Identification Of Soluble Leptin Receptor And It’s Interaction With Leptin In Crucian Carp

Leptin, a hormonal product of the obese (ob) gene, is a 16 kDa hydrophilic peptides secreted primarily by adipose tissue in mammals. Different from mammals, fish leptin mRNA expressed mainly in the liver. Mature leptin is secreted into the blood, playing a role in the central and peripheral. Leptin has been demonstrated to have pleiotropic effects in feed intake, energy homeostasis, reproduction, immune function. The physiological actions of leptin depend on signaling through the leptin receptor (LepR).LepR is a member of the class I cytokine-receptor family and widely distributed in the central and peripheral. There are multiple LepR isoforms, all of which are products of alternative mRNA splicing and/or proteolytic cleavage of membrane-bound forms of LepR. In teleosts, the LepR expression was first characterized in marine medaka (Oryzias melastigma) by wong et al. To date, only one LepR isofonn were found in most fish. The different receptor isoforms are just reported in Atlantic salmon, crucian carp, rainbow trout and tilapia. we have cloned 3 LepR isoform complementary DNAs (ie, the long form [cclpr-L], the short form [cclpr-S1], and the secreted form [cclpr-S2]) of the crucian carp.The main aim of the present study was to further detect and analyze crucian carp cclpr-S2 in protein levels and analyze the different binding affinity of cclpr-S2 to leptin-a and leptin-b. In addition, we constructed cclpr-S1 and cclpr-L eukaryotic fluorescent protein vectors for subsequent research on cclpr-S1 internalization and heterodimerization between cclpr-L and cclpr-S1.1. According to leptin-b sequences of carp, zebra fish and "Jian" carp, we cloned the complete coding sequence and 3’UTR of crucian carp’s leptin-b by RT-PCR and RACE. Phylogenetic analysis among vertebrate leptins showed that the similarity of crucian carp leptin-b with "Jian"carp was highest. Both carpsare clustered into a branch with grass carp and zebrafish. This result is accordance with the traditional classification of species.2. In order to further verify the existence of cclpr-S2 and analyze difference combination between cclpr-S2 and two leptin (leptin-a, leptin-b), Firstly we constructed pET32-leptin-b recombinant expression vector and induced pET32-leptin-a and pET32-leptin-b at 25 ℃ with 0.1 mM IPTG. and purified recombinant protein with Ni Resin. Then we extract the carp serum and conduct pull-down assay. We found that much more cclpr-S2 were pulled down by Trx-leptin-a than Trx-leptin-b. It is showed that leptin-a has a higher binding energy than leptin-b. We speculated that leptin-a may be the main ligand of LepR.3. We constructed eukaryotic fluorescent protein vectors, including pcDN A3.1-cclpr-S1-eGFP. pcDNA3.1-cclpr-S1-eYFP, pcDNA3.1-cclpr-S1-eCFP, pcDNA3.1-cclpr-L-eYFP, pcDNA3.1-cclpr-L-eCFP. Then we extract pcDNA3.1-cclpr-S1-eGFP plasmid and transiently transfect into Hela cells.