Research paperEffect of tributyltin chloride (TBT-Cl) exposure on expression of HSP90β1 in the river pufferfish (Takifugu obscurus): Evidences for its immunologic function involving in exploring process
Introduction
Heat shock proteins (HSPs) are a series of special proteins which could generate and be activated in the environment of heat stress and other biological stresses (Ritossa, 1962). HSPs are also called stress proteins (SPs) due to taking part in regulating other stress responses such as oxidative stress, heat, infection, toxicosis and so on (Erlejman et al., 2014a; Sørensen et al., 2003). It has been proved that HSPs have imperative roles in inhibiting protein aggregation, helping in folding the nascent proteins, and are considered to protect cells against oxidative stress (Fu et al., 2011; Jiang et al., 2012; Parsell and Lindquist, 1993). HSPs are a cluster of highly conserved molecular chaperones which were ubiquitously expressed in tissues. They are segmented into distinct multigenic families, like HSP110, HSP90, HSP70, HSP60, HSP40 and other small HSPs. Among them, HSP90 is often found in a constitutive dimmer, which participates in controlling multiple regulatory pathways such as stress defense and apoptosis (Rajeshkumar et al., 2013). In Crassostrea hongkongensis, HSP90 plays a vital role in response to both osmotic stress and bacterial invasion (Fu et al., 2011). For many fish species, the HSP90 have been connected to cytoprotection and cell survival (Csermely et al., 1998; Smith et al., 2015), performing a protective and inducible role (Xu et al., 2014; Zhang et al., 2015). HSP90 in the pufferfish liver was also found induced by ammonia stress, indicating that this kind of protein hammered at protecting body from oxidative stress and apoptosis (Cheng et al., 2015). It's worth noting that HSP90β1 (GP96), a subtype of HSP90 members, associated with major histocompatibility complex (MHC) class I molecule, which indicated it might be involved in immune response (Suto and Srivastava, 1995). The mRNA level of HSP90β1 preferentially expressed in hepatocellular carcinoma and significantly increased in hepatoma cell line. Its expression had a down-regulation when the oncocytes differentiation was inducted by sodium butyrate. This indicated that HSP90β1 had correlations with occurrence and development of cancer and cell differentiation (Cai et al., 1993; Heike et al., 2000b). Studies had shown that some stress factors made increases of expression level for GP96, in the meanwhile, its immunogenicity was also aggrandized and rose with the expression level increasing (Dai et al., 2003). In the process of autoimmunity, the cell surface expression level of an endoplasmic reticulum-dependent GP96 initiated systemic autoimmune diseases in the body (Liu et al., 2003).
Tributyltin chloride (TBT-Cl) is one of the most representative chemical compounds of Tributyltin (TBT). In view of its fatal toxicity to hydrobios, TBT-Cl was severed as a threat to water security (Antizar-Ladislao, 2008; Organization, 2001). TBT residual in water from various channels had become a noticeable problem, which made TBT contamination for aquatic ecosystems (Antizar-Ladislao, 2008; Tessier et al., 2007). TBT induced imposex in mollusks and fishes, which suggested that this toxic substance exerted a force on aquatic animal gonad function (Matthiessen, 2008; Mcallister and Kime, 2003; Nakayama et al., 2004; Shimasaki et al., 2003). Furthermore, TBT was found to be an inducer in the accumulation course of adipose and altered fatty acid levels in Marisa cornuarietis (Inadera and Shimomura, 2005; Janer et al., 2007; Meador et al., 2011). In zebra fish, TBT indeed altered multiple and complex activities of mRNA level in lipid metabolism and cell damage, which implied the underlying molecular mechanism of TBT on hepatic steatosis (Zhang et al., 2016).
Takifugu obscures, commonly known as river pufferfish, is an anadromous fish and an economic species. Studies on pufferfish aquaculture and its ecological environment have been regarded as a hot topic in the meanwhile (Kai et al., 2005; Van, 2004; Yamanoue et al., 2009). The pufferfish are important and scarce sources at the lower reach of the Yangtze River and the river mouth area in China. T. obscures was always chosen as a model to explore its adaptive and resisting mechanisms when exposed to different kinds of environmental stress factors (Kato et al., 2005; Kim et al., 2010b). However, the physiological function of the pufferfish under the TBT-Cl exposure keeps unclear. It's attractive to us that studying the mechanism of TBT-Cl exposure in T. obscures may have a profound meaning.
In this regard, HSP90β1 gene in T. obscures from transcriptome sequencing databases was characterized by bioinformatic analysis, and the phylogenetic tree was constructed based on HSP90 sequences of other species. Tissues expressions were detected by quantitative real-time PCR (qPCR) method. After exposing to different concentrations of TBT-Cl in the acute and chronic experiment, the HSP90β1 mRNA level was checked through qPCR. The histochemistry and IHC test were performed to verify the damaging effect of TBT-Cl to the pufferfish. This study may supply a deep understanding for the unique function of HSP90β1 in the course of fighting with the adverse effect of TBT-Cl and explain the conceivable mechanism in immunoreaction.
Section snippets
Animals
T. obscurus (two months old, average length = 10 ± 1.5 cm with average weight = 25.1 ± 2.23 g) were obtained from the aquaculture base in Freshwater Fisheries Research Center (FFRC, Wuxi, China). The pufferfish were kept in 100 L cylindrical opaque polypropylene aquaria and supplied with commercial feed twice a day at regular intervals. After 7-day acclimation, robust animals were chosen until 24 h feeding before the experimental treatment. The water was exposed to air for a week to remove
The characterization and phylogenetic analysis
After exploring the transcriptome libraries, a member of To-HSP90 was determined: To-HSP90β1 (GeneBank accession number: MG597234). The full length was obtained as 2775 bp in length; with a 2412 bp ORF, which encoded an 803 aa polypeptide with 92.36 kDa molecular mass and 4.72 pI. Besides, it contained a 112 bp of 5′-UTR (untranslated region) and a 251 bp of 3′-UTR (Fig. 1). After the InterPro sequence search, four homologous superfamilies were found in To-HSP90β1: two Histidine
Discussion
As a stress-sensitive molecular chaperone, HSPs played essential roles in a series of metabolism processes (Xie et al., 2015). A lot of treatments on cells activated the expression of HSP genes (Song et al., 2016). HSP90β1, a special actor in stress response, is an ER-enriched distributed protein which participates in associating with neonatal or abnormal proteins, assisting repair and thermo-resistance of cells (Berwin et al., 2002). Moreover, its expression on the cell surface was deemed to
Acknowledgments
This work was supported by funds from the National Infrastructure of Fishery Germplasm Resources (2017DKA3047-003) and the Fund from the Anhui Province Key Laboratory of Conservation and Utilization of Important Biological Resources (PKLCU201705).
Author contributions
Xu Dong-po was responsible for data scoring and analyses, and writing the manuscript. Hu Hao-yuan conceived and designed the experiments. Fang Di-an, Zhao Chang-sheng and Jiang Shu-lun helped selecting the pufferfish tissues sample, RNA extraction and data analysis during manuscript preparation. All authors have read and approved the final manuscript.
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