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Article

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

1
Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada
2
Brain and Mind Research Institute, University of Ottawa, Ottawa, ON K1H 8M5, Canada
3
Cancer Research Group, School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, London W1W 6 UW, UK
4
Electron Microscopy Suite, Faculty of Science, Technology, Engineering and Mathematics, Open University, Walton Hall, Milton Keynes MK7 6AA, UK
5
Tissue Architecture and Regeneration Research Group, School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, London W1W 6 UW, UK
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(21), 5378; https://doi.org/10.3390/ijms20215378
Received: 25 September 2019 / Revised: 17 October 2019 / Accepted: 28 October 2019 / Published: 29 October 2019

Abstract

:
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.

1. Introduction

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes that post-translationally convert arginine into citrulline in target proteins in an irreversible manner. This can cause structural changes in target proteins and affect protein function, gene regulation and generation of neoepitopes [1,2,3,4,5,6]. Such post-translational changes in proteins may also allow for protein moonlighting, an evolutionary acquired phenomenon facilitating proteins to exhibit several physiologically relevant functions from within one polypeptide chain [7,8].
PADs and associated protein deimination are crucial players in cancer, autoimmune and neurodegenerative diseases [4,5,6,9], with recent indications also for PAD-mediated mechanisms in ageing [10]. Furthermore, critical roles have been identified for PADs and PAD-mediated protein deimination in response to hypoxia and in CNS regeneration [11,12,13,14,15] as well as in tissue remodelling and immunity [16,17,18]. Importantly, PADs have been found to have key roles in the regulation of extracellular vesicle (EV) release [19,20,21,22]. EVs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material [6,23,24,25,26]. EVs isolated from serum, plasma and other body fluids can therefore be useful health biomarkers [27,28]. Hitherto, work on EVs has mainly been in the context of human pathologies and recent comparative immunology studies on EVs and EV cargo have been performed [29], including the assessment of deiminated EV protein cargo [30,31,32].
PADs have been identified throughout phylogeny from bacteria to mammals, with five tissue specific PAD isozymes in mammals, three in chicken, one in bony and cartilaginous fish [1,16,17,31,33] and PAD homologues in parasites [34], fungi [35] and bacteria [22]. While five PAD isozymes have been described in the naked mole-rat (PADI1, Gene ID: 101722077; PADI2, Gene ID: 101721485; PADI3, Gene ID: 101722435; PADI4, Gene ID: 101722785; PADI6, Gene ID: 101723122), no studies have been carried out on their deiminated protein products or the putative physiological relevance of such post-translational deimination in the physiology of the naked mole-rat.
The naked mole-rat is a burrowing rodent and eusocial mammal, and the only species in the genus Heterochephalus of the family Heterochepalidae [36,37]. They have a set of highly unusual physical traits, many of which are thought to derive from their highly-social and putatively hypoxic and hypercapnic subterranean lifestyle. For example, naked mole-rats are among the most hypoxia-tolerant mammal presently identified and tolerate minutes of anoxia, hours at 3% O2, and days to weeks at 8% O2 [38,39,40,41]. The key to tolerating prolonged hypoxia is to match metabolic demand to reduced energy (O2) supply [42,43,44,45], and in acute severe hypoxia (3% O2), the metabolic rate of adult naked mole-rats decreases up to 85% [40]. However, naked mole-rats remain conscious and active, albeit to a reduced degree [46,47,48]. These findings indicate that naked mole-rats are capable of significant metabolic plasticity within their natural environment. Conversely, naked mole-rats are largely non-responsive to hypercapnia and associated acidity-related pain responses are largely absent [49,50]. Naked mole-rats also have numerous adaptations that are not as obviously linked to their natural habitat, including a remarkable resistance to cancer [51,52], they are the only mammalian thermo-conformer and almost entirely ectothermic for regulation of body temperature [53,54] and they have remarkable longevity [55,56,57,58]. These traits make the naked mole-rat an important animal model for a range of human diseases and for furthering understanding of pathways underlying cancer resistance and longevity [59,60,61]. However, little is known about the immune system of naked mole-rats. As PAD-mediated pathways and EVs are increasingly recognized as key players in immune responses and metabolism, and related to a range of human inflammatory pathologies and cancer, a study on these parameters in mole-rat is warranted.
In the current study, plasma and plasma-derived EVs were profiled in naked mole-rats and assessed for deiminated protein profiles as well as three key microRNAs (miRs) related to inflammation and hypoxic resistance. For the first time we report on post-translational deimination of key immune and metabolic proteins in naked mole-rat and species-specific EV profiles.

2. Results

2.1. PAD Homologues in Naked Mole-Rat Plasma

Using PAD-isozyme specific antibodies, generated against human PADs, positive bands were observed by Western blotting and indicated PAD homologue proteins in mole-rat plasma at the expected size of approximately 70–75 kDa for PAD2, PAD3 and PAD4 (Figure 1A).

2.2. 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 (Table 1 and Table 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 (Figure 3 and Figure 4).

2.3. 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).

2.4. 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).

3. 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.

4. Materials and Methods

4.1. Sampling of Naked Mole-Rat Plasma

Naked mole-rats were group-housed in interconnected multi-cage systems at 30 °C and 21% O2 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.

4.2. 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.

4.3. 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).

4.4. Western Blotting Analysis

Mole-rat plasma and plasma-EVs (an EV pellet derived from 100 µL plasma, reconstituted in 100 µL PBS) were diluted 1:1 in 2 × Laemmli sample buffer, boiled for 5 min at 100 °C and separated by SDS-PAGE on 4–20% TGX gels (BioRad, Watford, UK). Approximately 5 μg protein was loaded per lane. Following SDS-PAGE, proteins were transferred to nitrocellulose membranes using semi-dry Western blotting; even transfer was assessed by PonceauS (Sigma-Aldrich, Gillingham, UK) staining. The membranes were blocked in 5% BSA in TBS-T for 1 h at room temperature (RT) and thereafter incubated with the following primary antibodies diluted in TBS-T: F95 pan-deimination antibody (MABN328, Merck, Watford, UK, 1/1000); PAD2 (ab50257, Abcam, Cambridge, UK, 1/1000); PAD3 (ab50246, Abcam, 1/1000); PAD4 (ab50247, Abcam, 1/1000); and two EV-specific markers: CD63 (ab216130, Abcam, 1/1000); Flot-1 (ab41927, Abcam, 1/2000). After primary antibody incubation overnight at 4 °C on a shaking platform, the membranes were washed for 3 × 10 min in TBS-T at RT and thereafter incubated with the appropriate HRP-conjugated secondary antibodies (anti-rabbit IgG BioRad or anti-mouse IgM BioRad, diluted 1/4000 in TBS-T) for 1 h, at RT. Membranes were washed for 5 × 10 min in TBS-T, followed by 1 × 10 min in TBS proteins bands were visualised using enhanced chemiluminescence (ECL, Amersham, Buckinghamshire, UK) and digital images were obtained using the UVP BioDoc-ITTM System (Thermo Fisher Scientific, Hemel Hempstead, UK).

4.5. 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).

4.6. 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 2ΔΔCt method [90] was used for calculating relative miR expression levels and for normalisation. Each experiment was performed in 3 individuals, in triplicate.

4.7. 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.

5. 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 https://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 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.

Author Contributions

Data curation, S.L.; Formal analysis, P.U.-O. and S.L.; Funding acquisition, M.E.P. and S.L.; Investigation, P.U.-O. and S.L.; Methodology, M.E.P.; P.U.-O.; K.W.H.; I.K. and S.L.; Project administration, S.L.; Resources, M.E.P.; I.K., P.U.-O. and S.L.; Supervision, S.L.

Funding

This work was supported in parts by a University of Westminster Start-up Grant to SL and a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and the Canada Research Chairs Program to MEP.

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.

Abbreviations

BSABovine Serum Albumin
CD63CD63 antigen; granulophysin; lysosomal-associated membrane protein 3
CNSCentral Nervous System
ECLEnhanced Chemiluminescence
EVsExtracellular Vesicles
F95Pan-deimination/citrullination antibody
FBSFoetal Bovine Serum
Flot-1Flotillin-1
HIFHypoxia-inducible Factor
KEGG Kyoto Encyclopedia of Genes and Genomes
kDaKilodalton
LC-MS/MSLiquid Chromatography Mass Spectrometry
miRmicroRNA
NTANanoparticle Tracking Analysis
PADPeptidylarginine Deiminase
SDS-PAGESodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
TBSTris Buffered Saline
TEMTransmission Electron Microscopy
WBWestern Blotting

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Figure 1. 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.
Figure 1. 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.
Ijms 20 05378 g001
Figure 2. 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.
Figure 2. 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.
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Figure 3. Protein-protein interaction networks of deiminated proteins identified in naked mole-rat plasma. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis. (A) Coloured nodes represent query proteins and first shell of interactors. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways relating to the identified proteins and reported in STRING are highlighted as follows: red = complement and coagulation cascade; dark green = Staphylococcus aureus infection; purple = pertussis; yellow = platelet activation; light blue = systemic lupus erythematosus (SLE); orange = prion diseases; dark blue = cholesterol metabolism; light green = vitamin digestion and absorption; dark red = ferroptosis; pink = drug metabolism. Coloured nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines included in the figure).
Figure 3. Protein-protein interaction networks of deiminated proteins identified in naked mole-rat plasma. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis. (A) Coloured nodes represent query proteins and first shell of interactors. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways relating to the identified proteins and reported in STRING are highlighted as follows: red = complement and coagulation cascade; dark green = Staphylococcus aureus infection; purple = pertussis; yellow = platelet activation; light blue = systemic lupus erythematosus (SLE); orange = prion diseases; dark blue = cholesterol metabolism; light green = vitamin digestion and absorption; dark red = ferroptosis; pink = drug metabolism. Coloured nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines included in the figure).
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Figure 4. Protein-protein interaction networks of deiminated proteins identified in plasma-EVs of naked mole-rat. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING analysis. (A) Coloured nodes represent query proteins and first shell of interactors. (B) KEGG pathways relating to the identified proteins and reported in STRING are highlighted as follows: red = complement and coagulation cascade; dark green = Staphylococcus aureus infection; purple = HIF-signalling pathway; yellow = platelet activation; light blue = systemic lupus erythematosus (SLE); orange = oestrogen signalling pathway; dark blue = cholesterol metabolism; light green = vitamin digestion and absorption; pink = glycolysis/gluconeogenesis. Coloured nodes represent query proteins and first shell of interactors, white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines included in the figure).
Figure 4. Protein-protein interaction networks of deiminated proteins identified in plasma-EVs of naked mole-rat. Reconstruction of protein-protein interactions based on known and predicted interactions using STRING analysis. (A) Coloured nodes represent query proteins and first shell of interactors. (B) KEGG pathways relating to the identified proteins and reported in STRING are highlighted as follows: red = complement and coagulation cascade; dark green = Staphylococcus aureus infection; purple = HIF-signalling pathway; yellow = platelet activation; light blue = systemic lupus erythematosus (SLE); orange = oestrogen signalling pathway; dark blue = cholesterol metabolism; light green = vitamin digestion and absorption; pink = glycolysis/gluconeogenesis. Coloured nodes represent query proteins and first shell of interactors, white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines included in the figure).
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Figure 5. Extracellular vesicle profiling in naked mole-rat plasma. (A) Nanoparticle tracking analysis (NTA) shows a size distribution of EVs from naked mole-rat in the range of mainly 50 to 300 nm, with representative NTA profiles of EVs from 3 different animals (NTA-1, NTA-2, NTA-3). (B) Western blotting analysis confirms that naked mole-rat EVs are positive for the phylogenetically conserved EV-specific markers CD63 and Flot-1. (C) Transmission electron microscopy (TEM) analysis of naked mole-rat plasma-derived EVs shows typical EV morphology; a composite figure is shown and the scale bar (100 nm) applies for all images in the panel. (D) EV yield in plasma of 12 individual naked mole-rats is shown. (E) EV modal size in plasma of 12 individual naked mole-rats is presented.
Figure 5. Extracellular vesicle profiling in naked mole-rat plasma. (A) Nanoparticle tracking analysis (NTA) shows a size distribution of EVs from naked mole-rat in the range of mainly 50 to 300 nm, with representative NTA profiles of EVs from 3 different animals (NTA-1, NTA-2, NTA-3). (B) Western blotting analysis confirms that naked mole-rat EVs are positive for the phylogenetically conserved EV-specific markers CD63 and Flot-1. (C) Transmission electron microscopy (TEM) analysis of naked mole-rat plasma-derived EVs shows typical EV morphology; a composite figure is shown and the scale bar (100 nm) applies for all images in the panel. (D) EV yield in plasma of 12 individual naked mole-rats is shown. (E) EV modal size in plasma of 12 individual naked mole-rats is presented.
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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).
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).
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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 included and total score is shown. Protein hits identified in whole plasma only (but not in plasma EVs) are highlighted in pink. For full LC-MS/MS data analysis see Supplementary Table S1.
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 included and total score is shown. Protein hits identified in whole plasma only (but not in plasma EVs) are highlighted in pink. For full LC-MS/MS data analysis see Supplementary Table S1.
Protein NameSymbolTotal Score (p < 0.05) #
Apolipoprotein B-100G5AZB7_HETGA6077
Alpha-2-macroglobulinG5BPM1_HETGA4974
Complement C3G5C0N5_HETGA4300
SerotransferrinG5BQA9_HETGA4058
Serum albuminG5B5P2_HETGA3394
FibronectinG5BHR4_HETGA3333
Fibrinogen beta chainG5BML3_HETGA2638
Kininogen-1G5BT86_HETGA2276
Fibrinogen alpha chainG5BML2_HETGA2094
CeruloplasminG5BSL1_HETGA2022
Histidine-rich glycoproteinG5BT87_HETGA1851
Complement C4-AG5C3H6_HETGA1759
Complement factor HG5BM71_HETGA1646
PlasminogenG5BC53_HETGA1548
Inter-alpha-trypsin inhibitor heavy chain H4G5BUN4_HETGA1455
Fibrinogen gamma chainG5BML1_HETGA1338
Complement factor BG5C3H1_HETGA1313
Plasma kallikreinG5BNV2_HETGA1167
Antithrombin-IIIG5ARS6_HETGA1093
Coagulation factor VG5CB46_HETGA1092
Complement C5G5AXS5_HETGA1002
Hemoglobin subunit betaG5BS33_HETGA941
Transcobalamin-2 isoform 1G5AVP0_HETGA913
HemopexinG5BBR0_HETGA899
Coagulation factor XIII B chainG5BM72_HETGA821
Protein AMBPG5B1Y4_HETGA817
N-acetylmuramoyl-L-alanine amidaseG5BYP3_HETGA812
Coagulation factor XIIG5BQ09_HETGA802
Apolipoprotein A-IAPOA1_HETGA798
Hemoglobin subunit alphaG5BXY1_HETGA784
Coagulation factor XIII A chainG5BAS8_HETGA779
Hemoglobin subunit betaG5BYJ8_HETGA759
Alpha-1-antiproteinase SG5B496_HETGA724
Inter-alpha-trypsin inhibitor heavy chain H3G5BUN3_HETGA682
VitronectinG5BVN8_HETGA652
Complement factor IG5AQM1_HETGA647
Hemoglobin subunit epsilon-1G5BS35_HETGA642
Inter-alpha-trypsin inhibitor heavy chain H2G5AXV8_HETGA584
Apolipoprotein EG5CBM7_HETGA581
Inhibitor of carbonic anhydraseG5BQB0_HETGA537
Inter-alpha-trypsin inhibitor heavy chain H1G5BUN2_HETGA482
Four and a half LIM domains protein 1G5CA61_HETGA469
HaptoglobinG5B5U6_HETGA464
C4b-binding proteinG5BP10_HETGA438
L-lactate dehydrogenaseG5AKA3_HETGA437
Insulin-like growth factor-binding protein complex acid labile chainG5BY64_HETGA403
CatalaseG5AXV0_HETGA382
Fetuin-BG5BT88_HETGA347
Alpha-2-HS-glycoproteinG5BT89_HETGA337
Selenoprotein PG5APA7_HETGA328
Vitamin D-binding proteinG5BE53_HETGA317
AdiponectinG5BT83_HETGA283
von Willebrand factorG5CAN6_HETGA274
Beta-2-glycoprotein 1G5BGY7_HETGA268
Basement membrane-specific heparan sulfate proteoglycan core proteinG5BI06_HETGA267
GelsolinG5AXS0_HETGA259
Aspartyl aminopeptidaseG5AKJ4_HETGA256
Complement C1q subcomponent subunit AG5BHZ8_HETGA246
Ficolin-3G5AUT5_HETGA241
Mannan-binding lectin serine protease 1G5BTD5_HETGA228
Alpha-1-antichymotrypsinG5B491_HETGA228
Carboxypeptidase N catalytic chainG5AYP3_HETGA210
Complement C1q subcomponent subunit CG5BHZ7_HETGA191
Mannan-binding lectin serine protease 2G5C521_HETGA188
ProthrombinG5ATC4_HETGA177
Complement C1q tumour necrosis factor-related protein 3G5BQ97_HETGA0177
Nucleoside diphosphate kinaseG5C4L3_HETGA168
L-lactate dehydrogenaseG5BEG2_HETGA167
Alpha-2-antiplasminG5BCV1_HETGA164
Keratin, type II cytoskeletal 6BG5ALS1_HETGA152
Carbonic anhydrase 2G5ATW7_HETGA143
Proprotein convertase subtilisin/kexin type 9G5APQ4_HETGA137
AngiotensinogenG5BQI5_HETGA121
6-phosphogluconate dehydrogenase, decarboxylatingG5C530_HETGA91
Nesprin-1G5C0E1_HETGA87
Complement C1q subcomponent subunit BG5BHZ6_HETGA87
Angiopoietin-related protein 6G5B341_HETGA85
Hepatocyte growth factor activatorG5BZF5_HETGA84
Calpain-1 catalytic subunitG5B6L3_HETGA84
Sulfhydryl oxidaseG5AYL7_HETGA80
Keratin, type II cytoskeletal 5G5ALS3_HETGA76
ProfilinG5BH50_HETGA75
40S ribosomal protein S27aG5B8W4_HETGA68
Xanthine dehydrogenase/oxidaseG5B3Z0_HETGA68
Plasma protease C1 inhibitorG5BLJ5_HETGA67
Collectin-11G5C7L1_HETGA66
Extracellular matrix protein 1G5BH40_HETGA64
Actin, gamma-enteric smooth muscleG5AXH0_HETGA61
Collectin-10G5C9R8_HETGA61
Keratin, type I cytoskeletal 17G5B0M4_HETGA58
Contactin-1G5BGJ6_HETGA55
Dynein heavy chain 1, axonemalG5BUL8_HETGA50
Leucine-rich repeat-containing protein 15G5ALE6_HETGA43
Transcriptional regulator ATRXG5C0I5_HETGA42
Transmembrane protein 127G5BMW1_HETGA41
TRAF3-interacting protein 1G5BHH8_HETGA41
Mannose-binding protein AG5C4H7_HETGA40
Hyaluronan-binding protein 2G5BKD5_HETGA39
Fer3-like proteinG5BZJ1_HETGA38
Complement component C7G5C4R4_HETGA38
PITH domain-containing proteinG5BHY3_HETGA38
1,4-alpha-glucan-branching enzymeG5BA56_HETGA37
tRNA pseudouridine synthaseG5BB54_HETGA37
SRRM2-like proteinG5BG61_HETGA34
Putative carboxypeptidase PM20D1G5BX50_HETGA34
Olfactory receptorG5C741_HETGA33
Protein CASC5G5BLZ4_HETGA33
Conserved oligomeric Golgi complex subunit 1G5B5M3_HETGA32
Small G protein signalling modulator 1G5BGN5_HETGA32
Eukaryotic translation initiation factor 3 subunit EG5BD61_HETGA31
HEAT repeat-containing protein 2G5AZ15_HETGA31
Proline-rich protein 11G5AXY5_HETGA31
Peroxiredoxin-1G5ARW1_HETGA30
Pre-mRNA-processing factor 17G5B0Y2_HETGA30
Ribonucleoside-diphosphate reductaseG5B9C2_HETGA30
# Ions score is −10 * Log(p), where p is the probability that the observed match is a random event. Individual ions scores > 30 indicate identity or extensive homology (p < 0.05). Protein scores are 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 pink.
Table 2. Deiminated proteins identified by F95 enrichment in plasma-derived EVs 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 included and total score is shown. Protein hits identified in plasma-EVs only are highlighted in blue. For full LC-MS/MS data analysis see Supplementary Table S2.
Table 2. Deiminated proteins identified by F95 enrichment in plasma-derived EVs 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 included and total score is shown. Protein hits identified in plasma-EVs only are highlighted in blue. For full LC-MS/MS data analysis see Supplementary Table S2.
Protein NameSymbolTotal Score (p < 0.05) #
Alpha-2-macroglobulinG5BPM1_HETGA2978
Serum albuminG5B5P2_HETGA2570
SerotransferrinG5BQA9_HETGA1976
Keratin, type II cytoskeletal 6BG5ALS1_HETGA1459
Complement C3G5C0N5_HETGA1440
Keratin, type II cytoskeletal 5G5ALS3_HETGA1211
Kininogen-1G5BT86_HETGA1196
Histidine-rich glycoproteinG5BT87_HETGA1186
Keratin, type I cytoskeletal 10G5AX66_HETGA1130
Keratin, type I cytoskeletal 14G5B0M6_HETGA1038
Keratin, type II cytoskeletal 1G5ALS8_HETGA1011
Fibrinogen beta chainG5BML3_HETGA974
Fibrinogen alpha chainG5BML2_HETGA956
Apolipoprotein B-100G5AZB7_HETGA834
Keratin, type I cytoskeletal 13G5B0N0_HETGA701
Keratin, type I cytoskeletal 17G5B0M4_HETGA673
DesmoplakinG5BAT4_HETGA670
Hemoglobin subunit alphaG5BXY1_HETGA591
Hemoglobin subunit betaG5BS33_HETGA581
Complement C4-AG5C3H6_HETGA571
Hemoglobin subunit betaG5BYJ8_HETGA568
Apolipoprotein A-IAPOA1_HETGA499
Keratin, type II cytoskeletal 79G5BJ37_HETGA468
Actin, cytoplasmic 1G5BI78_HETGA444
Keratin, type II cytoskeletal 4G5BJ36_HETGA443
Fibrinogen gamma chainG5BML1_HETGA382
CeruloplasminG5BSL1_HETGA350
Apolipoprotein EG5CBM7_HETGA338
Inter-alpha-trypsin inhibitor heavy chain H4G5BUN4_HETGA332
Protein AMBPG5B1Y4_HETGA280
Inhibitor of carbonic anhydraseG5BQB0_HETGA244
HemopexinG5BBR0_HETGA239
Histone H4G5BKL3_HETGA238
PlasminogenG5BC53_HETGA232
Alpha-1-antiproteinase SG5B496_HETGA217
Hemoglobin subunit epsilon-1G5BS35_HETGA213
Junction plakoglobinG5B0M0_HETGA202
N-acetylmuramoyl-L-alanine amidaseG5BYP3_HETGA190
Inter-alpha-trypsin inhibitor heavy chain H3G5BUN3_HETGA190
Keratin, type II cytoskeletal 7G5BL96_HETGA182
FibronectinG5BHR4_HETGA164
Glyceraldehyde-3-phosphate dehydrogenaseG5CAP7_HETGA154
Coagulation factor XIIG5BQ09_HETGA152
Antithrombin-IIIG5ARS6_HETGA150
Catenin beta-1G5ALX2_HETGA126
Plakophilin-1G5B3A4_HETGA120
AnnexinG5AWC0_HETGA119
Keratin, type II cytoskeletal 3G5ALT2_HETGA118
40S ribosomal protein S27aG5B8W4_HETGA116
Inter-alpha-trypsin inhibitor heavy chain H2G5AXV8_HETGA106
Alpha-2-antiplasminG5BCV1_HETGA86
Complement factor BG5C3H1_HETGA81
Elongation factor 1-alphaG5ALK7_HETGA78
AngiotensinogenG5BQI5_HETGA77
Histone H2BG5BH20_HETGA77
Plasma kallikreinG5BNV2_HETGA72
Beta-enolaseG5BW96_HETGA70
Vitamin D-binding proteinG5BE53_HETGA57
Alpha-1-antichymotrypsinG5B491_HETGA55
Keratin, type II cytoskeletal 80G5BL95_HETGA53
Heat shock cognate 71 kDa proteinG5B170_HETGA51
Leucine-rich repeat-containing protein KIAA1731G5C3Y1_HETGA50
Cathepsin DG5C2G1_HETGA49
Basement membrane-specific heparan sulfate proteoglycan core proteinG5BI06_HETGA47
Skin-specific protein 32G5BUY9_HETGA46
Desmocollin-1G5C312_HETGA46
Protein-arginine deiminase type-6G5BZN1_HETGA46
Cryptochrome-1G5C454_HETGA45
Complement C1q subcomponent subunit CG5BHZ7_HETGA45
p2Y purinoceptor 12G5C102_HETGA44
Alpha-1B-glycoproteinG5B7K8_HETGA44
Exportin-1G5C1Y9_HETGA44
Putative hexokinase HKDC1G5B183_HETGA43
Transcriptional regulator ATRXG5C0I5_HETGA42
Coiled-coil domain-containing protein 57G5BJ98_HETGA42
Microtubule plus-end tracking protein TIP150G5ATU1_HETGA40
Tubulin alpha-1C chainG5AQ00_HETGA38
Fer3-like proteinG5BZJ1_HETGA38
Amino acid transporterG5BYQ9_HETGA36
Ventricular zone-expressed PH domain-containing protein-like protein 1G5B253_HETGA35
SRRM2-like proteinG5BG61_HETGA35
N6-adenosine-methyltransferase 70 kDa subunitG5BFU9_HETGA33
Proline-rich protein 11G5AXY5_HETGA33
Carbonic anhydrase 2G5ATW7_HETGA32
26S proteasome non-ATPase regulatory subunit 3G5BRY1_HETGA31
Serine/threonine-protein kinase LATS1G5BMD1_HETGA31
Transmembrane gamma-carboxyglutamic acid protein 1G5C418_HETGA31
Collagen alpha-2(I) chainG5ANK8_HETGA31
Bcl-2-associated transcription factor 1G5C1A7_HETGA30
# 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.

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Pamenter, M.E.; Uysal-Onganer, P.; Huynh, K.W.; Kraev, I.; Lange, S. Post-Translational Deimination of Immunological and Metabolic Protein Markers in Plasma and Extracellular Vesicles of Naked Mole-Rat (Heterocephalus glaber). Int. J. Mol. Sci. 2019, 20, 5378. https://doi.org/10.3390/ijms20215378

AMA Style

Pamenter ME, Uysal-Onganer P, Huynh KW, Kraev I, Lange S. Post-Translational Deimination of Immunological and Metabolic Protein Markers in Plasma and Extracellular Vesicles of Naked Mole-Rat (Heterocephalus glaber). International Journal of Molecular Sciences. 2019; 20(21):5378. https://doi.org/10.3390/ijms20215378

Chicago/Turabian Style

Pamenter, Matthew E., Pinar Uysal-Onganer, Kenny W. Huynh, Igor Kraev, and Sigrun Lange. 2019. "Post-Translational Deimination of Immunological and Metabolic Protein Markers in Plasma and Extracellular Vesicles of Naked Mole-Rat (Heterocephalus glaber)" International Journal of Molecular Sciences 20, no. 21: 5378. https://doi.org/10.3390/ijms20215378

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