title: Molecular identification and functional delineation of a glutathione reductase homolog from disk abalone (Haliotis discus discus): Insights as a potent player in host antioxidant defense.
abstract: Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H2O2, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H2O2. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171-246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu+2and Cd+2ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: H M L P B
authors.initials: HMLPB
authors.lastname: Herath
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: P D S U
authors.initials: PDSU
authors.lastname: Wickramasinghe
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: S D N K
authors.initials: SDNK
authors.lastname: Bathige
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: R G P T
authors.initials: RGPT
authors.lastname: Jayasooriya
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: Gi-Young
authors.initials: GY
authors.lastname: Kim
authors.affiliation: Southeast Sea Fisheries Research Institute, National Institutie of Fisheries Science, Tongyeong-si, Gyoengsangnam-do, 53085, Republic of Korea.
authors.forename: Myoung Ae
authors.initials: MA
authors.lastname: Park
authors.affiliation: Informatics Development & Management Group, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon, 34054, Republic of Korea. Electronic address: chulnice@kiom.re.kr.
authors.forename: Chul
authors.initials: C
authors.lastname: Kim
authors.affiliation: Department of Marine Life Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea. Electronic address: jehee@jejunu.ac.kr.
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005260:Female; D049851:Gastropoda / Q000187:drug effects / Q000235:genetics* / Q000276:immunology* / Q000382:microbiology; D005980:Glutathione Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000494:pharmacology; D008089:Listeria monocytogenes / Q000502:physiology; D008297:Male; D019216:Metals, Heavy / Q000633:toxicity; D018384:Oxidative Stress; D010802:Phylogeny; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000662:veterinary; D014736:Vibrio parahaemolyticus / Q000502:physiology; D014874:Water Pollutants, Chemical / Q000633:toxicity
chemical_list: D000975:Antioxidants; D018076:DNA, Complementary; D008070:Lipopolysaccharides; D019216:Metals, Heavy; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014874:Water Pollutants, Chemical; D005980:Glutathione Reductase
keywords: Abalone; Antioxidant activity; Biochemical properties; Glutathione reductase; Transcriptional analysis