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.

Keywords: Mollusca; clam; extracellular vesicles (EVs); immunity; metabolism; oyster; peptidylarginine deiminase (PAD); protein deimination/citrullination.

Figure 1

Nanoparticle tracking analysis (NTA) of Mollusca hemolymph EVs from (A) blue mussel; (B) soft shell clam; (C) Eastern oyster; (D) Atlantic jacknife clam.

Figure 2

Transmission electron microscopy (TEM) analysis of Mollusca hemolymph EVs. (A) Blue mussel; (B) soft shell clam; (C) Eastern oyster; (D) Atlantic jacknife clam. (E) Western blotting (WB) of hemolymph EVs (representative figure showing EVs from soft Atlantic jacknife clam) shows strong CD63 positive (protein size standard is indicated in kilodaltons, kDa).

Figure 3

Mollusca PAD and deiminated proteins in hemolymph. (A) Western blotting analysis for PAD homologues in Mollusca, using the anti-human PAD2 antibody. (B) Silver stained SDS-PAGE gel (4–20% gradient TGX gel), showing F95-enriched fractions (F95_IP) from the four Mollusca species. All lanes show analysis of a pool from four individual animals, per species. The protein standard (std) is indicated in kilodaltons (kDa).

Figure 4

Deiminated protein hits in the four Mollusca species. The Venn diagram represents the number of deiminated proteins identified in and overlapping in blue mussel, soft shell clam, Eastern oyster, and Atlantic jacknife clam.

Figure 5

Local STRING network clusters and PFAM protein domains identified for deiminated proteins in blue mussel hemolymph. Protein–protein interaction network for blue mussel based on protein identifiers from Californian sea hare (Aplysia californica). PPI enrichment p-value: 0.000169. Color coding for network nodes and interaction lines is included in the figure.

Figure 6

STRING network for soft shell clam. Protein–protein interaction network for soft shell clam based on protein identifiers in California sea hare (Aplysia californica). PPI enrichment p-value: 0.00477. Color coding for network nodes and interaction lines is included in the figure.

Figure 7

STRING network for Eastern oyster. Protein–protein interaction network for Eastern oyster, based on protein identifiers in California sea hare (Aplysia californica). PPI enrichment p-value: 0.254. Color coding for network nodes and interaction lines is included in the figure.

Figure 8

STRING network for Atlantic jacknife clam. Protein-protein interaction network for Atlantic jacknife clam, based on protein identifiers in California sea hare (Aplysia californica). PPI enrichment p-value: 0.0069. Color coding for network nodes and interaction lines is included in the figure.

Figure 9

STRING protein interaction networks for deiminated protein hits identified in Mollusca, using human protein identifiers. (A) Local STRING network cluster; (B) PFAM and SMART protein domains; (C–F) biological GO processes: GO-1 (C), GO-2 (D), GO-3 (E), GO-4 (F). PPI enrichment p-value: 3.16 × 10⁻⁸. Color coding for network nodes and interaction lines is included in the figure.

Figure 9

STRING protein interaction networks for deiminated protein hits identified in Mollusca, using human protein identifiers. (A) Local STRING network cluster; (B) PFAM and SMART protein domains; (C–F) biological GO processes: GO-1 (C), GO-2 (D), GO-3 (E), GO-4 (F). PPI enrichment p-value: 3.16 × 10⁻⁸. Color coding for network nodes and interaction lines is included in the figure.

Figure 9

STRING protein interaction networks for deiminated protein hits identified in Mollusca, using human protein identifiers. (A) Local STRING network cluster; (B) PFAM and SMART protein domains; (C–F) biological GO processes: GO-1 (C), GO-2 (D), GO-3 (E), GO-4 (F). PPI enrichment p-value: 3.16 × 10⁻⁸. Color coding for network nodes and interaction lines is included in the figure.