Sulfide: Quinone Oxidoreductase From Urechis Unicinctus

Sulfide is a natural, widely distributed, poisonous substance. Urechis unicinctus which subjected to Echiuroidea, Echiurida, Xenopneusta, Urechidae, is a benthic organism, mostly living in sulfidic habitats including intertidal and subtidal zones. Previous study has proved the worm has strong sulfide endurance and detoxification ability. Mitochondrial sulfide oxidation is an important mechanism for reducing sulfide toxicity in sulfide-adapted animals. Sulfide:quinone oxidoreductase (SQR) is the first enzyme in mitochondrial sulfide oxidation enzyme system. In this study, we cloned the U. unicinctus SQR full-length cDNA by RACE techniques and obtianed the activated SQR by recombinant expression in Escherichia coli BL21 (DE3), and then analyzed its enzyme characteristic in vitro. Furthermore, we reported SQR gene expression at mRNA and protein level, and enzyme activity with and without sulfide exposure. The investigation of the U. unicinctus biological characteristic can deepen the understanding of sulfide metabolic molecular mechanism and provides more evidence to explore U. unicinctus as a model organism in sulfide metabolic research.The full-length of U. unicinctus SQR cDNA cloned by homology cloning approach combined with RT-PCR and 3'- and 5'-RACE was deposited in GenBank (accession number:EF487538) as a 2315 bp consisting of an open reading frame of 1356 bp encoding a putative protein of 451 amino acids, which had a theoretical pI of 8.98 and molecular weight of 50.5 kDa. The nucleotide and deduced amino acid sequence contained conserved FAD-binding domains (domainⅠ, DIVIVGGGCAGSAIANKFA PYLGQGKV; domainⅡ, GDKLKYDYLLVSMG; domainⅢ, NFVTVNRDTLQH TKYPNVFGMGD), cysteines (Cys202 and Cys380), conserved histidines (His80 and His294) and glutamic acid (Glu159).SQR mRNA was detected by real-time RT-PCR in all tissues examined, with the highest SQR mRNA expression in the mid-gut, followed by the anal sacs and coelomic fluid cells, finally, the body wall and hindgut with a relatively low level of expression.Prokaryocyte expression vector (pET28a-SQR/pET32a-SQR) was constructed by cloned U. unicinctus SQR open reading frame cDNA into pET28a/pET32a, and then transformed into E. coli BL21 (DE3) to get the recombinant protein. The SQR was expressed in the form of inclusion body in E. coli with the induction of IPTG by SDS-PAGE analysis. Recombinant protein was purified by Ni2+ -NTA affinity chromatography after the inclusion body dissolved in 8 mol/L urea. Dilution refolding was used to get the activated protein. The best refolding condition was determined as follow:pH 8.0, protein concentration 20μg/ml, L-arginine concentration 0.2 mol/L, reduced glutathione:oxidized glutathione=10:1, renaturation time 96 h. Activated SQR exhibited specific activity at 5.12μM/min/mg (25℃, pH 8.0), optimal temperature at 37℃, optimal pH of 8.5 and Km for ubiquinone and sulfide determined as 15.6μM and 40.3μM respectively from Lineweaver-Burk plotting. EDTA and GSH had an activating effect, whereas Zn2+ acted as an inhibitor decreasing the activity of this SQR.Rabbit antisera against the purified recombinant SQR were obtained with a titre of 1:96,000. Western blot showed that rabbit antisera reacted with the total protein and mitochondria from body wall samples yielding a single band of-60 kDa.SQR was detected by immunohistochemistry in all tissues examined which mainly detected in epithelium and in the free surfaces. In body wall samples, SQR was mainly detected in the free surfaces of simple columnar epithelium and underlying connective tissue. In hindgut, SQR was present in the free surfaces of pseudostratified columnar epithelium. Also, in mid-gut, SQR was present in the free surfaces of simple columnar epithelium. In anal sacs, SQR was localized in whole stratified epithelium. However, in all tissues, SQR was not detected in deep muscle and coelomic membranes or chorion.Real-time RT-PCR demonstrated that SQR mRNA expression was increased in a time-and concentration-dependent manner in body wall after sulfide exposure, and significantly increased at 12 h and continuously increased with time. In hindgut, the SQR mRNA expression showed a similar trend with body wall, but the expression level was higher.Indirect competitive ELISA was established to analysis SQR content in body wall, hindgut, anal sacs, mid-gut and coelomic fluid cells. The results revealed SQR content did not showed significantly difference from control in all tissue examined. SQR content in body wall showed a time dependent manner after sulfide exposure. After 50μM sulfide exposure, SQR content increased significantly at 12 h and reached the highest level at 24 h, and decreased at 48 h. After 150μM sulfide exposure, SQR content increased significantly at 2 h, and increased with time, then reached the highest level at 48 h. In hindgut, the SQR content showed a similar trend with body wall after sulfide exposure, but the expression level was higher. Exposed to 50μM sulfide, anal sacs SQR expression significantly increased until 6 h, and then, increased with time and reached the highest level at 24 h, and finally significantly decreased at 48 h. Exposed to 150μM sulfide, the SQR expression in anal sacs was not significantly increased from control. Exposed to sulfide in mid-gut, the SQR expression was not significantly increased. In coelomic fluid cells, after 50μM sulfide exposure, SQR expression significantly decreased until 6 h, and continued in low level at 48 h sulfide exposure. In 150μM sulfide group, at sulfide exposure for 2 and 24 h, the SQR expression significantly decreased.To further investigate the response of SQR to sulfide exposure, we determined SQR enzyme activity after sulfide exposure. The results indicated SQR enzyme activity was significantly increased in a time-dependent manner in body wall, hindgut and anal sacs after 50μM sulfide exposure. In body wall and hindgut, SQR enzyme activity significantly increased after initial 2 h exposure, subsequently, increased continuously and reached the highest level after 24 h, and then, decreased at 48 h. In anal sacs, SQR enzyme activity significantly increased after initial 6 h exposure, subsequently, increased with time and reached the highest level after 24 h, and then, decreased in 48 h. In mid-gut and coelomic fluid cells, SQR enzyme activity only increased after 50μM sulfide exposure for 48 h.Base on the results, we conclude sulfide oxidation is existed in several tissues in U. unicinctus. After sulfide exposure, body wall, hindgut and anal sacs play the major role in the mitochondrial sulfide adaptation, while mid-gut and coelomic fluid cells with the minor role. The results in this study provide valuable evidence for deep understanding of echiuran worm sulfide metabolic adaptation.