title: Characterization and functional analysis of a tandem-repeat galectin-9 in large yellow croaker Larimichthys crocea.
abstract: Galectins are a family of endogenous lectins with β-galactosides affinity, playing significant roles in the innate immunity of vertebrates and invertebrates. In this report, a new galectin-9 cDNA was identified and characterized in large yellow croaker Larimichthys crocea (designated as LcGal-9). 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 protein contained two carbohydrate recognition domains (CRDs) connected by a linker peptide, with each carrying two conserved β-galactoside binding motifs H-NPR and WG-EE-, and it possessed neither a signal peptide nor a transmembrane domain. LcGal-9 protein shared 43-74% identity with galectin-9 sequences from other species. The qRT-PCR analysis revealed that LcGal-9 mRNA was constitutively expressed in all tissues examined, predominately expressed in liver, spleen, gill, kidney, head-kidney and intestine. Western blot analysis showed that LcGal-9 protein was highly expressed in liver, spleen, intestine, kidney, head-kidney, skin, gill, and heart, but not detected in muscle and plasma. LcGal-9 mRNA transcripts were induced by poly I:C in the liver (from 6 h to 48 h), spleen (at 12 h) and head-kidney (at 12 h and 24 h). In contrast, Vibrio parahaemolyticus caused a significant down-regulation in these three tissues, except for in spleen of 48 h and head-kidney of 3 h. Post-infection with Cryptocaryon irritans, the transcripts were dramatically up-regulated in gill, skin, spleen and head-kidney during initial infection period, while significant down-regulation afterward was also observed both in spleen and head-kidney. The recombinant LcGal-9 (named as rLcGal-9) purified from Escherichia coli BL21 (DE3) demonstrated hemagglutination against human, rabbit and L. crocea in a Ca(2+)-independent manner, which was inhibited by α-Lactose and LPS. The results of bacterial agglutination assays showed that rLcGal-9 was able to agglutinate Gram-negative bacteria V. alginolyticus and Aeromonas hydrophila in a Ca(2+)-independent manner. By immunohistochemistry assay, significant increases of LcGal-9 protein appeared in the spleen stimulated with poly I:C (for 12 h) and V. parahaemolyticus (for 48 h) compared with the control. Based on the collective data, LcGal-9 might play an important role in innate immune responses, especially defense against Gram-negative bacteria in L. crocea.
authors.affiliation: Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen, 361021, PR China.
authors.forename: Dong Ling
authors.initials: DL
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen, 361021, PR China.
authors.forename: Chang Huan
authors.initials: CH
authors.lastname: Lv
authors.affiliation: South China Sea Resource Exploitation and Protection Collaborative Innovation Center, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510300, PR China.
authors.forename: Da Hui
authors.initials: DH
authors.lastname: Yu
authors.affiliation: Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Fisheries College, Jimei University, Xiamen, 361021, PR China. Electronic address: zywang@jmu.edu.cn.
authors.forename: Zhi Yong
authors.initials: ZY
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016798:Ciliophora / Q000502:physiology; D016770:Ciliophora Infections / Q000235:genetics / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D037161:Galectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D010473:Perciformes; D011070:Poly I-C / Q000494:pharmacology; D014735:Vibrio Infections / Q000235:genetics / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D014736:Vibrio parahaemolyticus / Q000502:physiology
chemical_list: D029941:Fish Proteins; D037161:Galectins; D011070:Poly I-C
keywords: Agglutination; Galectin; Immunohistochemistry; Marine fish; Secrete