Expression And Immune Function Of Galectins In Sinonovacula Constricta

Razor clam(Sinonovacula constricta)is a mudflat shellfish of economic importance in China.In recent years,bacterial infections have caused heavy losses to clam's production,prompting research into congenital immunity.Like other invertebrates,shellfish depend on cellular and humoral defences for protection against infection.The shellfish innate immune system recognises pathogen-associated molecular patterns(PAMPs)that are not present in higher animals and are essential and unique to almost all microorganisms.These are recognised by a series of wellconserved pattern recognition receptors(PRRs),and activated PRRs directly or indirectly trigger cellular or humoral responses such as phagocytosis,nodulation and encapsulation,and synthesis of antimicrobial peptide.1.Full-length cDNA cloning and sequence analysis of two galectinsScGal-1 comprises 1066 bp with a 531 bp coding sequence(CDS)including a stop codon,an 84 bp 5? untranslated region(UTR),and a 451 bp 3? UTR with a predicted polyadenylation signal sequence(1026AATAAA1031)and a poly(A)tail.The ORF encodes a protein of 176 amino acid residues without a signal peptide,with a calculated molecular mass of 19.77 kDa and the theoretical isoelectric point of 5.65.The sequence has only one CRD domain(residues 48-174);The 1,309 bp ScGal-2 gene comprises a 47 bp 5'-untranslated region(UTR),a 755 bp 3'-UTR with a polyadenylation signal,and a 507 bp ORF encoding a protein of 168 amino acid residues without a signal peptide(ScGal2)with a molecular mass of 23.49 kDa and the theoretical isoelectric point of 5.54.A galactose binding domain is present at the C-terminus(residues 44-166),and the eight residues that make up the sugar binding pocket were mapped in the conserved sequence.2.The analysis of tissue expression and bacterial challenge expressionScGal-1 mRNA transcripts were detected in all tested tissues,and expression was highest in hemolymph,followed by gill.Invertebrate hemolymph is involved in various aspects of surveillance and cellular immune responses,while gills are the first line of defence against aquatic microbial invasion.Hemolymph were selected for studying mRNA expression of ScGal-1 following microbial invasion.The results revealed rapid up-regulation that peaked at 12 h after S.aureus challenge,whereas ScGal mRNA transcripts increased gradually after challenge with V.anguillarum,and reached 3-fold up-regulation at 72 h.ScGal-2 is widely expressed in all seven adult tissues tested,with highest expression in hemolymph and liver.V.anguillarum induced significant upregulation of the ScGal2 gene at 8 h,and expression peaked at 24 h.By contrast,challenge with S.aureus did not affect ScGal-2 up-regulation.ScGal are clam immunerelated genes,and different regulatory patterns for different bacteria suggest that the two genes may have different reaction mechanisms.3.Recombinant protein expression and functional verificationThe recombinant proteins rScGal-1 and rScGal-2 of the two genes were obtained by inducing expression,we studied the ability of recombinant proteins to aggregate,bind carbohydrates and promote phagocytosis.ScGal-1 showed strong binding ability to selected bacteria,and produced different degrees of agglutination.We pre-incubated rScGal-1 with various carbohydrates prior to binding to bacteria to explore their inhibitory effects.The results indicate that rScGal-1 binds PGN most strongly,followed by D-galactose and LTA,and the other tested carbohydrates bound weakly.rScGal-2 exhibited much stronger binding to the three Gram-negative bacteria than the three Gram-positive bacteria,and LPS strongly inhibited the binding ability of rScGal-2,whereas PGN and LTA did not affect binding.PGN is the main component of many bacterial cell walls,and PGN in the cell wall of Gram-positive bacteria(G+)accounts for ~50% of the dry weight,while PGN in the cell wall of Gram-negative bacteria(G-)accounts for only ~10%.LTA is a specific component of Gram-positive bacteria.This result indicates that the ScGal is important immune gene,and its CRD recognizes a unique molecular structure to exert an immune function.