Cloning, Expression And Functional Characterization Of Galectin-3Binding Protein In Cvnonlossus Semilaevis

Galectin-3binding protein (G3BP) is a secreted glycoprotein with galectin-3binding activety in vertebrates. In human, it has been found to exist in various bodyfluids like serum、milk、urea etc., and participate in many pathophysiologic processesincluding cancers and viral infection. In fish, G3BP-like genes have been identified infew species (i.e. zebrafish and salmon), but their biological functions are entirelyunknown.In this work, we cloned a teleost G3BP, CsG3BP, from half-smooth tongue sole(Cynoglossus semilaevis), which is an important commercial fish species in NorthernChina. We analysed the domain architecture and conserved residues of importance inactivity of this protein, followed by expression and purification of recombinantCsG3BP which was subsequently subjected to functional characterization.The full-length cDNA of CsG3BP was of2016bp which encoded a polypeptideof565amino acids. CsG3BP shared medium similarity of30.6%-47.8%withG3BP-like protein from mammals and other teleost. The deduced amino acidssequence of CsG3BP possesses a Scavenger Receptor Cysteine-Rich (SRCR) domain.It contained six conserved cysteine residues (C65/C78/C109/C119/C129/C139) thatwere predicted to form three intramolecular disulfide bridges.Under normal physiological conditions, the mRNA of CsG3BP was detected inliver, spleen, kidney, gill, intestine, brain, heart, blood and muscle, which was mostabundant in blood and least abundant in intestine. After bacterial stimulation,expressions of CsG3BP were upregulated significantly in a time-dependent manner.In spleen, kidney, blood, the highest levels of their mRNAwere detected at12h (36.5,67.8,6.1-fold respectively, P<0.05); whereas in liver, that was detected at4h as 49.8-fold upregulated. Megalocytivirus infection caused similar effect on theexpression of CsG3BP though the folds were not that different.We expressed the recombinant G3BP-like protein (rCsG3BP) in Escherichia coliand prepared its multi-clone antibody. Purified recombinant CsG3BP (rCsG3BP)exhibited bacterial (G+/G-) binding ability in a dose-dependent manner and itexhibited a preference for Gram-negative bacteria. In contrast, the mutant forms ofrCsG3BP either lacking the SRCR domain or bearing mutationsserine substitutions atthree disulfide bond-forming cysteine residues, involved in disulfide bond formation,lost the capacity of bacterial interaction. Immunoblot analysis revealed that CsG3BPis an extracellular protein in Cynoglossus semilaevis, as it appeared in the culturemedium of peripheral blood leukocytes (PBL) and in serum. Further study showedthat when the CsG3BP produced by PBL was blocked by anti-rCsG3BP antibodies,the phagocytic activity of the cells was significantly reduced.Taken together, these results indicated that CsG3BP is a secreted proteindistributed widely in the tissues of Cynoglossus semilaevis. It takes part in host’simmune responses to infection as an important innate immune factor. It probablyfulfills its immune function by binding to bacterial cells via the SRCR domain andthereby facilitating the host phagocytosis. This research may further explore theteleost innate immune system and improve the understanding of piscine anti-infectiveimmunity as well.