Chlamys Farreri Lectin Family Genes

Zhikong scallop Chlamys farreri is one of the most important species culturedwidely in the northern coastal provinces in China. Since the summer in 1997, a largescale mortality of C. farreri scallop has caused catastrophic losses to scallopaquaculture. Understanding the immune defense mechanisms of scallop maycontribute to the development of management strategies for disease control andlong-term sustainability of scallop farming. Based on EST sequencing and anchoredcDNA cloning technology, five full length cDNAs of C. farreri C-type lectins werecloned.The full length cDNA of CFLec-1 was 1785bp, consisting of a 5'-terminaluntranslated region of 66 bp and an unusual long 3'-UTR of 1040 bp which containsseven polyadenylation signal sequences AATAAA and a poly (A) tail. The CFLec-1cDNA encoded a polypeptide of 221 amino acids with a putative signal peptide of 15amino acid residues and a mature protein of 206 amino acids. The deduced matureprotein was of 206 amino acids with a pI of 5.12 and a theoretical mass of 23.49 kDa.Analysis of the protein domain features indicated a typical long-formcarbohydrate-recognition domain (CRD) of 130 residues in the CFLec-1 deducedamino acid sequence which contained four cysteine residues (Cys¹⁰⁴, Cys¹⁷⁷, Cys¹⁹³,Cys²⁰²) forming the CRD internal disulfides bridges, the other two cysteine residues(Cys⁷⁴, Cys⁸⁵ ) at N-terminus and the EPD motif (Glu¹⁶⁹-Pro¹⁷⁰-Asp¹⁷¹) fordetermining ligand-binding specificity. The deduced amino acid sequence of CRDregion in CFLec-1 shared 28% identity and 47% positivity with the predictedmacrophage mannose receptor in Gallus gallus, which contains eight CTLDs, and31% identity with C-type lectin receptor C from Atlantic salmon Salmo salar. Thetissue specific expression of CFLec-1 was studied by RT-PCR. The CFLec-1transcripts were mainly detected in the tissues of gill and gonad, and marginallydetectable in haemolymph and mantle, while there seemed to be no signal in thekidney, hepatopancreas and adductor muscle of healthy scallops. However, in scallopsinjected with heat-killed Vibrio anguillarum, the CFLec-1 mRNA levels in all thedetected tissues were higher than those in the unchallenged scallops, especially in thetissues of haemolymph, gill, gonad and mantle. The expression levels of CFLec-1 inhemolymph after Gram-positive and Gram-negative bacteria challenge. The resultshowed that the expression levels of CFLec-1 were significantly increased in bothgroups. CFLec-1 was successfully recombinant expressed in E. coli in form of fusionprotein. rCFLec-1 could agglutinate Gram-negative E. coli JM109, and theagglutination was Ca2+ dependent. The antibacterial activity of rCFLec-1 was detectedby antibacterial assay. It was found that recombinant CFLec-1 harbored remarkable invitro inhibitive effect on tested Gram-positive bacteria M. Luteus and relatively lowinhibitive effect on Gram-negative bacteria E. coliJM109. No lytic effect wasobserved on V. anguillarum.The other four C-type lectins cloned in this study were CFLec-2, mCFLec-1,mCFLec-2 and mCFLec-3.The full length cDNA of CFLec-2 was 708bp, which contained a 5'UTR of 59bpand a 3'UTR of 217bp. The open reading frame of CFLec-2 was 432bp encoding apolypeptide of 160 amino acids, including an N-terminus 17 amino acid long singnalpeptide. The mature protein of CFLec-2 contained one C-type lectin domin. Thefragment coding the mature peptide of CFLec-1 was cloned by PCR reaction andinserted in pET32a expression vector, and CFLec-2 was successfully expressed in E.coli.The full length cDNA of mCFLec-1 was 2257bp, which contained a 5'UTR of17bp and a 3'UTR of 713bp. The open reading frame of mCFLec-1 was 1527bp,encoding a polypeptide of 508 amino acids, including an N-terminus signal peptide of17 amino acids. The mature protein of mCFLec-1 harbored three C-type lectindomains. The fragment coding mature protein of mCFLec-1 was amplified andinserted in pET30a expression vector and mCFLec-1 was successfully expressed in E.coli.The full length of mCFLec-2 was 2086bp, which contained a 5'UTR of 18bp anda 3'UTR of 238bp. The open reading frame of mCFLec-2 was 1776bp, encoding apolypeptide of 609 amino acids. The mature protein of mCFLec-2 harbored fourC-type lectin domains. The full length of mCFLec-3 was 1897bp, its 5'UTR andcoding region was identical to mCFLec-2, the 3'UTR of mCFLec-3 was only 49bp.