Functional Analysis Of Galectin-9 And Nattectin-like Protein In Large Yellow Croaker Larimichthys Crocea

Lectins are carbohydrate-binding proteins which function as important pattern recognition receptors(PRRs)in innate immunity.Two lectin genes,galecitn-9 and a novel C-type lecin(Nattectin-like protein),were identified from a comprehensive transcriptome of Larimichthys crocea in our laboratory during screening,and were named as LcGal-9 and LcNTC respecctively.The immune function study of LcGal-9 and LcNTC was conducted and the obtained results are as follows:LcGal-9 is a member of galectin superfamily.The complete cDNA sequence of LcGal-9 was 1795 bp with an open reading frame(ORF)of 1032 bp encoding 343 amino acids.The putative LcGal-9 contained two carbohydrate recognition domains(CRDs)connected by a linker peptide,with each carrying two conserved ?-galactoside binding motifs H-NPR and WG-EE-,and it possessed neither a signal peptide nor a transmembrane domain.The phylogenetic tree showed that LcGal-9 clustered to galectin-9s from other fishes,such as Stegastes partitus.The qRT-PCR analysis revealed that LcGal-9 mRNA was present in all examined tissues with the most abundant in liver.Western blot analysis showed that LcGal-9 protein was highly expressed in many tissues,including liver,spleen,and intestine,but not detected in muscle and plasma.LcGal-9 transcripts were significantly up-regulated at most time points in the liver,spleen and head-kidney challenged with PolyI:C.In contrast,LPS and Vibrio parahaemolyticus caused a significant down-regulation in these three tissues.Post-infection with Cryptocaryon irritans,LcGal-9 transcripts were dramatically up-regulated in gill,skin,spleen and head-kidney during the initial stages of infection.The recombinant LcGal-9(named as rLcGal-9)exhibited agglutinating activity against erythrocytes from human,rabbit and large yellow croaker in a Ca2+-independent manner.Meanwhile,rLcGal-9 was able to agglutinate Gram-negative bacteria V.alginolyticus and Aeromonas hydrophila in the absence of Ca2+.The hemagglutinating activity of rLcGal-9 was inhibited by ?-Lactose and LPS.By immunohistochemistry assay,significant increases of LcGal-9 protein appeared in the spleen infected with PolyI:C and V.parahaemolyticus compared with the control.The collective data could provide a basis for studying the function of LcGal-9 in innate immune responses in L.crocea.LcNTC belongs to C-type lectin superfamily.The full-length cDNA of LcNTC was 859 bp,containing an ORF of 465 bp which encodes a protein of 154 amino acids.A signal peptide and a transmembrane region were predicted in the N-terminas of the deduced LcNTC.LcNTC contained a single CRD in the C-terminas with characteristic mannose-binding motif EPN and four conserved disulfide-bonded cysteine residues.Blastp search and phylogenetic analysis indicated that LcNTC was homologous to C-type lectins and Nattectin-like proteins from other fishes.LcNTC mRNA was constitutively expressed in all tested tissues,predominately expressed in gill.After challenged with LPS,Poly I:C and V.parahaemolyticus,the temporal expression of LcNTC was induced in liver,spleen and head-kidney at different time points.LcNTC transcripts were also up-regulated in gill,skin,spleen and head-kidney after C.irritans stimulation.The recombinant LcNTC(rLcNTC)possessed agglutinating activity in a Ca2+-dependent manner.It demonstrated hemagglutination against human,rabbit and large yellow croaker and caused positive microbial agglutination against Gram-positive and Gram-negative bacteria.Carbohydrates including D-Mannose,D-Glucose,D-Fructose,?-Lactose,D-Maltose and LPS could inhibit the hemagglutination well.These findings illustrated that LcNTC might be involved in the immune defense against virus,bacteria and parasites infection in L.crocea as an important PRR.