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Gene Structure, Recombinant Expression and Functional Characterization of Grass Carp Leptin

Author: LiGuanGui
Tutor: LiangXuFang
School: Jinan University
Course: Animal Genetic Engineering
Keywords: leptin grass carp recombinant expression food intake energy expenditure
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 159
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Abstract

Leptin, the protein product of the obese gene, is a type-I cytokine hormone secreted primarily by adipocytes in mammals, which plays a key role in the regulation of food intake, energy expenditure and reproduction. However, information regarding its role in teleosts remains scant. In the present study, the gene structure of grass carp (Ctenopharyngodon idellus) leptin was obtained. The mature peptide of grass carp leptin was expressed in the prokaryotic expression system and purified. The acute and chronic effects of recombinant grass carp leptin (rgc-LEP) on food intake and energy metabolism, as well as their molecular mechanism were also charactered. These results would shed a new light on the biological functions of leptin in teleosts, and provide an essential foundation for elucidating the structural and functional evolution of this important hormone.Full-length cDNA sequence of grass carp leptin (gc-LEP) was obtained by RT-PCR and RACE methods. The full-length cDNA sequence of gc-LEP was 1096 bp in length, encoding a peptide of 173 amino acids. The deduced gc-LEP shared 57.2%-87.9% amino acid identity with cyprinid fish leptins, whereas only 20.0%-28.8% amino acid identity with other fish, amphibian and mammalian leptins. The predicted second and tertiary structures of gc-LEP was highly conserved with other fish, amphibian and mammalian leptins, which comprised of four helices and had two cysteine residues for the disulfide bonds of ahelices C and D. All the fish leptins clustered together in the phylogenetic tree, which was constructed based on the amino acid sequences.The genomic region of gc-LEP was isolated by PCR and genome walker methods. Like the leptin genes of amphibian and mammal, the gc-LEP gene consisted of three exons and two introns. The two introns appeared at similar positions as in the leptin genes of frog and human, as well as those of other fishes. The obtained 5’-flanking region of gc-LEP gene was 265 bp, including several predictive transcription elements, such as typical TATA box, activator protein 1 (API) site, CCAAT/enhancerbinding protein (C/EBP) site. These sites were located at similar positions in the 5’-flanking region of leptin gene as they are in mammals, suggesting that the regulative elements of leptin gene might be conserved between fish and mammals.The rgc-LEP was expressed in the prokaryotic expression system, using pET-3c vector and BL21 E. coli as the mediums. Almost all of the target protein was produced as IBs (inclusion bodies).75 mg of purified rgc-LEP was obtained from 1 L of bacterial culture after denatured, renatured and purified. Identification of rgc-LEP was performed by sequencing its partial amino acid sequences by mass spectrometric analysis. Analysis of the conformational space of the purified rgc-LEP was performed by SDS-PAGE electrophoresis, under reducing condition and nonreducing condition. The result revealed that the intramolecular disulfide bridge of purified rgc-LEP was properly reformed.The chronic effects of rgc-LEP on food intake and energy metabolism were assessed by chronic (13 days) intraperitoneal (I.P.) injection of rgc-LEP. A strong anorexic effect on food intake (compared to PBS injection group, PBS group) was observed in leptin injection group (LEP group) on the first day after I.P. injection of rgc-LEP, but not during the following days. Body weight of the LEP group and the pair-fed group (PF group) showed no difference throughout the experimental period.In order to further expose the molecular regulative mechanism of rgc-LEP on food intake and energy metabolism, partial cDNA sequences of eleven genes correlating to food intake (such as NPY), degestion (such as BSAL, TRY, AMY), energy metabolism (such as UCP1, UCP2) and lipid metabolism (such as HL, LPL, HSL, FAD, ELO) were also isolated form grass carp.The acute and chronic effects of rgc-LEP on the expression of the above 11 genes, as well as SCD1 gene and leptin gene itself were further characterized by real-time PCR. The mRNA levels of NPY, SCD1 and lipoprotein lipase LPL were significantly reduced whereas the mRNA levels of UCP2, BSAL and ELO were significantly elevated, and the expression of UCP1、HSL、HL、FAD、AMY and TRY genes had no significant differece on the first day after injection. No effect on the expression of these genes (except LPL) was observed on day 13. Our results suggest that leptin has an acute effect on the regulation of food intake, energy expenditure and lipid metabolism in grass carp, but the effect can be rapidly counteracted through mechanisms that are currently unknown.

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