The expression of superoxide dismutase in Mytilus coruscus under various stressors

Highlights

• Intracellular Cu/Zn SOD and Mn SOD were identified from Mytilus coruscus.

• The functional domains and some crucial amino acids in two SODs were conserved.

• They were constitutively expressed in different tissues but highest in gill.

• Their temporal expression were up-regulated under various stressors.

• The results might be helpful to comprehend immune response of SOD in bivalve.

Abstract

Superoxide dismutases (SODs), a by-product of antioxidative defence system, protects organisms for eliminating excess reactive oxygen species (ROS) and maintaining the redox balance of immune system. The complete open reading frames (ORFs) of Cu/Zn-SOD and Mn-SOD were identified from Mytilus coruscus (designated as McSOD and MnSOD) by homologous cloning. The sequence lengths were 474bp and 687bp, encoding 157 and 228 amino acids respectively. The deduced amino acid sequences of McSOD and MnSOD shared high identities with Cu/Zn-SOD and Mn-SOD from other mollusca. The distributions of McSOD and MnSOD were detected in six tissues including adductor, hemocyte, gill, gonad, mantle and hepatopancreas, and the highest expressions were both in gills. The temporal expression of McSOD and MnSOD were up-regulated in gills under a variety of stress factors, including Vibrio parahemolyticus, Aeromonas hydrophila, Cu²⁺ and Pb²⁺. After being challenged with V. Parahemolyticus, the expressions of McSOD and MnSOD were increased rapidly at the initial hours, reaching the peaks of 4.9-fold and 15.3-fold respectively, and got to the highest levels of 43.5-fold and 7.1-fold after being challenged with A. hydrophila. The highest point of McSOD mRNA appeared at 15 d after being exposed to copper (7-fold at 0.5 mg/L and 13.2-fold at 1.5 mg/L), except for 0.1 mg/L group of Cu²⁺ maintaining to the normal level, but plumbum at 1 d (2.4-fold at 1.0 mg/L and 4.4-fold at 3.0 mg/L) and at 15 d (2.1-fold at 0.2 mg/L). The temporal expression peaks of MnSOD appeared differently after exposing to copper of various concentrations (0.1 mg/L at 10 d with 4.7-fold, 0.5 mg/L at 1 d with 17.9-fold and 1.5 mg/L at 3 d with 13.2-fold). Whereas in plumbum exposing treatments, the 3.0 mg/L group jumped to the peak at 1 d (18.2-fold), the 0.2 mg/L and 1.0 mg/L groups had little change and maintained at the normal level throughout the experiment. The results provided several new evidences for further understanding of the regulatory mechanism of SOD on the innate immune system in bivalve.

Introduction

As an essential response formation of innate immunity, the reactive oxygen species (ROS) response is generated from large amounts factors such as diseases, pathogenic organism, physical or chemical contamination, as well produces following single electron reductions of molecular oxygen [1], [2]. The ROS plays an important role in regulating several physiological processes at low concentration, whereas at higher concentration they impair cellular functions by oxidative damages [3], [4]. In addition, the mass accumulated ROS will cause immune dysfunction and damage to cell structure including lipid, proteins and nucleic acids [5]. Consequently, for normal cell function and organism survival, several key enzymes, such as superoxide dismutases (SODs), catalases (CATs), glutathione S-transferases (GSTs), glutathione peroxidase (GSH-PX) and glutathione reductase (GSR) are employed to maintain the ROS at physiologically optimal levels in organisms [6].

Superoxide dismutases (SODs; EC 1.15.1.1) belong to a significant and ubiquitous family of metalloenzymes for eliminating excess ROS which can be found in virtually all oxygen-consuming organisms, aero-tolerant anaerobes and some obligate anaerobes [7], [8]. For preventing generation of highly toxic OH⁻, SODs catalyze the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide [9]. Among the antioxidant enzymes, both copper-zinc and manganese superoxide dismutases (Cu/Zn-SOD and Mn-SOD) are assumed to play key roles in protecting tissues from oxidative injuries [10], [11], [12], and they could be found in both prokaryotes and eukaryotes [13], [14]. Cu/Zn-SOD had been proved to be a considerably important type in SOD superfamily for its physiological function and therapeutic potential [15], which was one of the most important free radical scavengers responding to oxidative stress [16]. Mn-SOD was regarded as a precursor protein which was produced in mitochondria and played vital roles in cytoplasm after cleaving the signal peptide [17]. Many studies have demonstrated the importance of SODs to the immune response as well as the role in protecting host against virus [18], [19], bacteria [20], [21], parasites [20], physical and chemical challenges [22], [23].

In recent years, marine pollution has become more and more serious than before, aquatic organisms are now suffering from increasing environmental stresses [24]. For the sensitive reaction to marine or aquatic environment, marine bivalves in mollusks have been regarded as the ideal indicators in the assessment of environmental pollution because they are sedentary, ubiquitous, filter-feeders inhabiting in the coastal and estuarine areas [25]. The effects of pollutants on the immune and stress responses have been reported in many bivalve species, such as Mytilus edulis, Mytilus galloprovincialis, Crassostrea virginica, Crassostrea gigas, Ostrea edulis, Crassostrea angulata and Haliotis rufescens [26]. M. coruscus is an important economic shellfish among aquatic invertebrates along the eastern coast of China, especially in Zhoushan (China) coast [27]. After having flourished for several years, M. coruscus industry has become one of the mainstay maricultures in Zhejiang province of China. But recently, higher mortality rates caused by pathogenic bacterium and heavy metals have a serious effect on the production of M. coruscus. However, the research information of stress in response to marine pollution in this species is still limited. That understanding of immunologic mechanism of M. coruscus may contribute to the development of strategies for the management of diseases and long-term sustainability of its culture. In addition, much more attentions have been paid to SODs for its multiple functions than just for itself [11]. Therefore, the objectives of this study are: (1) to clone the ORFs of Cu/Zn-SOD and Mn-SOD from M. coruscus (named as McSOD and MnSOD); (2) to examine the expressions of McSOD and MnSOD in various tissues; (3) to investigate McSOD and MnSOD expressions after being stressed by V. parahemolyticus, A. hydrophila, Cu²⁺ and Pb²⁺.