Extracellular Vesicles and Post-Translational Protein Deimination Signatures in Mollusca—The Blue Mussel (Mytilus edulis), Soft Shell Clam (Mya arenaria), Eastern Oyster (Crassostrea virginica) and Atlantic Jacknife Clam (Ensis leei)

Abstract

Oysters and clams are important for food security and of commercial value worldwide. They are affected by anthropogenic changes and opportunistic pathogens and can be indicators of changes in ocean environments. Therefore, studies into biomarker discovery are of considerable value. This study aimed at assessing extracellular vesicle (EV) signatures and post-translational protein deimination profiles of hemolymph from four commercially valuable Mollusca species, the blue mussel (Mytilus edulis), soft shell clam (Mya arenaria), Eastern oyster (Crassostrea virginica), and Atlantic jacknife clam (Ensis leei). EVs form part of cellular communication by transporting protein and genetic cargo and play roles in immunity and host–pathogen interactions. Protein deimination is a post-translational modification caused by peptidylarginine deiminases (PADs), and can facilitate protein moonlighting in health and disease. The current study identified hemolymph-EV profiles in the four Mollusca species, revealing some species differences. Deiminated protein candidates differed in hemolymph between the species, with some common targets between all four species (e.g., histone H3 and H4, actin, and GAPDH), while other hits were species-specific; in blue mussel these included heavy metal binding protein, heat shock proteins 60 and 90, 2-phospho-D-glycerate hydrolyase, GTP cyclohydrolase feedback regulatory protein, sodium/potassium-transporting ATPase, and fibrinogen domain containing protein. In soft shell clam specific deimination hits included dynein, MCM3-associated protein, and SCRN. In Eastern oyster specific deimination hits included muscle LIM protein, beta-1,3-glucan-binding protein, myosin heavy chain, thaumatin-like protein, vWFA domain-containing protein, BTB domain-containing protein, amylase, and beta-catenin. Deiminated proteins specific to Atlantic jackknife clam included nacre c1q domain-containing protein and PDZ domain-containing protein In addition, some proteins were common as deiminated targets between two or three of the Bivalvia species under study (e.g., EP protein, C1q domain containing protein, histone H2B, tubulin, elongation factor 1-alpha, dominin, extracellular superoxide dismutase). Protein interaction network analysis for the deiminated protein hits revealed major pathways relevant for immunity and metabolism, providing novel insights into post-translational regulation via deimination. The study contributes to EV characterization in diverse taxa and understanding of roles for PAD-mediated regulation of immune and metabolic pathways throughout phylogeny.

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

“…In four species of whale, EV profiles were seen in the ranges of 50–500 (minke whale Balaenoptera acutorostrata ), 50–400 (fin whale Balaenoptera physalus ), 80–300 (humpback whale Megaptera novaeangliae ) and 90–300 nm (Cuvier’s beaked whale Ziphius cavirostris ), respectively, while orca serum-EVs ( Orcinus orca ; dolphin family) were reported at 30–500 nm [ 39 ]. Reports of EV profiling of haemolymph from species lower in the phylogeny tree include Crustacea (lobster Homarus americanus ) with EVs in the 10–500 nm size range (with the majority of EVs being small in the 22–115 nm size range) [ 22 ]; Mollusca haemolymph EVs at 50–300 nm (blue mussel, Mytilus edulis ), 30–300 nm (soft shell clam Mya arenaria ), 90–500 nm (Eastern oyster Crassostrea virginica ) and 20–300 nm (Atlantic jacknife clam Ensis leei ), respectively [ 24 ]; Arthropoda (horseshoe crab Limulus polyphemus ) EVs at 20–400 nm (with the majority of EVs falling within 40–123 nm) [ 23 ]. In the protozoa Giardia intestinalis , two distinct size populations of EVs have been described (20–80 nm and 100–400 nm, respectively), which display different functions in host–pathogen interactions [ 21 ].…”

“…While in fish, only one PAD form is present [ 11 , 12 , 13 , 14 ], mammals contain five tissue-specific PAD isozymes, with varying preferences for target proteins [ 3 , 4 , 5 ]. In other phyla, such as reptiles and birds, only three PAD forms are described [ 3 , 15 , 16 ], and PAD homologues are identified lower in the phylogeny tree [ 17 ], including in bacteria [ 18 , 19 ], fungi [ 20 ], parasites [ 21 ], as well as in Crustacea [ 22 ], Merostomata [ 23 ] and Mollusca [ 24 ]. PAD-mediated protein deimination has been reported in a range of taxa throughout the phylogeny tree, both in ontogeny, serum and plasma, as well as forming part of extracellular vesicle (EV) protein cargo [ 12 , 13 , 14 , 16 , 22 , 23 , 24 ].…”

“…While EV research has been an exponentially expanding field in the past decade in relation to human disease, less is known about EV communication in other taxa. The comparative field of EV research has recently been growing, including by studies from our group [ 14 , 16 , 19 , 22 , 23 , 24 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Therefore, there is currently great interest in expanding EV studies, also in relation to teleost fish and biomarker discovery for aquaculture [ 32 , 33 , 38 , 41 ].…”

Post-Translational Protein Deimination Signatures in Plasma and Plasma EVs of Reindeer (Rangifer tarandus)

“…While the reindeer genome has been sequenced [ 10 ], and genetic diversity and mitochondrial DNA have furthermore been studied [ 11 ], no studies have hitherto been performed into mechanisms relating to post-translational modifications such as deimination, which is caused by peptidylarginine deiminases (PADs). Furthermore, while research on extracellular vesicles (EVs) is a major field in relation to biomarker discovery in human pathologies, and recent comparatives studies have highlighted their value in a range of wild, domestic and commercially valuable land and aquatic animals throughout the phylogeny tree [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], the field is still in its infancy in relation to studies and biomarker development in wild animals.…”

“…A majority of studies on PADs and downstream deimination have related to human pathological mechanisms, but recently a comparative body of research has focused on identifying putative roles for PADs in physiological and immunological pathways in a wide range of taxa throughout the phylogenetic tree, including land and sea mammals, reptiles, birds, bony and cartilaginous fish, Mollusca and Crustacea [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 28 , 29 ]. PADs have furthermore been identified to have roles in mucosal, innate and adaptive immunity in a range of taxa [ 17 , 18 , 19 , 20 , 25 , 28 , 29 , 43 , 44 ].…”