Full length articleCgNrdp1, a conserved negative regulating factor of MyD88-dependent Toll like receptor signaling in oyster Crassostrea gigas
Introduction
As a family of evolutionary conserved pattern recognition receptors (PRRs), Toll like receptors (TLRs) play vital roles in early host defense against various pathogenic invasions through their reorganizations of conserved pathogen-associated molecular patterns (PAMPs) and the activations of downstream signaling pathways [[1], [2], [3], [4]]. However, TLRs and TLR signaling also have devastating effects on the host. Imbalanced TLR signaling especially the exaggerated release of the inflammatory cytokines is correlated to the genesis of several diseases, such as autoimmune, chronic inflammatory and many other diseases [3,5,6]. As an example, the sepsis induced by lipopolysaccharide (LPS), a strong agonist of TLR4, was considered to be the consequence of hyperactivation of TLR signaling [7]. Therefore, TLR signaling pathways are under tightly negative regulations.
So far, many regulators of TLR signaling pathway with different regulatory mechanisms have been reported majorly in vertebrates, such as soluble decoy TLRs, myeloid differentiation factor88 short (MyD88s), suppressor of cytokine signaling 1 (SOCS1), neuregulin receptor degradation protein-1 (Nrdp1) and others [[8], [9], [10], [11], [12]]. Nrdp1, also named as fetal liver ring finger (FLRF) protein, is an E3 ubiquitin ligase that promotes ubiquitination and proteasomal degradation of targeted proteins [[13], [14], [15]]. The biological functions of Nrdp1 are well studied in mammals. Nrdp1 can directly bind the epidermal growth factor receptor 3 (ErbB3) and prompt its ubiquitination and degradation, through which Nrdp1 serves as an important factor suppressing cell growth [15]. Meanwhile, Nrdp1 triggers the apoptosis by degrading the inhibitor of apoptosis protein (IAP) and BRUCE/apollon in human embryonic kidney (HEK) 293T cells [16]. Consequently, loss-of-function of Nrdp1 leads to serious diseases due to uncontrolled cell proliferation-apoptosis balance, such as breast tumors [17]. Recently, Nrdp1 was identified as a novel regulator of TLR signaling pathways in mammals. It inhibited LPS-activated synthesis and secretion of pro-inflammatory cytokines like tumor necrosis factor (TNF) through directly binding MyD88 [12].
Pacific oyster Crassostrea gigas (Thunberg, 1793), a marine bivalve belonging to the phylum Mollusca, is considered as an important economical fishery and aquiculture animal around the world as well as a classical model to study various biological issues [18]. Recently, the canonical and primary TLR signaling pathway has been identified in mollusks [19,20], raising the question that whether Nrdp1 functions as a key regulator of TLR signaling in an evolutionary conserved manner. In the present study, an Nrdp1 homologue, CgNrdp1, was cloned from Pacific oyster and its potential negative regulating function of TLR signaling pathway was revealed by dual luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA). This study shed light on understanding of the regulating mechanisms of TLR signaling in invertebrates.
Section snippets
Immune stimulation of oyster
Pacific oyster, C. gigas, averaging 110 mm in shell height, were collected from a farm in Qingdao, China and maintained in the aerated seawater at 18 °C for two weeks before processing.
Eighty oysters were employed to the LPS stimulation experiment. Briefly, they were equally divided into two groups and each group included 40 individuals. Oysters from the two different groups received an intramuscular injection of 100 μL phosphate buffered saline (PBS, 0.14 M sodium chloride, 3 mM potassium
Molecular character of the ORF of CgNrdp1 cDNA
Through the whole genome screening, a homologue of Nrdp1 (OYG_10012383) was found in oyster C. gigas. The open reading frame (ORF) of CgNrdp1 was verified by sequencing.
The ORF of CgNrdp1 contained 948 bp which encoded a polypeptide of 315 amino acids with the predicted molecular mass of 36.00 kDa and theoretical isoelectric point of 5.90. No signal peptide was predicted in CgNrdp1. A RING finger domain (from Cys18 to Asp56) and an USP8-interacting domain (from Glu137 to Glu315) were identified
Discussion
The activation of TLR pathway is a double-edged sword [6]. It is essential to induce the innate response and boost the adaptive immunity against pathogens. However, members of the TLR family are also involved in the pathogenesis of various diseases, such as autoimmune, chronic inflammatory and infectious diseases. Therefore, TLR signaling cascades are under precise regulations. To our knowledge, there are few reports about the negative regulators of TLR signaling in invertebrates, especially in
Acknowledgements
The authors are grateful to all the laboratory members for the technical advice and helpful discussion. Special thanks go to Prof. Jiahuai Han from Xiamen University for his kindly providing the plasmid encoding HsMyD88. This research was supported by a grant (No. U1706204) from National Science Foundation of China, Dalian High Level Talent Innovation Support Program (2015R020), Aoshan Talent Cultivation Program Supported by Qingdao National Laboratory for Marine Science and Technology (No.
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