title: Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus).
abstract: C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5' untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3' UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys(61)-Cys(158), Cys(134)-Cys(150)) and a Ca(2+)/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca(2+)-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca(2+)-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.
authors.affiliation: Ocean College, Shandong University (Weihai), Weihai 264209, PR China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Yu
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.forename: Yingmei
authors.initials: Y
authors.lastname: Chai
authors.forename: Yingying
authors.initials: Y
authors.lastname: Liu
authors.forename: Qiuxia
authors.initials: Q
authors.lastname: Zhang
authors.forename: Xinbiao
authors.initials: X
authors.lastname: Ding
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhu
mesh_terms: D000372:Agglutination Tests; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002681:China; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D020224:Expressed Sequence Tags; D005399:Fishes / Q000235:genetics* / Q000276:immunology; D040461:Gene Components; D005786:Gene Expression Regulation / Q000276:immunology*; D037181:Lectins, C-Type / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D037181:Lectins, C-Type; D008070:Lipopolysaccharides