Identification and characterization of an L-type lectin from obscure puffer Takifugu obscurus in response to bacterial infection.

L-type lectins (LTLs) contain a carbohydrate recognition domain homologous to leguminous lectins, and have functions in selective protein trafficking, sorting and targeting in the secretory pathway of animals. In this study, a novel LTL, designated as ToERGIC-53, was cloned and identified from obscure puffer Takifugu obscurus. The open reading frame of ToERGIC-53 contained 1554 nucleotides encoding 517 amino acid residues. The deduced ToERGIC-53 protein consisted of a signal peptide, a leguminous lectin domain (LTLD), a coiled-coil region, and a transmembrane region. Quantitative real-time PCR showed that ToERGIC-53 was expressed in all examined tissues, with the highest expression level in the liver. The expression of ToERGIC-53 was significantly upregulated after infection with Vibrio harveyi and Staphylococcus aureus. Recombinant ToERGIC-53-LTLD (rToERGIC-53-LTLD) protein could not only agglutinate and bind to one Gram-positive bacterium (S. aureus) and three Gram-negative bacteria (V. harveyi, V. parahaemolyticus and Aeromonas hydrophila), but also bind to glycoconjugates on the surface of bacteria such as lipopolysaccharide, peptidoglycan, mannose and galactose. In addition, rToERGIC-53-LTLD inhibited the growth of bacteria in vitro. All these results suggested that ToERGIC-53 might be a pattern recognition receptor involved in antibacterial immune response of T. obscurus.

Molecular cloning, characterization and gene expression analysis of twelve interleukins in obscure puffer Takifugu obscurus.

Interleukins (ILs) are a subgroup of secreted cytokines, which are molecules involved in the intercellular regulation of the immune system. In this study, 12 IL homologs were cloned and functionally identified from obscure puffer Takifugu obscurus, and they were termed as ToIL-1ß, ToIL-1, ToIL-6, ToIL-10, ToIL-11, ToIL-12, ToIL-17, ToIL-18, ToIL-20, ToIL-24, ToIL-27, and ToIL-34. Multiple alignment results showed that except for ToIL-24 and ToIL-27, other deduced ToIL proteins shared typical characteristics and structure with other known fish ILs. Phylogenetic analysis revealed that 12 ToILs were evolutionarily closely related to their counterparts in other selected vertebrates. Tissue distribution assay demonstrated that the mRNA transcripts of most ToIL genes were constitutively expressed in all tissues examined, with relatively high expression in immune tissues. Following Vibrio harveyi and Staphylococcus aureus infection, the expression levels of 12 ToILs in the spleen and liver were significantly upregulated, and their response over time varied. Taken together, these data were discussed accordingly with the ToIL expression and the immune response under the different situations tested. The results suggest that the 12 ToIL genes are involved in the antibacterial immune response in T. obscurus.

Three novel L-type lectins from obscure puffer Takifugu obscurus promote antimicrobial immune response.

L-type lectins (LTLs) have leguminous lectin domains that bind to high-mannose-type oligosaccharides. LTLs are involved in glycoprotein secretory pathways and associated with many immune responses. In the present research, three LTL homologs from obscure puffer Takifugu obscurus, designated as ToVIP36-1, ToVIP36-2, and ToVIP36-3, were first cloned and identified. The open reading frames of ToVIP36-1, ToVIP36-2, and ToVIP36-3 were 1068, 1002, and 1086 bp in length, respectively, and encode polypeptides with 355, 333, and 361 amino acids, respectively. Key conserved residues and functional domains, including lectin_leg-like domain (LTLD), transmembrane region, and C-terminal trafficking signal KRFY, were identified in all ToVIP36s. Quantitative real-time PCR analysis showed that the three ToVIP36s were widely expressed in six examined tissues and had relatively high expression levels in the liver and intestine. The expression levels of ToVIP36s were remarkably altered in the liver and kidney after induction by Vibrio harveyi and Staphylococcus aureus. Subsequently, the recombinant LTLDs of ToVIP36s (rToVIP36-LTLDs) were prepared by prokaryotic expression. Three rToVIP36-LTLD proteins agglutinated with S. aureus, V. harveyi, Vibrio parahaemolyticus, and Aeromonas hydrophila in a calcium-dependent manner. In the absence of calcium, rToVIP36-LTLD proteins bound to the bacteria by binding to lipopolysaccharides, peptidoglycans, d-mannose, and d-galactose and inhibited the growth of S. aureus and V. harveyi. Our results indicated that ToVIP36s function as pattern-recognition receptors in T. obscurus immunity, providing insights into the role of LTLs in the antibacterial immunity of fishes.