Molecular cloning, characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper, Epinephelus coioides.
C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell-cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys(71), Cys(152), Cys(167), Cys(175)) involved in the formation of disulphide bridges and the potential Ca(2+)/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca(2+)-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca(2+)-dependent manner.
Wei
Jingguang
J
State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, PR China.
Xu
Dan
D
Zhou
Jinggeng
J
Cui
Huachun
H
Yan
Yang
Y
Ouyang
Zhengliang
Z
Gong
Jie
J
Huang
Youhua
Y
Huang
Xiaohong
X
Qin
Qiwei
Q
D000372:Agglutination Tests; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001492:Bass / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D037181:Lectins, C-Type / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment
D018076:DNA, Complementary; D037181:Lectins, C-Type