Molecular characterization and functional analysis of two phospholipid hydroperoxide isoforms from Larimichthys crocea under Vibrio parahaemolyticus challenge

Highlights

• The full-length cDNA of LcGPx4a and LcGPx4b was identified in Larimichthys crocea.

• LcGPx4a and LcGPx4b have a wide distribution in all examined tissue and a high relative expression level in the liver.

• After Vibrio parahaemolyticus challenge in vivo, the relative expression level of LcGPx4a were significantly up-regulated in the spleen and kidney, and down-regulated in the liver, and displayed diverse changes in the head kidney.

• The relative expression level of LcGPx4b was significantly down-regulated in the liver and kidney, and up-regulated in the spleen, and also displayed diverse changes in the head kidney under the Vibrio parahaemolyticus challenge in vivo.

Abstract

Glutathione peroxidases family is a key role in the antioxidant system in oxybiotic organisms for cell redox homeostasis. One of their members, phospholipid hydroperoxide glutathione peroxidase (GPx4) have unique monomeric structure and can directly react with complex lipid and membrane-bound peroxides under the presence of glutathione(GSH). In this paper, two complete GPx4 cDNAs (designated as LcGPx4a and LcGPx4b) from Larimichthys crocea are identified by rapid amplification of cDNA ends. The cDNA of LcGPx4a was consisted of a 5′-untranslated region (UTR) of 258 bp, a 3′-UTR of 330 bp, and an open reading frame (ORF) of 561 bp encoding 186 amino acid (aa) polypeptides. And the full-length sequence of LcGPx4b was 1164 bp with a 5′-UTR of 34 bp, a 3′-UTR of 551 bp and an ORF of 576 bp encoding a polypeptide of 191 aa residues with a predicted signal peptide of 15 aa. The characteristic selenocysteine insertion (SECIS) sequence was detected in the 3′UTR of the two sequences with 78 bp in length. The conserved active site of selenocysteine (Sec) encoded by TGA was also identified and formed a tetrad functional structure with glutamine, tryptophan, and asparagine in LcGPx4a and LcGPx4b. Two signature site motifs (“LRILAFPSNQFGNQEPG” and “LRILGFPCNQFGGQEPG”) were both conserved in the deduced amino acid of LcGPx4a and LcGPx4b. The genomic structure analysis revealed that the two sequences both had 7 exons and 6 introns, and the Sec opal codon and SECIS element were located at the third and seventh exons, respectively. LcGPx4a and LcGPx4b both have a wide distribution in 9 tissues with various relative expression levels and a highest expression pattern in the liver. Under Vibrio parahaemolyticus challenge, their relative expression levels were altered in the liver, spleen, kidney, and head kidney but with different magnitudes and response time. LcGPx4a and LcGPx4b showed a significantly up-regulated trend in the spleen during experimental period. Above results suggested that LcGPx4a and LcGPx4b were two conserved immune molecules and might play a role in the immune response of fish with a tissue-depemdent manners.

Introduction

Glutathione peroxidases (GPxs) are a key component of antioxidant enzyme family that have been discovered in various forms of life [1]. They reduce hydrogen peroxide (H2O2) or organic hydroperoxides to water or corresponding alcohols typically utilizing glutathione(GSH), which acts as a reductant and is simultaneously oxidized to glutathione disulfide(GSSG). Nowadays, 8 members of GPxs have been identified in mammal on the basis of their structure, cellular expression and specific function [2,3]. These members can be classified into two types: selenium-dependent and selenium-independent forms [4]. Selenium-dependent GPxs (Se-GPxs), including GPx1, GPx2, GPx3, GPx4 and GPx6, have a vital enzymatic site: selenocysteine(Sec), whereas selenium-independent GPxs (GPx5, GPx7 and GPx8) use cysteine as the enzymatic site instead of Sec [5]. Notable is, Sec is encoded by an opal (UGA) codon which typically means stop and causes termination of translation [6]. Therefore, organisms had developed a mechanism that UGA meaning Sec recognized by a specific tRNA^(Ser)Sec when a particular stem loop, called selenocysteine insertion sequence (SECIS), forms in downstream or the 3′untranslated region (UTR) during the transcript [5]. In organisms development, Se-GPxs take a fundamental part in maintaining the homeostasis of reactive oxygen species(ROS), whereas selenium-independent GPxs function as a backup system [7].

Differ from GPx1, GPx2, GPx3 and GPx6, which are homotetramer, phospholipid hydroperoxide (GPx4) is monomeric and membrane-associated [8]. GPx4 had been initially characterized as lipid peroxidation inhibiting protein because it could react with, besides H₂O₂ and small hydroperoxides in general, hydroperoxides in complex lipids, including phospholipid, cholesterol and cholesterol ester hydroperoxides, even when they are inserted into bio-membranes or lipoproteins [9,10]. In particular, its substrate is that protein thiols can replace the function of GSH as reductants when the latter becomes limited [11,12]. Thus, compared to other members, GPx4 can either act as a GSH or a thiol peroxidase according to the availability of GSH. In mammals, GPx4 exists in three isoforms: cytosolic, mitochondrial and sperm nuclear GPx4, which are derived from the same gene [13]. Cytosolic GPx4 is widespread in cells and play a vital role in life, which had been proved that the knockout of it could cause mice death [14]. And mitochondrial and sperm nuclear GPx4 are mainly expressed in testis with less amounts in other tissues, which take a crucial structural role in spermatogenesis [[15], [16], [17]].

To date, GPx4 has been characterized in some fishes, such as Danio rerio [18], Thunnus maccoyii [19], Seriola lalandi [20], Cyprinus carpio [21], Oncorhynchus mykiss [22], Oncorhynchus kisutch [23] and Sparus aurata [24]. It was well-known that GPxs members could be used as an indicator, which evaluate the effect of different environmental factors to fishes [25]. For example, the GPx4 expression pattern of C. carpio and O. kisutch have occurred significantly varied under cadmium exposure [21,23]. Also, several physical factors could lead to the variant of GPx4 expression pattern in fish, like confinement and cold stress [21,24]. In microbial infected-organism, GPxs also exhibited an up-regulated expression patterns [26,27]. In fact, abundant ROS production could benefit to resist pathogens due to the raised activity of macrophage [[28], [29], [30]]. And the excessive ROS generation would damage to organisms, and GPxs acted as a regulator to maintain the ROS balance in organism [31]. Studies on the function of GPx4 suggested that it was not merely prevents lipid peroxidation, but participated in cellular inflammation, apoptosis and signal transduction [32]. The loss of GPx4 activity have a strong association with some diseases, such as male infertility and arteriosclerosis [33]. Thus, it was necessary to understand the role of GPx4 in the immune response of fish under pathogen challenge.

Larimichthys crocea, previous known as the large yellow croaker, belongs to Sciaenidae under Osteichthyes. It has a large economic marine market and been widely cultured in southeast China with good nutritional quality and palatability [34]. However, the production of L. crocea is now decreasing due to overfishing and diseases caused by pathogens such as vibrio, parasite and virus [[35], [36], [37], [38]]. Therefore, what the correlation between the antioxidative and immunity system against the pathogens need to be studied. In this study, two complete cDNA sequences of phospholipid hydroperoxide from L. crocea (designated as LcGPx4a and LcGPx4b, respectively) were cloned. Their molecular features, gene structures and motif regions were characterized compared to other known GPx4 genes. Also, their expression patterns of different tissues and temporal expression profiles at mRNA level after being challenged by Vibrio parahaemolyticus are investigated by real-time reverse transcriptase PCR. Above results can provide new perspectives into the role of two GPx4 isoforms in the potential regulatory mechanism of the antioxidative and immunity system.