A copper-zinc-superoxide dismutase (CuZnSOD) from redlip mullet, Liza haematocheila: Insights to its structural characteristics, immune responses, antioxidant activity, and potent antibacterial properties.

Copper-zinc superoxide dismutase (CuZnSOD) is a nuclear-encoded metalloenzyme responsible for scavenging harmful reactive oxygen species (ROS). In this study, the CuZnSOD homolog from redlip mullet (Liza haematochelia) (MuCuZnSOD) was structurally and functionally characterized to evaluate its antioxidant capacity, antibacterial properties, and protective level in various pathogenic stress conditions. Structural characteristics of MuCuZnSOD were evaluated using different bioinformatics tools. Pairwise sequence comparison and evolutionary tree structure revealed that the MuCuZnSOD sequence was closely related to the CuZnSOD sequence of Oplegnathus fasciatus with a 94.2% sequence identity. Sequence alignment analysis indicated that the CuZnSOD domain was well conserved. The highest transcriptional expression of MuCuZnSOD was identified in the blood. Immune challenge with lipopolysaccharide (LPS), Lactococcus garvieae, and polyinosinic-polycytidylic acid (poly I:C) exhibited an increased MuCuZnSOD mRNA expression in the blood and liver. Transfected green fluorescent protein-fused MuCuZnSOD was localized in the cytoplasm. Recombinant MuCuZnSOD (rMuCuZnSOD) was overexpressed in a bacterial system. The rMuCuZnSOD possessed significant antioxidant properties as determined by conventional xanthine oxidase assay. The optimum pH and temperature of rMuCuZnSOD were found to be pH 9 and 25 °C, respectively. rMuCuZnSOD enzyme activity increased in a concentration-dependent manner. Treatment with potassium cyanide highly inhibited the rMuCuZnSOD activity. rMuCuZnSOD possessed a significant peroxidation activity in the presence of HCO3- ions as demonstrated by the increased viability in cells treated with the enzyme in the presence of hydrogen peroxide. Antibacterial assays showed that rMuCuZnSOD had significant growth-inhibitory effects on both gram-positive and gram-negative bacteria. Collectively, our findings demonstrate that MuCuZnSOD is an essential antioxidant protein, which regulates the host defense mechanisms and innate immunity under oxidative stress.

The possible role of catalase in innate immunity and diminution of cellular oxidative stress: Insights into its molecular characteristics, antioxidant activity, DNA protection, and transcriptional regulation in response to immune stimuli in yellowtail clownfish (Amphiprion clarkii).

Catalase, a key enzyme in the antioxidant defense grid of organisms, scavenges free radicals to curtail their harmful effects on the host, supporting proper immune function. Herein, we report the identification and characterization of a catalase homolog from Amphiprion clarkii (ClCat), followed by its functional characterization. An open reading frame was identified in the cDNA sequence of ClCat at 1581 bp, which encodes a protein of 527 amino acids (aa) with a molecular mass of 60 kDa. In silico analyses of ClCat revealed characteristic features of the catalase family and a lack of a signal peptide. Multiple sequence alignment of ClCat indicated the conservation of functionally important residues among its homologs. According to phylogenetic analysis, ClCat was of vertebrate origin, positioned within the teleost clade. During native conditions, ClCat mRNA was highly expressed in blood, followed by the liver and kidney. Moreover, significant changes in ClCat transcription were observed after stimulation with LPS, poly I:C, and Vibrio harveyi, in a time-dependent manner. Recombinant ClCat (rClCat) was characterized, and its peroxidase activity was determined. Furthermore, the optimum temperature and pH for rClCat were determined to be 30-40 °C and pH 7, respectively. Oxidative stress tolerance and chromatin condensation assays indicated enhanced cell survival and reduced apoptosis, resulting from reactive oxygen species scavenging by rClCat. The DNA-protective function of rClCat was further confirmed via a metal-catalyzed oxidation assay. Taken together, our findings propose that rClCat plays an essential role in maintaining cellular oxidative homeostasis and host immune protection.

Quadruple domain-containing galectin from marine invertebrate disk abalone (Haliotis discus discus): Molecular perspectives in early development, immune expression, and potent antiviral responses.

Galectins are well-known ß-galactoside-binding proteins, which play vital roles in innate immune responses of both vertebrates and invertebrates. However, knowledge regarding invertebrate galectins is still in its infancy. With the intention of filling the knowledge gap, here we identified a quadruple domain-containing galectin from marine invertebrate disk abalone, Haliotis discus discus (AbGalec), and characterized it. AbGalec consisted of four distinct carbohydrate-recognition domains (CRDs) and lacked a signal peptide. Expression analysis revealed AbGalec to be ubiquitously expressed in all the examined early embryonic stages of abalone, with highest expression in the 16-cell stage, suggesting the importance of AbGalec in early developmental processes. Tissue distribution analysis revealed the highest expression of AbGalec in abalone mantle, followed by that in gills and hemocytes. Immune challenge experiments revealed significant upregulation of AbGalec at 24 h and 48 h post injection (p.i.) with bacterial and viral components. These results suggested the possible involvement of AbGalec in host defense mechanisms. Polyinosinic: polycytidylic acid (Poly I:C) and viral hemorrhagic septicemia virus (VHSV) injections were capable of inducing AbGalec transcript expression more prominently than bacterial stimulants, thus providing evidence for its role in viral infections. We determined the virus-neutralizing ability of a quadruple domain-containing galectin for the first time, by analyzing the downregulation of VHSV transcripts during the overexpression of AbGalec. Significant downregulation of VHSV transcripts was observed after 24 h and 48 h of post infection. Collectively, our findings reveal the potent antiviral responses of molluscan quadruple domain-containing galectin, AbGalec, along with its involvement in innate immunity.