Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris.
C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity. The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues. The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively. But its expression level did not change significantly during peptidoglycan (PGN) stimulation. The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3). The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way. The agglutinating activity could be inhibited by d-mannose, LPS and glucan, but not by d-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs.
Zhang
Huan
H
The Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China.
Kong
Pengfei
P
Wang
Lingling
L
Zhou
Zhi
Z
Yang
Jialong
J
Zhang
Ying
Y
Qiu
Limei
L
Song
Linsheng
L
D000372:Agglutination Tests; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020224:Expressed Sequence Tags; D005936:Glucans / Q000276:immunology; D007113:Immunity, Innate / Q000235:genetics / Q000276:immunology*; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology; D008969:Molecular Sequence Data; D049897:Pectinidae / Q000235:genetics / Q000276:immunology*; D010457:Peptidoglycan / Q000276:immunology; D010843:Pichia / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D005936:Glucans; D037181:Lectins, C-Type; D008070:Lipopolysaccharides; D010457:Peptidoglycan; D011994:Recombinant Proteins; D012313:RNA