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

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.

Deiminated proteins and extracellular vesicles - Novel serum biomarkers in whales and orca.

Peptidylarginine deiminases (PADs) are a family of phylogenetically conserved calcium-dependent enzymes which cause post-translational protein deimination. This can result in neoepitope generation, affect gene regulation and allow for protein moonlighting via functional and structural changes in target proteins. Extracellular vesicles (EVs) carry cargo proteins and genetic material and are released from cells as part of cellular communication. EVs are found in most body fluids where they can be useful biomarkers for assessment of health status. Here, serum-derived EVs were profiled, and post-translationally deiminated proteins and EV-related microRNAs are described in 5 ceataceans: minke whale, fin whale, humpback whale, Cuvier's beaked whale and orca. EV-serum profiles were assessed by transmission electron microscopy and nanoparticle tracking analysis. EV profiles varied between the 5 species and were identified to contain deiminated proteins and selected key inflammatory and metabolic microRNAs. A range of proteins, critical for immune responses and metabolism were identified to be deiminated in cetacean sera, with some shared KEGG pathways of deiminated proteins relating to immunity and physiology, while some KEGG pathways were species-specific. This is the first study to characterise and profile EVs and to report deiminated proteins and putative effects of protein-protein interaction networks via such post-translationald deimination in cetaceans, revealing key immune and metabolic factors to undergo this post-translational modification. Deiminated proteins and EVs profiles may possibly be developed as new biomarkers for assessing health status of sea mammals.

Deiminated proteins and extracellular vesicles as novel biomarkers in pinnipeds: Grey seal (Halichoerus gryptus) and harbour seal (Phoca vitulina).

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, leading to functional and structural changes in target proteins. Protein deimination can cause the generation of neo-epitopes, affect gene regulation and also allow for protein moonlighting and therefore facilitate multifaceted functions of the same protein. PADs are furthermore a key regulator of cellular release of extracellular vesicle (EVs), which are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and EVs were assessed in sera of two seal species, grey seal and harbour seal. We report a poly-dispersed population of serum-EVs, which were positive for phylogenetically conserved EV-specific markers and characterised by transmission electron microscopy. A number of deiminated proteins critical for immune and metabolic functions were identified in the seal sera and varied somewhat between the two species under study, while some targets were in common. EV profiles of the seal sera further revealed that key microRNAs for inflammation, immunity and hypoxia also vary between the two species. Protein deimination and EVs profiles may be useful biomarkers for assessing health status of sea mammals, which face environmental challenges, including opportunistic infection, pollution and shifting habitat due to global warming.

Complement component C4-like protein in Atlantic cod (Gadus morhua L.) - Detection in ontogeny and identification of post-translational deimination in serum and extracellular vesicles.

The complement system is a critical part of teleost immune defences, with complement component C4 forming part of the classical and lectin pathways. Cod C4-like protein was isolated from plasma, specific antibodies generated and C4-like protein was assessed in cod sera, mucus and in extracellular vesicles (EVs) from serum and mucus. Higher levels of C4-like protein were detected in serum- than mucus-derived EVs. Post-translational deimination, caused by conversion of arginine into citrulline, can affect protein structure and function. Here we detected deiminated forms of C4-like protein in cod serum and at lower levels in mucus. C4-like protein was also found in deiminated form at low levels in EVs from both serum and mucus. C4-like protein was assessed by immunohistochemistry in cod larvae and detected in a range of organs including in liver, kidney, gut, muscle, skin and mucus, as well as in neuronal tissues of the brain, spinal cord and eye. This abundance of C4-like protein during early development may indicate roles in tissue remodelling, in addition to immune defences. The presence of deiminated C4-like protein in serum and mucosa, as well as in EVs, may suggest C4 protein moonlighting via post-translational deimination.

Extracellular vesicles from cod (Gadus morhua L.) mucus contain innate immune factors and deiminated protein cargo.

Extracellular vesicles are released from cells and participate in cell communication via transfer of protein and genetic cargo derived from the parent cells. EVs play roles in normal physiology and immunity and are also linked to various pathological processes. Peptidylarginine deiminases (PADs) are phylogenetically conserved enzymes with physiological and pathophysiological roles. PADs cause post-translational protein deimination, resulting in structural and, in some cases, functional changes in target proteins and are also linked to EV biogenesis. This study describes for the first time EVs isolated from cod mucosa. Mucosal EVs were characterised by electron microscopy, nanoparticle tracking analysis and EV-specific surface markers. Cod mucosal EVs were found to carry PAD, complement component C3 and C-reactive proteins. C3 was found to be deiminated in both whole mucus and mucosal EVs, with some differences, and further 6 deiminated immune and cytoskeletal proteins were identified in EVs by LC-MS/MS analysis. As mucosal surfaces of teleost fish reflect human mucosal surfaces, these findings may provide useful insights into roles of EVs in mucosal immunity throughout phylogeny.

Peptidylarginine deiminase and deiminated proteins are detected throughout early halibut ontogeny - Complement components C3 and C4 are post-translationally deiminated in halibut (Hippoglossus hippoglossus L.).

Post-translational protein deimination is mediated by peptidylarginine deiminases (PADs), which are calcium dependent enzymes conserved throughout phylogeny with physiological and pathophysiological roles. Protein deimination occurs via the conversion of protein arginine into citrulline, leading to structural and functional changes in target proteins. In a continuous series of early halibut development from 37 to 1050° d, PAD, total deiminated proteins and deiminated histone H3 showed variation in temporal and spatial detection in various organs including yolksac, muscle, skin, liver, brain, eye, spinal cord, chondrocytes, heart, intestines, kidney and pancreas throughout early ontogeny. For the first time in any species, deimination of complement components C3 and C4 is shown in halibut serum, indicating a novel mechanism of complement regulation in immune responses and homeostasis. Proteomic analysis of deiminated target proteins in halibut serum further identified complement components C5, C7, C8 C9 and C1 inhibitor, as well as various other immunogenic, metabolic, cytoskeletal and nuclear proteins. Post-translational deimination may facilitate protein moonlighting, an evolutionary conserved phenomenon, allowing one polypeptide chain to carry out various functions to meet functional requirements for diverse roles in immune defences and tissue remodelling.

A novel ladder-like lectin relates to sites of mucosal immunity in Atlantic halibut (Hippoglossus hippoglossus L.).

A novel 27 kDa ladder-lectin-like protein, showing a multimeric structure under non-reducing conditions, was isolated from halibut serum by binding to N-acetyl glucosamine. Mass-spectrometry analysis did not show significant homology with known proteins. Specific antibodies were produced and used in immunohistochemistry on tissue sections of early halibut ontogeny from 119 until 1050 °d post hatching. A strong positive response was detected in the mucosal cells of the skin, gills and gut, indicating a role in the mucosal immune defence at these sites. Further immunopositivity was detected in liver, myeloma of kidney and the brain at different developmental stages but predominant expression was found in mucosal surfaces at later stages of development tested (1050 °d). It is still uncertain whether this ladder-like lectin forms part of the complement pathway, as a lectin or ficolin, or if it belongs to galectins. A strong detection in mucosal surfaces on skin, gills and gut, show similar patterns of expression as both mucosal lectins and galectins in other fish. Detection in neuronal tissue may indicate putative roles in tissue remodelling of brain and in ongoing neurogenesis in the fish eye.

Post-translational protein deimination in cod (Gadus morhua L.) ontogeny novel roles in tissue remodelling and mucosal immune defences?

Peptidylarginine deiminases (PADs) are calcium dependent enzymes with physiological and pathophysiological roles conserved throughout phylogeny. PADs promote post-translational deimination of protein arginine to citrulline, altering the structure and function of target proteins. Deiminated proteins were detected in the early developmental stages of cod from 11 days post fertilisation to 70 days post hatching. Deiminated proteins were present in mucosal surfaces and in liver, pancreas, spleen, gut, muscle, brain and eye during early cod larval development. Deiminated protein targets identified in skin mucosa included nuclear histones; cytoskeletal proteins such as tubulin and beta-actin; metabolic and immune related proteins such as galectin, mannan-binding lectin, toll-like receptor, kininogen, Beta2-microglobulin, aldehyde dehydrogenase, bloodthirsty and preproapolipoprotein A-I. Deiminated histone H3, a marker for anti-pathogenic neutrophil extracellular traps, was particularly elevated in mucosal tissues in immunostimulated cod larvae. PAD-mediated protein deimination may facilitate protein moonlighting, allowing the same protein to exhibit a range of biological functions, in tissue remodelling and mucosal immune defences in teleost ontogeny.

Pentraxins CRP-I and CRP-II are post-translationally deiminated and differ in tissue specificity in cod (Gadus morhua L.) ontogeny.

Pentraxins are fluid phase pattern recognition molecules that form an important part of the innate immune defence and are conserved between fish and human. In Atlantic cod (Gadus morhua L.), two pentraxin-like proteins have been described, CRP-I and CRP-II. Here we show for the first time that these two CRP forms are post-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect to tissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed in liver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II is strongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types in immune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. The presence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possibly facilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions and thus meeting different functional requirements in different tissues. The presented findings may further current understanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as in various human pathologies, including autoimmune diseases, amyloidosis and cancer.

The acute phase response of cod (Gadus morhua L.): expression of immune response genes.

An acute phase response (APR) was experimentally induced in Atlantic cod (Gadus morhua L.) by intramuscular injection of turpentine oil. The change in the expression of immune related genes was monitored in the anterior kidney and the spleen over a period of 7 days. The genes examined were two types of pentraxins, apolipoprotein A1 (ApoA-I), the complement component C3, interleukin-1ß (IL-1ß), transferrin, cathelicidin, and hepcidin. All genes were constitutively expressed in both organs and their expression amplified by the turpentine injection. A pattern of response was observed both with respect to the organ preference and to the timing of a maximum response. The increased gene expression of the pentraxins, ApoA-I and C3 was restricted to the anterior kidney, the gene expression of IL-1ß, cathelicidin, and transferrin increased in both organs, while hepcidin gene expression was only significantly increased in the spleen. The pentraxins and ApoA-I appear to be early mediators of APR in cod, possibly stimulating C3 and IL-1ß response, while the antimicrobial peptides may play a minor role. The increase in transferrin gene expression in both organs, and apparent indifference to cortisol release associated with the turpentine injection, suggests that this could be a typical acute phase protein in cod.

The acute phase response of Atlantic cod (Gadus morhua): humoral and cellular response.

Intra-muscular injection of turpentine oil was used to induce acute phase response (APR) in Atlantic cod (Gadus morhua L.). The effects on the serum cortisol, total protein, IgM and pentraxin concentration were examined as well as the effects on natural antibody, anti-trypsin and leukocyte respiratory burst activity. The turpentine injection resulted in a 26 fold increase in the cortisol level after 72 h. Slightly reduced serum protein level in both groups was attributed to the restricted feeding during the experimental period. The IgM serum concentration was significantly reduced after 168 h in the turpentine treated fish while the natural antibody activity was not affected. The anti-trypsin activity was initially suppressed but recovered to normal levels at the end of the experiment. The turpentine injection had little effect on the serum level of the pentraxins, CRP-PI and CRP-PII. The respiratory burst activity was significantly suppressed after 72 h. It is concluded that 1) cod shows a relatively slow humoral and cellular response to APR induction, 2) the increase in serum cortisol level may be the key modulator of the mainly suppressive effects on the immune parameters and 3) pentraxins are not typical acute phase proteins in cod.

Immunological control of fish diseases.

All metazoans possess innate immune defence system whereas parameters of the adaptive immune system make their first appearance in the gnathostomata, the jawed vertebrates. Fish are therefore the first animal phyla to possess both an innate and adaptive immune system making them very interesting as regards developmental studies of the immune system. The massive increase in aquaculture in recent decades has also put greater emphasis on studies of the fish immune system and defence against diseases commonly associated with intensive fish rearing. Some of the main components of the innate and adaptive immune system of fish are described. The innate parameters are at the forefront of immune defence in fish and are a crucial factor in disease resistance. The adaptive response of fish is commonly delayed but is essential for lasting immunity and a key factor in successful vaccination. Some of the inherent and external factors that can manipulate the immune system of fish are discussed, the main fish diseases are listed and the pathogenicity and host defence discussed. The main prophylactic measures are covered, including vaccination, probiotics and immunostimulation. A key element in the immunological control of fish diseases is the great variation in disease susceptibility and immune defence of different fish species, a reflection of the extended time the present day teleosts have been separated in evolution. Future research will probably make use of molecular and proteomic tools both to study important elements in immune defence and prophylactic measures and to assist with breeding programmes for disease resistance.

Natural antibodies of cod (Gadus morhua L.): specificity, activity and affinity.

Natural antibodies are present in the serum of vertebrates regardless of antigenic stimulation. Characteristic activity is commonly detected against haptenated proteins, single stranded DNA and thyroglobulin. Natural antibodies are believed to provide an instant protection against pathogens of a broad specificity and to participate in homeostasis. Cod is a poor antibody responder but shows a relatively high level of natural antibodies against haptenated proteins. In this project the specificity, activity and affinity of natural antibodies was studied in different groups of cod and the effects of age/size, environmental temperature, immunisation and infection examined. Antigen driven selection of natural antibodies was also studied in one group of cod. The results demonstrated a broad and yet characteristic specificity, primarily directed against haptenated proteins and possible food antigens. The antibody activity increased with increasing age and at higher temperature whereas immunostimulation by immunisation or infection resulted in variable response. The affinity index of natural antibodies of cod generally did not correlate with changes in the antibody activity but it was in the same range as the affinity index of acquired cod antibodies and that of some mammalian monoclonal acquired antibodies. Analysis of antigen driven antibody selection showed that the natural antibody repertoire of individual cod was heterogeneous with respect to its affinity for haptenated protein.

Isolation of two C-reactive protein homologues from cod (Gadus morhua L.) serum.

Pentraxins are important molecules in innate defence and play a role in the acute phase response of both mammals and fish. Isolation of cod pentraxins by affinity chromatography using phosphorylcholine agarose revealed two pentraxin-like proteins, referred to as PI and PII proteins. These varied in their overall charge, pentameric and subunit molecular size, glycosylation and N-terminal amino acid sequences. The PI protein was homologous with the CRP-like pentraxin previously described in cod whereas the PII protein was a new CRP homologue, which was characterized by substantial individual heterogeneity with regard to subunit size and relative density. The results indicate considerable genetic variations in the cod pentraxins.

CRIT peptide interacts with factor B and interferes with alternative pathway activation.

Complement C2 receptor inhibitor trispanning (CRIT) inhibits the classical pathway (CP) C3 convertase formation by competing with C4b for the binding of C2. The C-terminal 11-amino-acid of the first CRIT-extracellular domain (CRIT-H17) has a strong homology with a sequence in the C4beta chain, which is responsible for the binding of C2. Since the CP and alternative pathway (AP) C3 convertases have many functional and structural similarities, we further investigated the effects of CRIT-H17 on the AP. The factor D-mediated cleavage of factor B (FB) was blocked by CRIT-H17. By ELISA and immunoblot, CRIT-H17 was shown to bind FB. CRIT-H17 had no decay activity on the C3bBb complex as compared to decay-accelerating factor. Binding of CRIT-H17 to FB did not interfere with the assembly of C3bB complex. In a haemolytic assay using C2-deficient serum, CRIT-H17 interfered with AP complement activation.

Innate immunity of fish (overview).

The innate immune system is the only defence weapon of invertebrates and a fundamental defence mechanism of fish. The innate system also plays an instructive role in the acquired immune response and homeostasis and is therefore equally important in higher vertebrates. The innate system's recognition of non-self and danger signals is served by a limited number of germ-line encoded pattern recognition receptors/proteins, which recognise pathogen associated molecular patterns like bacterial and fungal glycoproteins and lipopolysaccharides and intracellular components released through injury or infection. The innate immune system is divided into physical barriers, cellular and humoral components. Humoral parameters include growth inhibitors, various lytic enzymes and components of the complement pathways, agglutinins and precipitins (opsonins, primarily lectins), natural antibodies, cytokines, chemokines and antibacterial peptides. Several external and internal factors can influence the activity of innate immune parameters. Temperature changes, handling and crowding stress can have suppressive effects on innate parameters, whereas several food additives and immunostimulants can enhance different innate factors. There is limited data available about the ontogenic development of the innate immunological system in fish. Active phagocytes, complement components and enzyme activity, like lysozyme and cathepsins, are present early in the development, before or soon after hatching.

Complement component C3 transcription in Atlantic halibut (Hippoglossus hippoglossus L.) larvae.

The complement systems of fish are well developed and play an important role in the innate immune response. Complement C3 is the central protein of all three activation pathways and is the major opsonin of the complement system and essential for the generation of the membrane attack complex. A 1548 bp part of complement component C3 was isolated from a halibut liver cDNA library by immunoscreening. The deduced amino acid sequence showed that this part of halibut C3 contained key amino acids for factor H, I and properdin binding as well as two N-glycosylation sites. Digoxigenine labelled mRNA probes were synthesised and the transcription of C3 was monitored in three larval stages at 206, 430 and 1000 degrees d (30, 50 and 99 days post hatching), by in situ hybridisation. C3 mRNA was detected in muscle, liver, brain, chondrocytes, spinal cord, eye, intestines, oesophagus and kidney. These findings are in accordance with a former immunohistochemical study on halibut C3 protein ontogeny, indicating that C3 is indeed locally expressed in many organs from the youngest stages on. Complement may thus be linked to the formation and generation of different organs during development and play an important role in the early immune response of halibut larvae.

The ontogenic transcription of complement component C3 and Apolipoprotein A-I tRNA in Atlantic cod (Gadus morhua L.)--a role in development and homeostasis?

The complement system is important both in the innate and adaptive immune response, with C3 as the central protein of all three activation pathways. Apolipoprotein A-I (ApoLP A-I), a high-density lipoprotein (HDL), has been shown to have a regulatory role in the complement system by inhibiting the formation of the membrane attack complex (MAC). Complement has been associated with apoptotic functions, which are important in the immune response and are involved in organ formation and homeostasis. mRNA probes for cod C3 and ApoLP A-I were synthesized and in situ hybridisation used to monitor the ontogenic development of cod from fertilised eggs until 57 days after hatching. Both C3 and ApoLP A-I transcription was detected in the central nervous system (CNS), eye, kidney, liver, muscle, intestines, skin and chondrocytes at different stages of development. Using TUNEL staining, apoptotic cells were identified within the same areas from 4 to 57 days posthatching. The present findings may suggest a role for C3 and ApoLP A-I during larval development and a possible role in the homeostasis of various organs in cod.

Expression of functional recombinant von Willebrand factor-A domain from human complement C2: a potential binding site for C4 and CRIT.

CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the beta-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224-437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 alpha, beta and gamma chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4beta chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2-CRIT and C2-C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.

The ontogenic development of innate immune parameters of cod (Gadus morhua L.).

The aim of this study was to monitor the ontogenic development of innate immune parameters of cod (Gadus morhua L.) and to determine the presence of maternal IgM. The general protein composition and enzyme activity was also studied. At intervals, samples were collected of fertilized cod eggs and larvae from 3 days after fertilization until 57 days after hatching. Cell lysates were prepared and analysed by Western blotting using antibodies prepared against cod IgM, the complement component C3 and C-reactive protein (CRP) as well as against cod serum proteins and haemoglobin. Antibodies against salmon cathepsins and against several mammalian proteins of immunological significance were also used. Maternal IgM was not detected but C3 and the closely associated apolipoprotein A-I were present from the time of embryo organogenesis. C-reactive protein was not detected and none of the antibodies against mammalian immune parameters cross-reacted with the cod material. Protein and proteomic analysis showed that the major proteins of the egg samples were vitellogenin derived maternal proteins. Other non-vitellogenin maternal proteins, not yet identified, were also detected in the fertilized eggs. Cathepsin was present in all samples, but other enzyme activity was restricted to larval samples from 4 days after hatching when feeding had commenced. Haemoglobin was not detected until 10 days after hatching.