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. 2021 Jan 16;22(2):875.
doi: 10.3390/ijms22020875.

The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles-Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways

Bergljót Magnadóttir  1 , Igor Kraev  2 , Alister W Dodds  3 , Sigrun Lange  4
Affiliations

The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles-Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways

Bergljót Magnadóttir et al. Int J Mol Sci. .

Abstract

Extracellular vesicles (EVs) are lipid bilayer vesicles which are released from cells and play multifaceted roles in cellular communication in health and disease. EVs can be isolated from various body fluids, including serum and plasma, and are usable biomarkers as they can inform health status. Studies on EVs are an emerging research field in teleost fish, with accumulating evidence for important functions in immunity and homeostasis, but remain to be characterised in most fish species, including halibut. Protein deimination is a post-translational modification caused by a conserved family of enzymes, named peptidylarginine deiminases (PADs), and results in changes in protein folding and function via conversion of arginine to citrulline in target proteins. Protein deimination has been recently described in halibut ontogeny and halibut serum. Neither EV profiles, nor total protein or deiminated protein EV cargos have yet been assessed in halibut and are reported in the current study. Halibut serum EVs showed a poly-dispersed population in the size range of 50-600 nm, with modal size of EVs falling at 138 nm, and morphology was further confirmed by transmission electron microscopy. The assessment of EV total protein cargo revealed 124 protein hits and 37 deiminated protein hits, whereof 15 hits were particularly identified in deiminated form only. Protein interaction network analysis showed that deimination hits are involved in a range of gene regulatory, immune, metabolic and developmental processes. The same was found for total EV protein cargo, although a far wider range of pathways was found than for deimination hits only. The expression of complement component C3 and C4, as well as pentraxin-like protein, which were identified by proteomic analysis, was further verified in EVs by western blotting. This showed that C3 is exported in EVs at higher levels than C4 and deiminated C3 was furthermore confirmed to be at high levels in the deimination-enriched EV fractions, while, in comparison, C4 showed very low detection in deimination-enriched EV fractions. Pentraxin was exported in EVs, but not detected in the deimination-enriched fractions. Our findings provide novel insights into EV-mediated communication in halibut serum, via transport of protein cargo, including post-translationally deiminated proteins.

Keywords: citrullinome; complement; deimination/citrullination; extracellular vesicles; gene regulation; immunity; metabolism; pentraxin; peptidylarginine deiminase; proteome.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Halibut serum-extracellular vesicles (EV)s were characterised by: (A) Nanoparticle tracking analysis (NTA), showing size distribution profiles of EVs in the size range of 50–600 nm, with the modal size of vesicles at 138 nm; (B) Western blotting (WB) analysis shows that the EVs are positive for CD63 and Flotillin-1; (C) Transmission electron microscopy (TEM) showing EV morphology—see arrows pointing at EVs (scale bar is indicated at 20 nm).
Figure 2
Figure 2
The proteome and citrullinome of halibut serum-EVs. Silver-stained gels for: (A) total protein cargo in EVs and (B) F95 enriched (deiminated/citrullinated) proteins from EVs. The protein standard (std) is indicated in kilodaltons (kDa). (C) Venn diagram shows the number of candidate protein hits identified as cargo in total serum EVs (“The serum EV proteome”) as well as deiminated protein hits in EV cargo (the serum “EV citrullinome”).
Figure 3
Figure 3
Complement component C3, C4 and pentraxin-like protein in halibut EVs and F95 enriched EV fractions. Western blotting showing (A) complement component C3 detection in total protein cargo of halibut serum-EVs (“EVs”) and in F95-enriched protein fractions from serum-EVs (“EVs_F95”), C3 α- and β-chains, as well as α-fragment (α-f) are indicated; (B) complement component C4 detection in total protein cargo of serum-EVs (“EVs”) and lower detection observed in F95-enriched EV protein fractions (“EVs_F95”), C4 α-, β- and γ-chains are indicated; (C) pentraxin-like protein detection in total EV protein cargo (“EVs”), which was not detected in the F95-enriched EV protein fractions (“EVs_F95”).
Figure 4
Figure 4
(A) Protein interaction networks for deiminated proteins in halibut EVs. Local network clusters and UniProt keywords are indicated by the colour coded nodes. See colour key for nodes and interaction networks in the figure. (B) Reactome protein interaction networks for deiminated proteins in halibut EVs. Reactome pathways are indicated by the coloured nodes, as shown in the figure. (C,D) PFAM and SMART protein interaction networks for deiminated proteins in halibut EVs. PFAM and SMART protein domains are indicated by the coloured nodes, see colour code in the figure. (E) InterPro protein interaction networks for deiminated proteins in halibut EVs. InterPro protein domains and features are indicated by the coloured nodes; see colour code in the figure.
Figure 4
Figure 4
(A) Protein interaction networks for deiminated proteins in halibut EVs. Local network clusters and UniProt keywords are indicated by the colour coded nodes. See colour key for nodes and interaction networks in the figure. (B) Reactome protein interaction networks for deiminated proteins in halibut EVs. Reactome pathways are indicated by the coloured nodes, as shown in the figure. (C,D) PFAM and SMART protein interaction networks for deiminated proteins in halibut EVs. PFAM and SMART protein domains are indicated by the coloured nodes, see colour code in the figure. (E) InterPro protein interaction networks for deiminated proteins in halibut EVs. InterPro protein domains and features are indicated by the coloured nodes; see colour code in the figure.
Figure 5
Figure 5
(A) Protein interaction networks for total protein cargo in halibut EVs, showing local network clusters. The coloured nodes indicate the different networks, respectively. (B) Reactome protein interaction networks for total proteins in halibut EV cargo, showing reactome pathways. Specific reactome pathways are indicated by the coloured nodes, respectively. (C) UniProt protein interaction networks for total proteins in halibut EV cargo, showing UniProt keywords. UniProt keywords are indicated by the coloured nodes, respectively. (D) PFAM protein interaction networks for total proteins in halibut EV cargo. The specific PFAM protein domains are indicated by the coloured nodes, respectively. (E) Protein interaction networks for total proteins in halibut EVs, showing SMART protein domains. The specific SMART protein domains are indicated by the coloured nodes, respectively. (F) InterPro protein interaction networks for total proteins in halibut EVs. The specific protein domains and features (InterPro) are indicated by the coloured nodes, respectively.
Figure 5
Figure 5
(A) Protein interaction networks for total protein cargo in halibut EVs, showing local network clusters. The coloured nodes indicate the different networks, respectively. (B) Reactome protein interaction networks for total proteins in halibut EV cargo, showing reactome pathways. Specific reactome pathways are indicated by the coloured nodes, respectively. (C) UniProt protein interaction networks for total proteins in halibut EV cargo, showing UniProt keywords. UniProt keywords are indicated by the coloured nodes, respectively. (D) PFAM protein interaction networks for total proteins in halibut EV cargo. The specific PFAM protein domains are indicated by the coloured nodes, respectively. (E) Protein interaction networks for total proteins in halibut EVs, showing SMART protein domains. The specific SMART protein domains are indicated by the coloured nodes, respectively. (F) InterPro protein interaction networks for total proteins in halibut EVs. The specific protein domains and features (InterPro) are indicated by the coloured nodes, respectively.
Figure 5
Figure 5
(A) Protein interaction networks for total protein cargo in halibut EVs, showing local network clusters. The coloured nodes indicate the different networks, respectively. (B) Reactome protein interaction networks for total proteins in halibut EV cargo, showing reactome pathways. Specific reactome pathways are indicated by the coloured nodes, respectively. (C) UniProt protein interaction networks for total proteins in halibut EV cargo, showing UniProt keywords. UniProt keywords are indicated by the coloured nodes, respectively. (D) PFAM protein interaction networks for total proteins in halibut EV cargo. The specific PFAM protein domains are indicated by the coloured nodes, respectively. (E) Protein interaction networks for total proteins in halibut EVs, showing SMART protein domains. The specific SMART protein domains are indicated by the coloured nodes, respectively. (F) InterPro protein interaction networks for total proteins in halibut EVs. The specific protein domains and features (InterPro) are indicated by the coloured nodes, respectively.
Figure 5
Figure 5
(A) Protein interaction networks for total protein cargo in halibut EVs, showing local network clusters. The coloured nodes indicate the different networks, respectively. (B) Reactome protein interaction networks for total proteins in halibut EV cargo, showing reactome pathways. Specific reactome pathways are indicated by the coloured nodes, respectively. (C) UniProt protein interaction networks for total proteins in halibut EV cargo, showing UniProt keywords. UniProt keywords are indicated by the coloured nodes, respectively. (D) PFAM protein interaction networks for total proteins in halibut EV cargo. The specific PFAM protein domains are indicated by the coloured nodes, respectively. (E) Protein interaction networks for total proteins in halibut EVs, showing SMART protein domains. The specific SMART protein domains are indicated by the coloured nodes, respectively. (F) InterPro protein interaction networks for total proteins in halibut EVs. The specific protein domains and features (InterPro) are indicated by the coloured nodes, respectively.
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