Elsevier

Fish & Shellfish Immunology

Volume 74, March 2018, Pages 386-392
Fish & Shellfish Immunology

Full length article
CgNrdp1, a conserved negative regulating factor of MyD88-dependent Toll like receptor signaling in oyster Crassostrea gigas

https://doi.org/10.1016/j.fsi.2017.12.057Get rights and content

Highlights

  • A highly evolutionary conserved Nrdp1 homologous gene was identified from Crassostrea gigas.

  • CgNrdp1 could directly bind CgMyD88 in an assay using recombinant proteins.

  • CgNrdp1 negatively controlled MyD88-dependent TLR signaling upon over-expression of CgMyD88.

  • CgNrdp1 also negatively regulated HsMyD88 dependent TLR signaling in HEK 293T cells.

Abstract

Toll like receptor (TLR) signaling cascades are under precise regulations to ensure the proper immune responses during various pathogen invasions. The neuregulin receptor degradation protein-1 (Nrdp1) has been demonstrated to be a novel negative regulator of TLR signaling by targeting MyD88 to induce degradation in mammals. In the present study, an Nrdp1 homologue, CgNrdp1, was identified from the genome of Pacific oyster Crassostrea gigas. It contained an open reading frame encoding a polypeptide of 315 amino acids which shared high identities with other homologues from different species. There was a conserved RING domain in CgNrdp1, indicating the functional E3 ubiquitin ligase activity. The bacterially expressed recombinant CgNrdp1 and CgMyD88 showed much stronger affinity compared to control groups in the ELISA assay, showing the interacting ability between CgNrdp1 and CgMyD88. When CgMyD88 or HsMyD88 was co-transfected with CgNrdp1 into HEK293T cells, the luciferase activities of NF-κB were significantly decreased compared to those in MyD88 single-transfection groups, indicating the conserved negative regulating function of CgNrdp1 on the MyD88 induced TLR signaling. These results indicated that CgNrdp1 was a negative regulator of TLR signaling in oyster and the Nrdp1-MyD88 axis was functional and highly conserved from mollusks to mammals in the negative regulation of TLR signaling.

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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