Oysters belong to the second largest animal

Oysters belong to the second largest animal phylum Mollusca, and they are globally distributed aquaculture animals (Wang et al., 2018). A number of studies have demonstrated that oysters have evolved an integrated and complex innate immune system to recognize and eliminate various invaders. An array of orchestrated immune reactions are also found in C. gigas, including immune recognition, signal transduction, synthesis of antimicrobial peptides, as well as encapsulation and phagocytosis of the circulating hemocyte (Wang et al., 2018). However, the involvement and function of ubiquitnation in immune response are still not well known in C. gigas. In the present study, an E2 UBC2G1 homolog CgUbe2g1 was identified from C. gigas. Its distribution in various tissues and its role in regulating the innate immune response were also determined, and these results would be valuable for understanding the function of ubiqitination pathway in the immune system of mollusc.
Materials and methods
Results
Discussion Ubiquitination is an important post-translational protein modification, which involves in a broad array of basic cellular processes including regulation of the cell cycle, differentiation and development, the cellular response to extracellular stress, DNA repair, and the immune and inflammatory responses (Ciechanover et al., 2000; Cheng et al., 2016; Finley and Chau, 1991; Ciechanover et al., 2000; Li et al., 2016). In mammals, ubiquitin system can activate the host innate immune responses by encompassing inflammatory signaling, phagosomal maturation, autophagy and apoptosis to eradicate the invading pathogens and reduce concomitant host damage (Zinngrebe et al., 2014; Li et al., 2016). Recently, an array of orchestrated immune reactions, including maternal immune transfer, immune priming, hematopoiesis, apoptosis, autophagy as well as the neuroendocrine-immune (NEI) regulatory work, have been well demonstrated in oyster (Wang et al., 2018). Although there are more ubiquitin-related molecules identified in oysters (Zhang et al., 2012; Cheng et al., 2016), the regulation function of ubiquitin system in immune responses of mollusc is still not well understood. In the genome of Pacific oyster C. gigas, there are four E1s, 31 E2s and 199 E3s, which is less than that in human genome (Li et al., 2016). In our previous study, the KN-92 phosphate of an E2 UBC2G1 homolog CgUbe2g1 was found to be up-regulated after pathogen infection. CgUbe2g1 was comprised with a conserved ‘Ubc’ domain and shared similar secondary structure with members of E2G1 family in mammals (Huang et al., 2009; Ye et al., 2013; Stewart et al., 2016). The 3D structural conformation of CgUbe2g1 was similar to that of E2G1 from H. sapiens except the residues Gly98-Trp111 and Asp130-Leu168. In the present study, in vitro ubiquitination assays revealed that CgUbe2g1 could bind Ub, indicating that the ‘Ubc’ domain in CgUbe2g1 had the conservative Ub binding activity. Additionally, CgUbe2g1 was observed to co-localize with Ub in the hemocyte of oyster, suggesting that CgUbe2g1 could bind to Ub in oyster. These results suggested that CgUbe2g1 had a more complex protein structure than that from higher animals, which endowed it with the activity to bind both E1-activated and un-activated Ub. As the critical part of ubiquitination, E2s also play important roles in innate immune response regulated by ubiquitin system (Zinngrebe et al., 2014; Li et al., 2016). It has been reported that E2s is necessary to activate the cytokine TNF and NF-κB signaling pathway, and then induce the inflammation response (Tokumoto et al., 2011; Zinngrebe et al., 2014). In the present study, CgUbe2g1 was found to mainly distribute in hemocyte, mantle, hepatopancreas, and male gonad. As most of these tissues were related with pathogen elimination and immune defense of mollusc (Song et al., 2015; Wang et al., 2018), the higher expression of CgUbe2g1 mRNA indicated that E2 molecule was involved in the immune defense of oyster. In mammals, TNF-α is one of important pro-inflammatory cytokines to activate several immune signaling cascades, such NF-κB and mitogen-activated protein kinases, MAPK. Recently, there are many reports employing human cell models to explore and verify the functions of genes from other lower organisms (De et al., 2011; Subbarayan et al., 2016; Wu et al., 2014; Xin et al., 2015; Zhang et al., 2016). In order to further confirm the critical function of CgUbe2g1, the expressions of TNF-α and NF-κB were examined after it was overexpressed in HEK293T cell line. The expression levels of TNF-α and NF-κB in HEK293T cell with CgUbe2g1 overexpression were up-regulated significantly after LPS stimulation. It has been reported that E2s regulate the expression of TNF-α to prime the inflammasome in mammals (Blackwell and Christman, 1997; Li et al., 2016). The present results indicated that CgUbe2g1 could regulate the expressions of TNF-α and NF-κB in mammal cells, and it was suspected that CgUbe2g1 might also be involved in the inflammatory response in mollucs.