Molecular cloning, characterization and expression profiling of galectin-9 gene from Labeo rohita (Hamilton, 1822) (original) (raw)
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Fish & Shellfish Immunology, 2019
Galectins are β-galactoside-binding lectins, which are involved in pattern recognition, cell adhesion, and stimulation of the host innate immune responses against microbial pathogens. In spite of several functional studies on different galectins isolated from vertebrates and invertebrates, this is the first report to present functional studies for galectin-8 from the marine teleost tissues. In the present study, we characterized galectin-8 homolog from black rockfish (Sebastes schlegelii), in molecular and functional aspects. Rockfish galectin-8 (SsGal8) was found to consist of a 969 bp long open reading frame (ORF), encoding a protein of 322 amino acids and the predicted molecular weight was 35.82 kDa. In silico analysis of SsGal8 revealed the presence of two carbohydrate binding domains (CRDs), at both N and C-termini and a linker peptide of 40 amino acids, in between the two domains. As expected, the phylogenetic tree categorized SsGal8 as a tandem-repeat galectin, and ultimately positioned it in the sub-clade of fish galectin-8. rSsGal8 was able to strongly agglutinate fish erythrocytes and the inhibition of agglutination was successfully exhibited by lactose and D-galactose. Bacterial agglutination assay resulted in agglutination of both Gram (+) and Gram (-) bacteria, including Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus parauberis, Lactococcus garvieae, Streptococcus iniae and Vibrio tapetis. The tissue distribution analysis based on qPCR assays, revealed a ubiquitous tissue expression of SsGal8 for the examined rockfish tissues, with the most pronounced expression in blood, followed by brain, intestine, head kidney and kidney. Furthermore, the mRNA transcription level of SsGal8 was significantly up-regulated in spleen, liver and head kidney, upon immune challenges with Streptococcus iniae, LPS and poly I:C, in a time dependent manner. Taken together, these findings strongly suggest the contribution of SsGal8 in regulating innate immune responses to protect the rockfish from bacterial infections.
Molecular Immunology, 2015
Galectins belong to the family of galactoside-binding proteins and play a major role in the immune and inflammatory responses of vertebrates and invertebrates. The galectin family is divided into three subtypes based on molecular structure; prototypes, chimera types, and tandem-repeated types. We isolated and characterized the cDNA of galectin-8 (OnGal-8) in Nile tilapia (Oreochromis niloticus). OnGal-8 consisted of a 966 bp open reading frame (ORF) that encoded a 321 amino acid protein (43.47 kDa). Homology and phylogenetic tree analysis suggested the protein was clustered with galectin-8s from other animal species and shared at least 56.8 % identity with salmon galectin-8. Structurally, the amino acid sequence included two distinct N-and C-terminus carbohydrate recognition domains (CRDs) of 135 and 133 amino acids, respectively, that were connected by a 39 amino acid polypeptide linker. The N-and C-CRDs contained two conserved WG-E-I and WG-E-T motifs, suggesting they have an important role in mediating the specific interactions between OnGal-8 and saccharide moieties such as -galactoside. The structure of OnGal-8 was characterized by a twofold symmetric pattern of 10-and 12-stranded antiparallel ß-sheets of both N-and C-CRDs, and the peptide linker presumably formed a random coil similar to the characteristic tandem-repeat type galectin. The expression of OnGal-8 in healthy fish was highest in the skin, intestine, and brain. Experimental challenge of Nile tilapia with S. agalactiae resulted in significant up-regulation of OnGal-8 in the spleen after 5 d. Our results suggest that OnGal-8 is involved in the immune response to bacterial infection.
Glycoconjugate Journal, 2004
Cell surface glycans, such as glycocoproteins and glycolipids, encode information that modulates interactions between cells, or between cells and the extracellular matrix, by specifically regulating the binding to cell surface-associated or soluble carbohydrate-binding receptors, such as lectins. Rapid modifications of exposed carbohydrate moieties by glycosidases and glycosyltransferases, and the equally dynamic patterns of expression of their receptors during early development, suggest that both play important roles during embryogenesis. Among a variety of biological roles, galectins have been proposed to mediate developmental processes, such as embryo implantation and myogenesis. However, the high functional "redundancy" of the galectin repertoire in mammals has hindered the rigorous characterization of their specific roles by gene knockout approaches in murine models. In recent years, the use of teleost fish as alternative models for addressing developmental questions in mammals has expanded dramatically, and we propose their use for the elucidation of biological roles of galectins in embryogenesis and innate immunity. All three major galectin types, proto, chimera, and tandem-repeat, are present in teleost fish, and phylogenetic topologies confirm the expected clustering with their mammalian orthologues. As a model organism, the zebrafish (Danio rerio) may help to overcome limitations imposed by the murine models because it offers substantial advantages: external fertilization, transparent embryos that develop rapidly in vitro, a diverse toolbox of established methods to manipulate early gene expression, a growing collection of mutations that affect early embryonic development, availability of cell lines, and most importantly, an apparently less diversified galectin repertoire.
Manipulating Galectin Expression in Zebrafish (Danio rerio)
Methods in Molecular Biology, 2014
Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role(s) of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafi sh (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafi sh has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are β-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), which comprise an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic infl ammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafi sh, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.
The Journal of Immunology, 2009
To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1, TNF-␣, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection. Abbreviations used in this paper: SBNNV, Sea bass nervous necrosis virus; CRD, carbohydrate-recognition domain; CTLD, C-type-like domain; EST, expressed sequence tag; ORF, open reading frame; RLU, relative luminescence unit; SSH, suppression subtractive hybridization; qPCR, quantitative PCR.
2023
Galectin 9 (Gal9) is a tandem repeat type ß-galactoside-binding galectin that mediates various cellular biochemical and immunological functions. Many studies have investigated the functional properties of Gal9 in mammals; however, knowledge of fish Gal9 is limited to antibacterial studies. In this context, our aim was to clone Gal9 from Planiliza haematocheilus (PhGal9) and investigate its structural and functional characteristics. We discovered the PhGal9 open reading frame, which was 969 base pairs long and encoded a 322 amino acid protein. PhGal9 had a projected molecular weight of 35.385 kDa but no signal peptide sequence. PhGal9 mRNA was ubiquitously produced in all investigated tissues but was predominant in the intestine, spleen, and brain. Its mRNA expression was increased in response to stimulation by Poly(I:C), LPS, and L. garvieae. The rPhGal9 exhibited a dose-dependent agglutination potential toward gram-positive and gram-negative bacteria at a minimum concentration of 50 μg/mL. Overexpression of PhGal9 promoted M2-like phenotype changes in mouse macrophages, and RT-qPCR analysis of M1 and M2 marker genes confirmed M2 polarization with upregulation of M2 marker genes. In the antiviral assay, the expression levels of Viral Hemorrhagic Septicemia Virus (VHSV) glycoproteins, phosphoproteins, nucleoproteins, non-virion proteins, matrix proteins, and RNA polymerase were significantly reduced in PhGal9-overexpressed cells. Furthermore, the mRNA expression of autophagic genes (sqstm1, tax1bp1b, rnf13, lc3, and atg5) and antiviral genes (viperin) were upregulated in PhGal9 overexpressed cells. For the first time in teleosts, our study demonstrated that PhGal9 promotes M2 macrophage polarization by upregulating M2-associated genes (egr2 and cmyc) and suppressing M1-associated genes (iNOS and IL-6). Furthermore, our results show that exogenous and endogenous PhGal9 prevented VHSV attachment and replication by neutralizing virion and autophagy, respectively. Gal9 may be a potent modulator of the antimicrobial immune response in teleost fish.
Glycobiology, 2019
Galectins, highly conserved β-galactoside-binding lectins, have diverse regulatory roles in development and immune homeostasis and can mediate protective functions during microbial infection. In recent years, the role of galectins in viral infection has generated considerable interest. Studies on highly pathogenic viruses have provided invaluable insight into the participation of galectins in various stages of viral infection, including attachment and entry. Detailed mechanistic and structural aspects of these processes remain undetermined. To address some of these gaps in knowledge, we used Zebrafish as a model system to examine the role of galectins in infection by infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that is responsible for significant losses in both farmed and wild salmonid fish. Like other rhabdoviruses, IHNV is characterized by an envelope consisting of trimers of a glycoprotein that display multiple N-linked oligosaccharides and play an integral role ...
Developmental & Comparative Immunology, 2015
The infectious hematopoietic necrosis virus (IHNV; Rhabdoviridae, Novirhabdovirus) infects teleost fish, such as salmon and trout, and is responsible for significant losses in the aquaculture industry and in wild fish populations. Although IHNV enters the host through the skin at the base of the fins, the viral adhesion and entry mechanisms are not fully understood. In recent years, evidence has accumulated in support of the key roles played by protein-carbohydrate interactions between host lectins secreted to the extracellular space and virion envelope glycoproteins in modulating viral adhesion and infectivity. In this study, we assessed in vitro the potential role(s) of zebrafish (Danio rerio) proto type galectin-1 (Drgal1-L2) and a chimera galectin-3 (Drgal3-L1) in IHNV adhesion to epithelial cells.
Microbiological Research, 2014
In this study, we reported a molecular characterization of a novel proto-type galectin-1 from the striped murrel Channa striatus (named as CsGal-1). The full length CsGal-1 was identified from an established striped murrel cDNA library and further we confirmed the sequence by cloning. The complete cDNA sequence of CsGal-1 is 590 base pairs (bp) in length and its coding region encoded a poly peptide of 135 amino acids. The polypeptide contains a galactoside binding lectin domain at 4-135. The domain carries a sugar binding site at 45-74 along with its signatures (H 45 -X-Asn 47 -X-Arg 49 and Trp 69 -X-X-Glu 72 -X-Arg 74 ). CsGal-1 shares a highly conserved carbohydrate recognition domain (CRD) with galectin-1 from other proto-type galectin of teleosts. The mRNA expressions of CsGal-1 in healthy and various immune stimulants including Aphanomyces invadans, Aeromonas hydrophila, Escherchia coli lipopolysaccharide and poly I:C injected tissues of C. striatus were examined using qRT-PCR. CsGal-1 mRNA is highly expressed in kidney and is up-regulated with different immune stimulants at various time points. To understand its biological activity, the coding region of CsGal-1 gene was expressed in an E. coli BL21 (DE3) cloning system and its recombinant protein was purified. The recombinant CsGal-1 protein was agglutinated with mouse erythrocytes at a concentration of 4 g/mL in a calcium independent manner. CsGal-1 activity was inhibited by d-galactose at 25 mM −1 and d-glucose and d-fructose at 100 mM −1 . The results of microbial binding assay showed that the recombinant CsGal-1 protein agglutinated only with the Gramnegative bacteria. Interestingly, we observed no agglutination against Gram-positive bacteria. Overall, the study showed that CsGal-1 is an important immune gene involved in the recognition and elimination of pathogens in C. striatus.