An iodothyronine deiodinase from Chlamys farreri and its induced mRNA expression after LPS stimulation
Highlights
► An iodothyronine deiodinase (CfDx) from scallop Chlamys farreri. ► The CfDx mRNA transcripts and concentration of T3 increased significantly after LPS stimulation. ► The ratio of T4/T3 rose significantly after the inhibition of CfDx gene. ► CfDx involved in the immunomodulation of scallop via regulating the concentration of T3 and T4.
Introduction
The neuroendocrine and immune systems constitute the neuroendocrine-immune regulatory network to maintain host homeostasis in vertebrates through neurotransmitter, hormone and cytokine [1]. In the network, the neuroendocrine system releases neurotransmitter and hormone to modulate the immune system, while the immune system influences the neuroendocrine system through cytokines [2]. In vertebrates, the central nervous system is able to modulate immune response to limit inflammation-induced damage [3]. To date, some analogous neuroendocrine-immune regulatory networks have been also reported in invertebrate [4], [5], [6], and the molecules and basic mechanism involved in the network are found to be well conserved and fundamentally similar throughout evolution.
In the neuroendocrine-immune regulatory network, thyroid hormones play important roles in regulating the immune response including inflammation, natural killer cell activity, antiviral action of IFN, and the proliferation of T and B lymphocytes [7], [8]. Thyroid hormones are released by the thyroid gland or immunocytes after immune stimulation to bind thyroid hormone receptors located in immunocytes to regulate the expression of the immune-related genes in vertebrates [9]. Thyroid hormones have also been detected in some invertebrates, such as echinoid and anthozoan [10]. There are abundant evidence to support that thyroid hormones could modulate metamorphose and development of invertebrates [10], [11]. However, the modulation of thyroid hormones to the immune response is far from well understood [12], [13].
The iodothyronine deiodinase is responsible for the deiodination of thyroid hormone T4 to T3, and plays central role for the regulation of thyroid hormones level. According to the biochemical characteristic of iodothyronine deiodinase, the iodothyronine deiodinase can be divided into three types (D1, D2, and D3). In all the reported three types, there is an in-frame UGA codon to encode a selenocysteine (SeC) codon in the catalytic site [14]. D1 and D2 produce receptor-active T3 in the outer ring of the T4 molecule, and inner ring deiodination is catalyzed by D3. D3 can also deiodinate T3 and thereby terminate thyroid hormone action [15]. Iodothyronine deiodinase has been considered to be involved in the immune responses of vertebrates [16]. For example, these iodothyronine deiodinases can be expressed in immunocytes, and their expression level is able to be modulated by a wide variety of cytokines and immune factors such as IL-1, IL-6 and NF-κB [17], [18]. Iodothyronine deiodinase genes have been identified in vertebrates such as fish, amphibians, and mammals [19], [20], whereas only a homologue of iodothyronine deiodinase (HrDx) has been found in invertebrate Halocynthia roretzi. The knowledge is quite meagre on the characteristics of iodothyronine deiodinase and its possible immunomodulation in invertebrates.
Zhikong scallop Chlamys farreri is one of the important cultured mollusc species in North China. Severe mortality of scallop has resulted from disease in recent years. The investigation of neuroendocrine and immune system could provide more efficient knowledge for the management of scallop health during stress or infection. The purposes of this study were to (1) clone iodothyronine deiodinase from C. farreri (designated as CfDx); (2) detect CfDx mRNA distribution in different tissues and its temporal expression in haemocytes as well as the change of T4, T3 concentration in haemolymph after LPS stimulation; (3) investigate the modulation of CfDx on T4 and T3, and to provide information about the function of CfDx in the neuroendocrine-immune regulatory network of scallops.
Section snippets
Scallop, tissue collection, LPS and dsRNA stimulation
Scallops C. farreri with an average shell length of 55 mm, were collected from a farm in Qingdao, Shandong Province, China, and maintained in the aerated seawater at 20 °C for 10 days before processing.
Six tissues including hepatopancreas, kidney, adductor muscle, gonad, gill and mantle were collected from six healthy adult scallops to examine the mRNA transcript of CfDx. Haemolymph from these six scallops was also collected from the adductor muscle and then immediately centrifuged at 800 g,
Molecular characteristics of CfDx cDNA
A full-length CfDx cDNA of 1404 bp was obtained by overlapping the amplified segment with EST rscag0_008458, and deposited in GenBank under accession number JN604668. It included a 5′ untranslated region (UTR) of 391 bp, a 3′ UTR of 113 bp with a poly (A) tail, and an open reading frame (ORF) of 900 bp encoding a polypeptide of 299 amino acids. The nucleotide of the CfDx cDNA and its deduced amino acid sequences were shown in Fig. 1, which contained an in-frame TGA stop codon probably encoding
Discussion
In the present study, an iodothyronine deiodinase gene was cloned and characterized from scallop C. farreri, and its full-length cDNA was of 1404 bp. There was a conserved in-frame TGA stop codon which could code the essential Sec residue for the activity of iodothyronine deiodinase. This region might serve as the main active centre of CfDx, and it was highly conserved in all the known iodothyronine deiodinase from vertebrate and invertebrate. CfDx shared low similarity 19.1%–23.9% with
Acknowledgements
The authors were grateful to all the laboratory members for continuous technical advice and helpful discussion. This research was supported by 973 National Key Fundamental Research Program (No. 2010CB126404) from the Chinese Ministry of Science and Technology, and grants from NSFC (No. 31072192 to L.W., 30925028 to L.S.) and Shandong Provincial Natural Science Foundation (No. JQ201110 to L.W.).
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