Full length articleMolecular characterization of p38 MAPK from blunt snout bream (Megalobrama amblycephala) and its expression after ammonia stress, and lipopolysaccharide and bacterial challenge
Introduction
Mitogen-activated protein kinase (MAPK) superfamily are members of serine/threonine protein kinase that play important roles in cellular response to extracellular stimuli [1]. This superfamily has four main subgroups including extracellular signal-regulated kinases (ERKs), c-jun N-terminal or stress-activated protein kinases (JNK/SAPK), ERK/big MAP kinase 1 (BMK1), and p38 MAPK [2,3]. p38 MAPK is involved in inflammation, environmental stresses and microbial infections in various organisms [[4], [5], [6]]. It has been demonstrated that p38 MAPK is activated during viral infection and replication in mammals [[7], [8], [9]]. Overexpression of p38 MAPK inhibits viral gene transcription and protein synthesis as well as apoptosis in fish cells [10]. So, p38 MAPK is probably involved in cellular immune responses to microbial infection in fish. On the other hand, fish is subjected to various environmental stresses such as ammonia stress which adversely impact fish immune function [11]. To date, there is little available information about the role of p38 MAPK in ammonia stress in fish.
Blunt snout bream (Megalobrama amblycephala) is an herbivorous freshwater fish native to China. Due to its fast growth, tender flesh, and high disease resistance, it has been a favorable candidate for aquaculture in China. However, it is prone to bacterial and viral diseases and is relatively sensitive to ammonia exposure [12]. Also, induction of oxidative stress and inflammation by dietary oxidized lipids has been a common issue in its culture [13]. Therefore, to overcome such issues in M. amblycephala culture research on immune-related and anti-stress genes is imperative. The goals of the present study were to: (1) achieve the molecular characterization of p38 MAPK; (2) examine p38 MAPK expression following ammonia exposure and Aeromonas hydrophila challenge; and (3) explore the role of p38 MAPK in inflammation induced by lipopolysaccharide (LPS). The outcome of this research could contribute to our understanding on fish resistance against environmental stresses and bacterial infection.
Section snippets
Experimental fish and sample collection
Juvenile blunt snout bream (mean body weight, 50 g) were used to clone p38 MAPK sequence and study its expression after ammonia stress and bacterial challenge. Fish were obtained from a commercial farm in Wuhan (Hubei province, China) and reared in a recirculating aquaculture system in laboratory under the following conditions: water temperature, 25–27 °C; DO, 5.0–6.0 mg l−1; pH, 7.2–7.6; photoperiod, 12: 12 h (dark: light). During the adaptation period fish were fed a commercial diet twice
Cloning and characterization of p38 MAPK
The full length cDNA of p38 MAPK is 2419 bp containing 5′ UTR of 307 bp, open reading frame (ORF) of 1086 bp and 3′UTR of 1026 bp. This cDNA was submitted to GenBank with accession number of MH791036. The initiation codon (ATG) and stop codon (TAA) present at positions 308 bp and 1394 bp. The ORF encodes a predicted protein of 361 amino acids with no signal peptide. The calculated molecular mass is 41.48 kDa and the estimated pI is 5.10. There are three catalytic sites at residues 72, 151, 153.
Discussion
In the present study, p38 MAPK was cloned from liver of M. amblycephala. The ORF of p38 MAPK encodes a predicted protein of 361 amino acids with no signal peptide. As reported for all the p38 subfamily members, the dual phosphorylation site of TGY motif and substrate binding site of ATRW are highly conserved [20,21]. The dual phosphorylation of both Thr and Tyr in TGY motif is required for all p38 MAPK activation. The ATRW domain is the kinase interaction motif (KIM) docking site binding to the
Acknowledgments
This work was funded by Nature Science Foundation of Fujian Province (2017J05056), Central Public-interest Scientific Institution Basal Research & Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, P. R. China, CAFS (NO. 2018HY-XKQ01) and PHD Support Program from Henan University of Science and Technology (China, 13480074). Also, the financial support of the Ministry of Education, Youth and Sports of the Czech Republic – projects “CENAKVA” (No.
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