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

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.

Keywords: anti-viral; bovine (Bos taurus); extracellular vesicles (EVs); immunity; metabolism; peptidylarginine deiminases (PADs); protein deimination.

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 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 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 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).

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 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 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 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).