Functional Characterization Of A Galectin-8 Of Cynoglossus Semilaevis

Galectins are a family of lectins with a high affinity for β-galactosides.In mammals,galectins have been found to have multiple biological functions.In fish,galectins have been found to be important pattern recognition receptors and recognize a wide range of pathogens.However,other immune functions of fish galectins have rarely been reported.In the present study,we identified a galectin-8 gene from genome of Cynoglossus semilaevis(designated CsGal-8),and investigated the immune functions of CsGal-8.The open reading frame of CsGal-8 contains 939 bp nucleotides,which encodes312 amino acids.CsGal-8 belongs to the tandem repeat type of galectins which has two carbohydrate recognition domains(CRDs)connected by a linker peptide of 48 amino acids residues.The amino acids sequence similarities between CsGal-8 and the galectin-8 of other teleost species range from ～70% to ～80%.CsGal-8 m RNA was constitutively expressed in many tissues and highly expressed in kidney and spleen,which are immune associated organs.Expressions of CsGal-8 m RNA in kidney,spleen and blood were up-regulated after Edwardsiella tarda challenge.Subcellular distribution analysis showed that CsGal-8 was mainly distributed in the cytoplasm,and CsGal-8 could be secreted into the extracellular milieu in the form of microvesicles.Recombinant CsGal-8(rCsGal-8)showed high affinity for lactose,galactose,glucose and trehalose.The affinity of rCsGal-8 to carbohydrates had a positive relation with the dose of rCsGal-8.rCsGal-8 bound to a wide range of grampositive and gram-negative bacteria.The binding ability of rCsGal-8 toward Vibrio harveyi and Vibrio anguillarum was especially strong.Colocalization of CsGal-8 with V.anguillarum was observed in RAW264.7 cells expressing CsGal-8 and infected with V.anguillarum.Binding of rCsGal-8 to bacteria could be inhibited by carbohydrates.rCsGal-8 exhibited potent bactericidal ability against three gramnegative bacteria(V.harveyi,V.anguillarum and Escherichia coli).rCsGal-8disrupted the membrane integrity of V.anguillarum,and the morphology of V.anguillarum treated with rCsGal-8 changed greatly.In addition,rCsGal-8 inhibited reactive oxygen species(ROS)production in peripheral blood lymphocytes.These results indicated that CsGal-8 plays an important role in immune recognition,antibacterial infection and immune regulation processes.Galectins have been found to have direct bactericidal activities in fish for the first time.The results provide new insights for controlling fish diseases with lectins.