoyl-L-alanine amidase  G5BYP3_HETGA  812  Coagulation factor XII  G5BQ09_HETGA  802  Apolipoprotein A-I  APOA1_HETGA  798  Hemoglobin subunit alpha  G5BXY1_HETGA  784  Coagulation factor XIII A chain  G5BAS8_HETGA  779  Hemoglobin subunit beta  G5BYJ8_HETGA  759  Alpha-1-antiproteinase S  G5B496_HETGA  724  Inter-alpha-trypsin inhibitor heavy chain H3  G5BUN3_HETGA  682  Vitronectin  G5BVN8_HETGA  652  Complement factor I  G5AQM1_HETGA  647  Hemoglobin subunit epsilon-1  G5BS35_HETGA  642  Inter-alpha-trypsin inhibitor heavy chain H2  G5AXV8_HETGA  584  Apolipoprotein E  G5CBM7_HETGA  581  Inhibitor of carbonic anhydrase  G5BQB0_HETGA  537  Inter-alpha-trypsin inhibitor heavy chain H1  G5BUN2_HETGA  482  Four and a half LIM domains protein 1  G5CA61_HETGA  469  Haptoglobin  G5B5U6_HETGA  464  C4b-binding protein  G5BP10_HETGA  438  L-lactate dehydrogenase  G5AKA3_HETGA  437  Insulin-like growth factor-binding protein complex acid labile chain  G5BY64_HETGA  403  Catalase  G5AXV0_HETGA  382  Fetuin-B  G5BT88_HETGA  347  Alpha-2-HS-glycoprotein  G5BT89_HETGA  337  Selenoprotein P  G5APA7_HETGA  328  Vitamin D-binding protein  G5BE53_HETGA  317  Adiponectin  G5BT83_HETGA  283  von Willebrand factor  G5CAN6_HETGA  274  Beta-2-glycoprotein 1  G5BGY7_HETGA  268  Basement membrane-specific heparan sulfate proteoglycan core protein  G5BI06_HETGA  267  Gelsolin  G5AXS0_HETGA  259  Aspartyl aminopeptidase  G5AKJ4_HETGA  256  Complement C1q subcomponent subunit A  G5BHZ8_HETGA  246  Ficolin-3  G5AUT5_HETGA  241  Mannan-binding lectin serine protease 1  G5BTD5_HETGA  228  Alpha-1-antichymotrypsin G5B491_HETGA 228   Collagen alpha-2(I) chain G5ANK8_HETGA 31 Bcl-2-associated transcription factor 1 G5C1A7_HETGA 30 # Ions score is −10 * Log(p), where p is the probability that the observed match is a random event. Individual ions scores > 30 indicated identity or extensive homology (p < 0.05). Protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. Cut-off was set at Ions score 30. Protein hits identified in whole plasma only (but not in plasma EVs) are highlighted in blue.   Deiminated proteins identified in naked mole-rat plasma and plasma-EVs. Species specific hits identified for deiminated proteins in naked mole-rat plasma and EVs showed 112 total proteins identified in plasma and 80 in EVs, respectively. Of these, 48 protein hits were overlapping, while 64 proteins were specific for whole plasma and 32 for plasma-EVs only, respectively.

Characterisation of Extracellular Vesicles in Naked Mole-Rat Plasma
EVs isolated from naked mole-rat plasma were characterised for size distribution using nanoparticle tracking analysis (NTA; Figure 5A), by Western blotting using EV-specific markers ( Figure 5B) and by morphological analysis using transmission electron microscopy (TEM; Figure 5C). A poly-dispersed population of EVs mainly in the size range of 50 to 300 nm was observed, with some variations between individual animals ( Figure 5A), and also with respect to EV yield from plasma ( Figure 5D) and modal EV size ( Figure 5E). Overall, the main EV peaks were detected at approximately 100-140 nm ( Figure 5A,E), with the modal size of EVs falling mainly in the range of 90-115 nm, although this varied somewhat between animals and some outliers were detected ( Figure 5E). Western blotting confirmed that the naked mole-rat EVs were positive for the EV-specific markers CD63 and Flot-1 ( Figure 5B). Typical EV morphology was confirmed by TEM ( Figure 5C).

Characterisation of Extracellular Vesicles in Naked Mole-Rat Plasma
EVs isolated from naked mole-rat plasma were characterised for size distribution using nanoparticle tracking analysis (NTA; Figure 5A), by Western blotting using EV-specific markers ( Figure 5B) and by morphological analysis using transmission electron microscopy (TEM; Figure  5C). A poly-dispersed population of EVs mainly in the size range of 50 to 300 nm was observed, with some variations between individual animals ( Figure 5A), and also with respect to EV yield from plasma ( Figure 5D) and modal EV size ( Figure 5E). Overall, the main EV peaks were detected at approximately 100-140 nm ( Figure 5A,E), with the modal size of EVs falling mainly in the range of 90-115 nm, although this varied somewhat between animals and some outliers were detected ( Figure 5E). Western blotting confirmed that the naked mole-rat EVs were positive for the EV-specific markers CD63 and Flot-1 ( Figure 5B). Typical EV morphology was confirmed by TEM ( Figure 5C).

MicroRNA Analysis of Naked Mole-Rat EVs
EVs isolated from naked mole-rat plasma were assessed for the relative expression of the immune-and cancer-related miR21, the inflammatory-related miR155 and the metabolic-and hypoxia-related miR210. The highest relative miR levels of the three miRs tested, were observed for miR21 in naked mole-rat plasma-EVs, 394-fold higher than for miR155 and 153-fold higher than for miR210, respectively ( Figure 6A). The relative expression of the hypoxia-and metabolic-related miR210 was 2.6-fold higher than the inflammatory miR155 ( Figure 6B), which overall showed comparatively the lowest relative levels of expression of the three miRs tested ( Figure 6A,B).

MicroRNA Analysis of Naked Mole-Rat EVs
EVs isolated from naked mole-rat plasma were assessed for the relative expression of the immuneand cancer-related miR21, the inflammatory-related miR155 and the metabolic-and hypoxia-related miR210. The highest relative miR levels of the three miRs tested, were observed for miR21 in naked mole-rat plasma-EVs, 394-fold higher than for miR155 and 153-fold higher than for miR210, respectively ( Figure 6A). The relative expression of the hypoxia-and metabolic-related miR210 was 2.6-fold higher than the inflammatory miR155 ( Figure 6B), which overall showed comparatively the lowest relative levels of expression of the three miRs tested ( Figure 6A,B). . MicroRNA (miR) analysis of three key miRs related to cancer, inflammation, metabolism and hypoxia, in naked mole-rat plasma-derived EVs. A. The onco-related miR21 showed the highest relative expression of the three miRs tested, being 394-fold higher than miR155 and 153-fold higher than miR210, respectively (p < 0.0001; n = 3). B. The relative expression of the hypoxia and metabolic related miR210 was 2.6-fold higher than the inflammatory miR155 (p = 0.0002; n = 3); (miR21, miR155 and miR210 expression is represented as red circles, orange squares and blue triangles, respectively).

Discussion
For the first time, post-translationally deiminated proteins are described in naked mole-rat (Heterocephalus glaber) plasma, unravelling novel aspects of post-translational deimination of key proteins involved in immune defences and metabolism. PAD homologues were identified in naked mole-rat plasma by Western blotting via cross reaction with human PAD2, which is the phylogenetically most conserved form of PAD [1,16,31]. PAD-positive protein bands were also observed for PAD3 and PAD4, via cross-reaction with PAD isozyme-specific human antibodies, at an expected size of approximately 70 kDa, similar to that reported for other mammalian PADs. Such protein detection corresponds with PAD isozymes that have been identified in the naked mole-rat genome (PADI1, Gene ID: 101722077; PADI2, Gene ID: 101721485; PADI3, Gene ID: 101722435; PADI4, Gene ID: 101722785; PADI6, Gene ID: 101723122). Deiminated proteins were detected and identified both in whole plasma and in plasma-derived EVs. The identity of specific deimination protein candidates in plasma and plasma-EVs was assessed using F95-enrichment in tandem with LC-MS/MS analysis. The F95 mono-specific antibody used in the current study was developed against a deca-citrullinated peptide and has been verified to detect deiminated/citrullinated proteins in diverse taxa [11,12,16,18,19,21,22,31]. In the naked mole-rat, species-specific protein hits revealed 48 common deiminated proteins in plasma and EVs by F95-enrichment, which included some key immune and metabolic related proteins, while 65 deiminated protein hits were specific for plasma and 42 deiminated protein hits specific for EVs only.
As assessed by STRING analysis, the PPI enrichment p-value for deiminated proteins identified in naked mole-rat plasma and as cargo in plasma-derived EVs was found to be < 1.0 × 10 −16 for both. Such an enrichment value indicates that the identified network of proteins has significantly more interactions than expected. Therefore, these deiminated proteins have more interactions among themselves than would be expected for a random set of proteins of similar size, drawn from the genome. Such an enrichment indicates that the proteins, as a group, are at least partially biologically connected. Deiminated target proteins identified in whole naked mole-rat plasma belonged to the following KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways: complement coagulation cascade, platelet activation, cholesterol metabolism, vitamin digestion and adsorption, drug metabolism as well as bacterial infection (Staphylococcus aureus and pertussis), autoimmunity (systemic lupus erythematosus) and prion diseases ( Figure 3B). Deiminated target proteins Figure 6. MicroRNA (miR) analysis of three key miRs related to cancer, inflammation, metabolism and hypoxia, in naked mole-rat plasma-derived EVs. (A). The onco-related miR21 showed the highest relative expression of the three miRs tested, being 394-fold higher than miR155 and 153-fold higher than miR210, respectively (p < 0.0001; n = 3). (B). The relative expression of the hypoxia and metabolic related miR210 was 2.6-fold higher than the inflammatory miR155 (p = 0.0002; n = 3); (miR21, miR155 and miR210 expression is represented as red circles, orange squares and blue triangles, respectively).

Discussion
For the first time, post-translationally deiminated proteins are described in naked mole-rat (Heterocephalus glaber) plasma, unravelling novel aspects of post-translational deimination of key proteins involved in immune defences and metabolism. PAD homologues were identified in naked mole-rat plasma by Western blotting via cross reaction with human PAD2, which is the phylogenetically most conserved form of PAD [1,16,31]. PAD-positive protein bands were also observed for PAD3 and PAD4, via cross-reaction with PAD isozyme-specific human antibodies, at an expected size of approximately 70 kDa, similar to that reported for other mammalian PADs. Such protein detection corresponds with PAD isozymes that have been identified in the naked mole-rat genome (PADI1, Gene ID: 101722077; PADI2, Gene ID: 101721485; PADI3, Gene ID: 101722435; PADI4, Gene ID: 101722785; PADI6, Gene ID: 101723122). Deiminated proteins were detected and identified both in whole plasma and in plasma-derived EVs. The identity of specific deimination protein candidates in plasma and plasma-EVs was assessed using F95-enrichment in tandem with LC-MS/MS analysis. The F95 mono-specific antibody used in the current study was developed against a deca-citrullinated peptide and has been verified to detect deiminated/citrullinated proteins in diverse taxa [11,12,16,18,19,21,22,31]. In the naked mole-rat, species-specific protein hits revealed 48 common deiminated proteins in plasma and EVs by F95-enrichment, which included some key immune and metabolic related proteins, while 65 deiminated protein hits were specific for plasma and 42 deiminated protein hits specific for EVs only.
As assessed by STRING analysis, the PPI enrichment p-value for deiminated proteins identified in naked mole-rat plasma and as cargo in plasma-derived EVs was found to be <1.0 × 10 −16 for both. Such an enrichment value indicates that the identified network of proteins has significantly more interactions than expected. Therefore, these deiminated proteins have more interactions among themselves than would be expected for a random set of proteins of similar size, drawn from the genome. Such an enrichment indicates that the proteins, as a group, are at least partially biologically connected. Deiminated target proteins identified in whole naked mole-rat plasma belonged to the following KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways: complement coagulation cascade, platelet activation, cholesterol metabolism, vitamin digestion and adsorption, drug metabolism as well as bacterial infection (Staphylococcus aureus and pertussis), autoimmunity (systemic lupus erythematosus) and prion diseases ( Figure 3B). Deiminated target proteins identified in EVs of naked mole-rat plasma belonged to KEGG pathways of the complement coagulation cascade, platelet activation, HIF-(hypoxia-inducible factor) signalling pathway, glycolysis/gluconeogenesis, cholesterol metabolism, vitamin digestion and adsorption, oestrogen signalling pathway, as well as bacterial infection (Staphylococcus aureus) and autoimmunity (systemic lupus erythematosus) ( Figure 4B).
Of interest is that deiminated protein candidates involved in KEGG-pathways for HIF-1-signalling, the master regulator of oxygen homeostasis, seem enriched in the plasma-EVs, indicating a role for EV-mediated transport of such proteins in hypoxic signalling. It is worth noting that naked mole-rats have a high endogenous expression of HIF due to mutation in the VHL (Von Hippel-Lindau disease tumor suppressor) domain [62], which could possibly explain the elevated HIF-1 signalling related targets identified here. Furthermore, deiminated proteins identified in EVs were also enriched for glycolysis and gluconeogenesis KEGG pathways. Our findings indicate that protein deimination may play hitherto unidentified roles in the unusual hypoxia resistance and metabolism of the naked mole-rat, including via EV-transport in cellular communication, also under normal physiological conditions. In addition, the presence of deiminated histone H2B and H4 in EVs may be of some interest as histone deimination is well known to contribute to epigenetic regulation including in cancer [4,6], and the naked mole-rat has been found to have a particularly stable epigenome, which may contribute to the cancer resistance and longevity observed in these animals [63]. Furthermore, an abundance of deiminated complement components identified in both plasma and plasma-EVs may indicate roles for functional diversity of the complement system via post-translational deimination in the naked mole-rat. This may play various roles in naked mole-rat immune responses as recent studies have identified some unusual characteristics, including atypical immune surveillance and a greater reliance on myeloid-biased innate immunity [64]. Also noteworthy is the identification of deiminated adiponectin in naked mole-rat plasma identified here, as adiponectin in humans is the most abundant secreted adipokine with pleiotropic roles in metabolism [65,66], glucose regulation [67][68][69], longevity [70], regeneration and cancer [71][72][73]. Such deimination of adiponectin has not been studied and may add to some of its protein moonlighting function and be of relevance in the context of the unusual metabolism of the naked mole-rat.
As part of EV-mediated cellular communication in physiology and pathologies, the transport of microRNAs (miRs) is well acknowledged. There is increasing interest, reflected in a range of studies, in furthering our understanding of how such EV-mediated transport may play a part in physiological and pathophysiological processes. MiRs are highly conserved small non-coding RNAs that control gene expression and regulate biological processes by targeting messenger RNAs (mRNAs). MiRs can, for example, inhibit post-transcriptional translation of mRNA as well as enhance mRNA degradation [74]. Some expression profiling has been carried out in naked mole-rats, mainly at the transcriptome level [75,76], although no studies have assessed miRs in EVs of naked mole-rat plasma. This study focused on assessing three key miRs known to be involved in cancer, inflammation and hypoxia due to the unusual resistance of naked mole-rats to cancer, ageing and hypoxia. MiR21 is a main immunoregulatory and onco-related miR and is also associated with chronic diseases [77][78][79]. MiR21 is strongly conserved throughout evolution and while many experimentally verified targets of miR21 are tumour suppressors, miR21 is also linked to cardiac disease and oxidative stress [80]. Less is known about the physiological roles of miR21. In the current study, miR21 was found to be by far the highest miR expressed in EVs of naked mole-rat out of the three miRs tested. Roles for miR21 in immune responses of naked mole-rat have not been reported in detail and the expression of miR21 in EVs has not been assessed in naked mole-rat before.
In mammals, miR155 is known to be a major inflammatory related miR, linked to inflammatory and stress responses [81]. Here, miR155 was found to be the least expressed of the three miRs tested in naked mole-rat plasma-EVs, possibly indicating that this miR may be a contributing factor to the "anti-inflammatory" state of mole-rats, which may have some relation to their longevity and cancer resistance. MiR210 is known to be a major miR induced under hypoxia and has an important role in mitochondrial metabolism, DNA damage response, cell proliferation and apoptosis [74]. MiR210 has an important role in regulating mitochondrial metabolism [82] and cell glycolytic activity, as well as being linked to inflammation [83]. MiR210 has been identified as a regulator of the hypoxia pathway and was found to have pro-apoptotic functions under normoxic conditions, but anti-apoptotic effects under hypoxic conditions [84,85]. In the current study, miR210 was found to be more highly expressed in mole-rat plasma than the inflammatory miR155. As naked mole-rats are known to be hypoxia tolerant animals and to exhibit marked changes in their metabolic substrate use and metabolic demand in hypoxia [41,86], miR210 may have functional roles in metabolic control, possibly contributing to the well-known longevity of these animals. As this is the first study to assess the expression of these three onco-, inflammatory-and metabolic-related miRs in naked mole-rat plasma-EVs, it remains to be fully understood what specific functions the EV-mediated transport of these miRs play in the unusual physiology of naked mole-rats.
Here, for the first time, we report the protein deimination profiles of plasma and plasma-derived EVs in naked mole-rats. Post-translational deimination of major key immune and metabolic factors in naked mole-rats was identified and related to key KEGG pathways of inflammation, metabolism and oxygen transport. Our findings highlight novel aspects of protein moonlighting via post-translational deimination, including via EV-mediated transport. Research on EVs is a relatively new field in comparative animal models, and to our knowledge this is the first characterisation of EVs and associated protein and selected miR cargo markers in naked mole-rats. Furthermore, as PADs have been found to play major roles in the regulation of EV release [19][20][21][22]34], their contribution to EV-mediated cell communication in response to physiological and pathophysiological changes in naked mole-rats remains to be further investigated. Findings in long-lived mammals that display cancer resistance, including naked mole-rats, may be of considerable translational value for furthering our understanding of the mechanisms underlying cancer resistance for improved development of human cancer therapies [59].
In continuation of the current study, the assessment of changes in deiminated proteins and EV profiles, including protein and genetic EV-cargo, may be of great interest in studies using this unique animal model to further understanding of the hitherto novel and understudied mechanisms involved in cancer and ageing.

Sampling of Naked Mole-Rat Plasma
Naked mole-rats were group-housed in interconnected multi-cage systems at 30 • C and 21% O 2 in 50% humidity with a 12L:12D light cycle. Animals were fed fresh tubers, vegetables, fruit and Pronutro cereal supplement ad libitum. Animals were not fasted prior to experimental trials. All experimental procedures were approved by the University of Ottawa Animal Care Committee in accordance with the Animals for Research Act and by the Canadian Council on Animal Care (protocol # 2535). Non-breeding (subordinate) naked mole-rats do not undergo sexual development or express sexual hormones and thus we did not take sex into consideration when evaluating our results [87]. Blood was collected from 12 adult (~1-2 years old) subordinate naked mole-rats following live cervical dislocation and rapid decapitation. Blood was collected in Eppendorf tubes pre-coated with a 10% EDTA (ethylenediaminetetraacetic acid) solution. Plasma was isolated by centrifugation at 5000× g for 5 min. The isolated plasma was aliquoted and immediately frozen at −80 • C until further use.

Extracellular Vesicle Isolation and Nanoparticle Tracking Analysis (NTA)
EVs were isolated by step-wise centrifugation according to our established protocols using ultracentrifugation and the recommendations of MISEV2018 (the minimal information for studies of extracellular vesicles 2018; [88]). Mole-rat plasma were diluted 1:4 in ultra-filtered (using a 0.22 µm filter) Dulbecco's PBS (100 µL plasma added to 400 µL DPBS) and then centrifuged at 4000× g for 30 min at 4 • C for removal of aggregates and apoptotic bodies. The supernatants were collected and centrifuged further at 100,000× g for 1 h at 4 • C. The EV-enriched pellets were washed in 1 mL DPBS and ultra-centrifuged at 100,000× g for 1 h at 4 • C. The final EV pellets were resuspended in 100 µL DPBS and frozen at −80 • C until further use. For NTA, based on Brownian motion of particles in suspension, the EV pellets were diluted 1/100 in DPBS and applied to the NanoSight NS300 system (Malvern Panalytical Ltd., Malvern, UK) in conjunction with a syringe pump to ensure continuous flow of the sample. Five 60 sec videos were recorded for each sample, with approximately 40-60 particles per frame, and the replicate histograms generated were averaged.

Transmission Electron Microscopy (TEM)
EVs were isolated from individual plasma as described above. For TEM, the EV pellets were fixed with 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0) for 1 h at 4 • C. The EVs were then resuspended in 100 mM sodium cacodylate buffer (pH 7.0), placed on to a grid with a glow discharged carbon support film and stained with 2% aqueous Uranyl Acetate (Sigma-Aldrich, Gillingham, UK). Individual EVs were imaged by TEM using a Morada CCD camera (EMSIS GmbH, Münster, Germany) and processed via iTEM (EMSIS).

Immunoprecipitation and Protein Identification
Deiminated proteins in plasma and in plasma-derived EVs were immunoprecipitated by enrichment with the F95 pan-deimination antibody (MABN328, Merck, Watford, UK), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination [89]. The mono-specific F95 antibody is predicted to react with all deiminated/citrullinated proteins based on 100% sequence homology and has for example been used to identify deiminated proteins in human, mouse, rat, chicken and teleost fish tissue [11,12,16,18,19,21,22,31,89]. The Catch and Release immunoprecipitation kit (Merck, Watford, UK) was used according to the manufacturer's instructions. For F95 enrichment, plasma was pooled from 5 individual animals (5 × 20 µL), while for EVs, total protein was first extracted from the EV pellets derived from 100 µL plasma per animal, using 100 µL RIPA + buffer on ice for 2 h followed by centrifugation at 16,000× g for 30 min to collect the supernatant containing the proteins. The immunoprecipitation was carried out on a rotating platform overnight at 4 • C, and the F95 bound proteins were eluted using denaturing elution buffer according to the manufacturer's instructions (Merck). The F95 enriched eluates were then either analysed by Western blotting or by liquid chromatography with tandem mass spectrometry (LC-MS/MS; Cambridge Proteomics, Cambridge, UK). Peak files obtained were submitted to Mascot (Matrix Science). An in-house database (Cambridge proteomics) for naked mole-rat was used for the identification of species-specific protein hits (CCP_Heterocephalus_glaber Heterocephalus_glaber_20190911; 21449 sequences; 10466552 residues).

MicroRNA Analysis
EV isolates from individual naked mole-rat plasma (from 100 µL plasma as before) were assessed for relative expression of 3 key microRNAs (miRs) related to oncogenic, inflammatory and metabolic activity. These selected miRs included two cancer and immune-related miRs, miR21 and miR155, and miR210 for hypoxia and metabolic activity. Total RNA was extracted from mole-rat plasma EVs (prepared as before) using Trizol (Sigma-Aldrich, Gillingham, UK). The purity and concentration of the isolated RNA were measured using the NanoDrop Spectrophotometer at 260 nm and 280 nm absorbance. The cDNA was produced using the qScript microRNA cDNA Synthesis Kit (Quantabio, Beverly, MA, USA) according to the manufacturer's instructions and used to assess the expression of miR21, miR155 and miR210. Reference RNAs used for the normalization of miR expression levels were U6-snRNA and has-let-7a-5p. The PerfeCTa SYBR Green SuperMix (Quantabio, Beverly, MA, USA) was used together with MystiCq microRNA qPCR primers for the miR21 (hsa-miR-21-5p), mir155 (hsa-miR-155-5p) and miR210 (hsa-miR-210-5p). All miR primers were obtained from Sigma-Aldrich (UK). Thermocycling conditions were used as follows: denaturation at 95 • C for 2 min, followed by 40 cycles of 95 • C for 2 s, 60 • C for 15 s, and extension at 72 • C for 15 s. The 2 ∆∆Ct method [90] was used for calculating relative miR expression levels and for normalisation. Each experiment was performed in 3 individuals, in triplicate.

Statistical Analysis
The histograms and graphs were prepared using the Nanosigh NS300 software (Malvern Panalytical Ltd., Malvern, UK) and GraphPad Prism version 7 (GraphPad Software, San Diego, CA, USA). Experiments were repeated in triplicate, histograms represent mean of data and standard error of mean (SEM) is indicated by the error bars. Significant differences were considered as p ≤ 0.05, following one-way ANOVA or Student's t-test.

Conclusions
Here, for the first time, we report the protein deimination profiles of plasma and plasma-derived EVs in naked mole-rats. Post-translational deimination of major key immune and metabolic proteins in naked mole-rats was identified and related to key KEGG pathways of inflammation, metabolism and oxygen transport. Our findings highlight novel aspects of protein moonlighting via post-translational deimination, including via EV-mediated transport of such proteins in cellular communication. Three key microRNAs for oncogenic, inflammatory and metabolic/hypoxia function were also assessed in mole-rat plasma EVs. In continuation of the current study, the assessment of changes in deiminated proteins and EV profiles, including protein and microRNA EV-cargo may be of great interest in studies using this unique animal model to further understanding of hitherto novel and understudied mechanisms involved in cancer, inflammatory diseases and ageing.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/21/5378/ s1. Supplementary Table S1. Deiminated proteins identified by F95 enrichment in total plasma of naked mole-rat (Heterocephalus glaber). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95 enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptide sequences for the protein hits, their m/z values and individual scores are listed. Supplementary Table  S2. Deiminated proteins identified by F95 enrichment in plasma-EVs of naked mole-rat (Heterocephalus glaber). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The

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

Conflicts of Interest:
The authors declare no conflict of interest.