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Journal of Animal Science - Animal Genetics

Nonsynonymous natural genetic polymorphisms in the bovine leptin gene affect biochemical and biological characteristics of the mature hormone1

 

This article in JAS

  1. Vol. 90 No. 2, p. 410-418
     
    Received: June 17, 2011
    Accepted: Sept 12, 2011
    Published: January 20, 2015


    2 Corresponding author(s): gertler@agri.huji.ac.il
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doi:10.2527/jas.2011-4378
  1. S. Reicher*†,
  2. J. M. Ramos-Nieves,
  3. S. M. Hileman§,
  4. Y. R. Boisclair,
  5. E. Gootwine and
  6. A. Gertler 2
  1. The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel;
    Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel;
    Department of Animal Science, Cornell University, Ithaca, NY 14853; and
    Department of Physiology and Pharmacology, West Virginia University, Morgantown 26506

ABSTRACT

Leptin (LEP) is a cytokine-like hormone proven to be involved in diverse biological processes. In livestock, it regulates feed intake, BW homeostasis, and energy balance, among other traits. Natural nonsynonymous genetic polymorphisms in the ovine leptin (oLEP) alter the biochemical and physiological characteristics of its gene products. Here we studied in vitro and in vivo the biochemical and physiological characteristics of recombinant hormones representing the oLEP and bovine leptin (bLEP) reference sequences of wild-type (WT) leptins (GenBank accession No. U84247 and U50365, respectively), oLEP and bLEP recombinant muteins carrying the R4C mutation, and oLEP recombinant hormones carrying the A59V and Q62R mutations, which were detected in bLEP. All proteins were purified to homogeneity as monomers and formed 1:1 molar ratio complexes with the chicken leptin-binding domain (LBD). Surface plasmon resonance experiments revealed that all protein variants exhibit reduced (P < 0.05) affinity to chicken (ch) and human (h) LBD compared with the WT oLEP and bLEP recombinant proteins. The ovine and bovine R4C muteins exhibited significantly (P < 0.05) greater induction of cell proliferation in a Baf/3 cell line bioassay, despite lower affinity toward both hLBD and chLBD. Intra-third cerebral ventricle infusion of oLEP and its 3 muteins in sheep resulted in reduced feed intake. However, the 3 tested muteins had a decreased (P < 0.05) inhibitory effect than the WT LEP. It was concluded that natural genetic polymorphisms in the bLEP are associated with variation in the biochemical and physiological properties of the protein.



INTRODUCTION

Leptin (LEP) is a 16-kDa cytokine-like hormone translated from the obese gene and regulates BW homeostasis and energy balance (Zhang et al., 1994). It is synthesized and secreted mainly by white adipocytes (Zhang et al., 1994), but also by other tissues (Margetic et al., 2002). In farm animals, LEP gene polymorphisms have been shown to be involved in variation of traits such as feed intake, feed efficiency, energy balance, fertility, and reproductive efficiency (van der Lende et al., 2005; Wylie, 2010).

The LEP gene contains 3 exons, separated by 2 introns (Green et al., 1995). The LEP gene encodes a protein of 167 AA with exon 2 contributing the first 48 AA and exon 3 the remaining 119 AA. The mature circulating form of the protein is obtained after removal of the 21 AA signal peptide. The ovine (o) LEP and bovine (b) LEP mature proteins differ by only 2 AA (A66T and I74V in the ovine reference sequence, GenBank accession No. U84247, and bovine reference sequence, GenBank accession No. U50365, respectively), representing 98.8% identity. In humans, 4 nonsynonymous naturally occurring genetic polymorphisms have been detected in the LEP gene (van der Lende et al., 2005). Existence of nonsynonymous polymorphism has also been documented in the oLEP; 5 nonsynonymous polymorphisms, N78S, R84Q, P99Q, V123L, and R138Q, were detected in exon 3 of the gene (Zhou et al., 2009; Reicher et al., 2011). In our recent work (Reicher et al., 2011), we produced 6 recombinant oLEP protein muteins that represented products of all known genetic polymorphisms of the oLEP gene, and we showed that although most of those proteins have reduced affinity toward the soluble LEP receptor, some show greater biological activity than the wild type (WT) hormone.

In bovine, mutations in both exonic and intronic regions were associated with economically important traits, as reviewed by van der Lende et al. (2005). In addition, mutations in the bLEP promoter (Liefers et al., 2005; Nkrumah et al., 2005) as well as in bLEP receptor (Liefers et al., 2004; Guo et al., 2008) were also associated with performance traits in bovine. Altogether, 4 nonsynonymous genetic polymorphisms have been detected in the coding region of the bLEP gene (the numbering corresponds to the mature protein): R4C, A59V, Q62R, and N78S (van der Lende et al., 2005; Orrừ et al., 2006; Figure 1). The R4C variation was associated with increased milk yield, milk somatic cell count, carcass fat content, fat deposition rate, and body fat reserves, as well as increased LEP mRNA expression abundance and decreased serum LEP concentration during late pregnancy. The A59V variation was found to be associated in cattle with increased serum LEP concentrations during late pregnancy and increased ADG (van der Lende et al., 2005), with greater calving interval and number of days open, as well as with a reduced number of inseminations per conception (Komisarek and Antkowiak, 2007).

Figure 1.
Figure 1.

All known bovine leptin protein variants are shown on the mature protein structure of human leptin (PDB ID:1AX8). Color version available in the online PDF.

 

Whereas DNA polymorphism in the bLEP gene has been investigated in the context of its association with the phenotypic variation of several traits, there is no information on the significance of the genetic polymorphism for the biochemical and physiological properties of the LEP molecule, as was shown for oLEP (Reicher et al., 2011).

To address this question, we extended our work on natural polymorphisms in LEP (Reicher et al., 2011) and mutated the WT oLEP to carry the bovine alterations R4C, A59V, and Q62R, and tested the purified recombinant proteins for their biochemical and biological activities in vitro as well as in vivo, using sheep as the experimental animal model.


MATERIALS AND METHODS

Animal procedures were approved by the local Institutional Animal Care and Use Committee (West Virginia University for surgeries and Cornell University for experiments).

Materials

Plasmids carrying the published oLEP sequence (GenBank accession No. U84247; ovine reference sequence, WT), the published bLEP sequence (GenBank accession No. U50365; bovine reference sequence, WT), recombinant human and chicken leptin binding domain (termed hLBD and chLBD, respectively), prepared according to Niv-Spector et al. (2005), were obtained from Protein Laboratories Rehovot Ltd. (Rehovot, Israel). The Baf/3 cells stably expressing the long form of hLBD were obtained from R. Devos (Roche, Gent, Belgium). Restriction enzymes used in the molecular biology experiments were from Fermentas (Vilnius, Lithuania). High-purity DNA primers were ordered from Syntezza Bioscience Ltd. (Jerusalem, Israel). The RPMI-1640 medium and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue, MTT) were purchased from Sigma (St. Louis, MO). Superdex 75 HR 10/30 column and Q-Sepharose were from Pharmacia LKB Biotechnology AB (Uppsala, Sweden). Antibiotic-antimycotic solution (5 × 104 U/mL of penicillin, 50 mg/mL of streptomycin, 0.125 mg/mL of fungisone), NaCl, Tris-base, and fetal calf serum were purchased from Bio-Lab Ltd. (Jerusalem, Israel). Bacto-tryptone, Bacto-yeast extract, glycerol, EDTA, HCl, Triton X-100, and urea were from ENCO Diagnostics Ltd. (Petah-Tikva, Israel). Molecular markers for SDS-PAGE were purchased from Bio-Rad (Hercules, CA). A research-grade CM5 sensor chip, NHS (N-hydroxysuccinimide), EDC [N-ethyl-N′-(3-dimethylaminopropyl)-carbodi-imide hydrochloride], ethanolamine/HCl, and HBS-EP running buffer [10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% (vol/vol) surfactant P20 at pH 7.4] were purchased from Biacore AB (Uppsala, Sweden). All other materials were of analytical grade.

Preparation of oLEP Muteins

All LEP muteins were prepared using pMon3401 expression plasmids encoding either WT oLEP or WT bLEP (Gertler et al., 1998; Raver et al., 2000). The plasmid encoding WT bLEP was used as the backbone to prepare the bovine R4C variant, whereas the plasmid encoding WT oLEP was used for all other muteins. The LEP DNA inserts were modified with the QuickChange mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions, using 2 complementary primers (Table 1). The primers were designed to contain base changes (marked in bold, italicized letters) yielding the intended AA substitution and to modify specific restriction sites (underlined) for colony screening. The procedure included 18 PCR cycles with Pfu polymerase. To digest the template and select for sequences containing synthesized DNA, the altered construct was then digested with the restriction enzyme DpnI. The plasmids were transfected into Mon-105 expression-competent cells. Competent cells were grown in 5 to 10 mL of Luria-Bertani broth medium, and the plasmids were isolated. Six colonies for each mutein were screened for the desired alteration, using the specifically designed restriction site (Table 1). Two colonies were sequenced for each mutein and confirmed to contain the intended mutation variation with no change in other nucleotides.

Table 1.

Please see the pdf to view this table.

 

Expression, Refolding, and Purification of LEP Muteins

Preliminary experiments were performed for all 6 clones (WT oLEP, WT bLEP, ovine and bovine R4C, ovine A59V, and Q62R) in a 250-mL flask containing 50 mL of Terrific broth medium [1.2% (wt/vol) Bacto-tryptone, 2.4% (wt/vol) Bacto-yeast extract, 0.4% (vol/vol) glycerol in double-distilled H2O] and salts [0.023% (wt/vol) KH2PO4, 0.125% (wt/vol) K2HPO4] cultured at 37°C. After the bacterial culture reached an absorption at 600 nm (A600) of 0.9 to 1.1, nalidixic acid was added to a final concentration of 0.1 mM. The cells were harvested 3 to 4 h later and assayed for LEP expression by SDS-PAGE. From each group of colonies expressing one of the desired muteins, 1 colony was selected for large-scale preparation as described elsewhere (Reicher et al., 2011). Protein concentration was determined at A280 and monomer content by gel-filtration chromatography on a Superdex 75 HR 10/30 column. Fractions containing the monomeric protein were pooled, dialyzed at 4°C against NaHCO3 (pH 10) in a 4:1 (wt/wt) protein-to-salt ratio, and lyophilized.

Determination of Purity and Monomer Content

The SDS-PAGE analysis was performed in a 15% (wt/vol) polyacrylamide gel under reducing conditions, as described elsewhere (Reicher et al., 2011). Gel-filtration chromatography was performed on a Superdex 75 HR 10/30 column with 0.2-mL aliquots of the Q-Sepharose-column-eluted fraction using TN buffer (25 mM Tris-HCl and 150 mM NaCl, pH 10). Reverse-phase chromatography was carried out on a Symmetry 300 C4 4.6/250 column connected to an HPLC using the following gradient: 5 to 30% (vol/vol) acetonitrile with 0.1% (vol/vol) trifluoroacetate (TFA; 5 min), 30 to 60% acetonitrile with 0.1% TFA (30 min), and re-equilibration with 5% acetonitrile and 0.1% TFA (5 min).

Determination of Circular Dichroism Spectra

Circular dichroism (CD) spectroscopy technique was used to determine the secondary structure of the muteins that were infused in vivo into sheep (ovine WT LEP, R4C, A59V, and Q62R). The CD spectra were measured with an Aviv model 62A DS CD spectrometer (Aviv Associates, Lakewood, NJ) using a 0.020-cm rectangular QS Hellma cuvette as described previously (Niv-Spector et al., 2005).

Detection of oLEP Complexes with chLBD by Gel Filtration

To determine the impact of AA substitutions on binding stoichiometry, LEP and the respective muteins were mixed in different molar ratios with chLBD, incubated for 20 min at room temperature (25°C), and then separated under nondenaturing conditions by gel filtration using an analytical Superdex 75 column, as described previously (Salomon et al., 2006). The experiments were performed using a constant 5 to 10 μM of the respective ligand and 5 μM chLBD.

Kinetics Measurements of LEP-hLBD and LEP-chLBD Interactions

The kinetics and equilibrium constants for the interactions between hLBD or chLBD and all oLEP or bLEP muteins were determined by surface plasmon resonance (SPR) methodology using a Biacore 3000 instrument (Neuchatel, Switzerland) as described previously (Reicher et al., 2011). The experiments were analyzed using the Kinetics Wizard (Biacore control software, obtained from the supplier, Neuchatel, Switzerland). The resultant binding curves were fitted to the association and dissociation phases at all hLBD or chLBD concentrations simultaneously, using Biacore evaluation software.

In Vitro Biological Activity in Baf/3 Bioassay

The proliferation rate of LEP-sensitive Baf/3 cells stably expressing the long form of hLEP receptor was used to estimate the activities of the WT LEP and LEP mutein activities. Cell maintenance and experimental protocol were as described previously (Raver et al., 2000). Cell proliferation was determined by the MTT assay method as described previously (Hansen et al., 1989), 48 h after hormone addition. Each concentration for each protein was tested in triplicate. The growth curves for each experiment were drawn using the Prism (4.0) nonlinear regression sigmoidal dose-response curve (GraphPad Software Inc., San Diego, CA), and the EC50 values were calculated. The relative ability to induce cell proliferation was determined by calculating the ratio between the respective EC50 and the EC50 obtained for the WT oLEP. Seven cell-proliferation experiments were conducted.

In Vivo Feed Intake Study

Suffolk ewes (n = 4, age 2 to 4 yr) were prepared surgically with intracerebral ventricular (ICV) stainless-steel cannula and ovariectomized as described previously (Anderson et al., 2001). Ewes were allowed to recover for 3 to 4 wk. They were then transported to Cornell University and housed indoors in metabolic cages under a controlled environment (12 h light/dark photoperiod, 18 to 20°C). Ewes were randomly assigned to a Latin square design with treatments consisting of ICV infusion of the following oLEP variants: WT, R4C, A59V, and Q62R. At the start of the experiment, sheep had an average BW of 89.4 ± 4.3 kg and were in good body condition. Each experimental period lasted 7 d, with the first 4 d serving to measure preinfusion feed intake. Each ICV infusion was initiated at 1400 h on the fifth day and continued for 72 h. Each LEP variant was infused at a dose of 10 µg/d. This was done by diluting each LEP variant to a final concentration of 0.10 µg/µL in artificial cerebrospinal fluid (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2). Each variant was then infused continuously at 100 µL/h for 72 consecutive hours using a syringe pump (model SE 400, Vial Medical, Grenoble, France) connected to the ICV stainless-steel cannula via sterile polyvinyl chloride tubing. The diet was offered ad libitum and delivered semi-continuously (every 2 h) using automatic feeders. The diet was a total mixed ration consisting of chopped grass hay, cracked corn, molasses, and mineral supplement in the ratio 67:30:2.4:0.6, and contained 122 g of CP and 2.43 Mcal of ME/kg of DM. Feed intake was recorded every 24 h throughout the experimental period. An intervening period of at least 14 d separated each treatment.

Statistical Analysis

The dissociation constant (KD) of the different muteins to hLBD and chLBD in the SPR experimental system was analyzed using the GLM procedure of the Jump In computer package (SAS Inst. Inc., Cary, NC). The model included the effects of receptor type (hLBD, chLBD) and mutein type (n = 6). The relative biological activity of the different muteins compared with the biological activity of the WT oLEP in the Baf/3 bioassay was analyzed in a similar way. The model included the effects of experiment number and mutein type.

Daily feed intake results of the in vivo experiment were subjected to ANOVA. The model included the effects of ewe (n = 4), experiment within ewe (n = 4 for each ewe), experimental period (preinfusion or infusion), LEP type injected within experimental period (one class of “none” for the preinfusion period; 4 classes of WT oLEP, R4C, A59V, and Q62R for the infusion period), and day within experimental period (d −4, −3, −2, −1 for the preinfusion period; d 1, 2, 3, for the infusion period). All data are expressed as least squares means ± SEM. Differences were considered significant at P < 0.05.


RESULTS

Expression, Purification, and Characterization of LEP Muteins

The respective WT oLEP, WT bLEP, and all muteins (ovine R4C, bovine R4C, ovine A59V, and ovine Q62R) were purified by anion-exchange chromatography as described previously (Raver et al., 2000). Fractions containing monomers that were eluted with 50 mM NaCl were pooled, dialyzed in the presence of NaHCO3 to ensure a 4:1 protein-to-salt ratio, and lyophilized. The yields varied from 75 to 180 mg from 1 L of bacterial culture. The purity and homogeneity of all of the proteins were documented by SDS-PAGE. Only 1 band of ~16 kDa was obtained for all LEP under both reducing and nonreducing conditions (Figure 2).

Figure 2.
Figure 2.

The SDS-PAGE of the reference wild-type ovine leptin (WT oLEP) and the ovine muteins R4C, A59V, and Q62R. Lanes: 1 and 5, WT oLEP; 2 and 6, R4C; 3 and 7, A59V; 4 and 8, Q62R; middle lane, prestained molecular-mass markers (in kDa). Lanes 1 to 4 are without and lanes 5 to 8 are with β-mercaptoethanol. Aliquots of 1 μg of each protein were applied per lane. The gel was stained with Coomassie Brilliant Blue R. Color version available in the online PDF.

 

As expected, in the absence of reducing agent, the mobility of all proteins was slightly greater, indicating a globular structure. Gel filtration at pH 8 under native conditions yielded a main monomeric peak consisting of at least 95% of the total protein and corresponding to a molecular mass of ~16 kDa (not shown). Reverse-phase chromatography also yielded a single peak (not shown).

Stability and Determination of CD Spectra

The stability of all ovine and bovine muteins in solution was tested at 4 and 37°C. All 6 proteins could be stored at both temperatures as sterile 0.2 mM solutions for at least 30 d at pH 8 or 9 without undergoing any change in their monomeric content and retaining their full activity in a Baf/3 bioassay. The secondary structures of all ovine muteins, namely, WT oLEP, R4C, A59V, and Q62R, were calculated from the CD spectra as shown in Table 2. A high content of α-helix (53 to 61%), 0 to 3% β-sheets, and 16 to 17% β-turns were clearly characteristic of all proteins, similar to hLEP, indicating proper refolding.

Table 2.

Please see the pdf to view this table.

 

Detection of LEP Complexes with chLBD by Gel Filtration

The binding stoichiometry of WT and mutated LEP was assessed by mixing them in different molar ratios with chLBD, and separating by gel filtration using an analytical Superdex 75 column to determine the molecular mass of the binding complex under nondenaturing conditions. As expected, all proteins formed 1:1 molar ratio complexes with chLBD, as shown previously by us (Reicher et al., 2011) and others (Raver et al., 2002; Mistrík et al., 2004). This was evidenced by the appearance of a single main peak representing the complex eluting at 12.81 to 12.89 min, compared with 14.10 to 14.18 min for chLBD and 15.36 to 15.52 min for the various recombinant LEP. The greatest main peak appeared when the components were mixed at a 1:1 molar ratio, whereas an additional peak appeared when there was an excess of LEP or chLBD. The calculated molecular mass of the complex, based on peak retention times, was ~41 kDa in all cases, close to the predicted value of 40.5 kDa.

Kinetic Measurements of oLEP-hLBD and oLEP-chLBD Interactions

The SPR technique using hormones immobilized on a sensor chip was employed to characterize the binding capacities of hLBD and chLBD to WT oLEP, WT bLEP, and their muteins. The most acceptable interactions were obtained from comparison with a 1:1 theoretical model using χ2 analysis. The χ2 values were very low in all cases. The calculated data and χ2 values are presented in Table 3.

Table 3.

Please see the pdf to view this table.

 

On average, binding capacities of the LEP muteins to hLBD and chLBD did not differ (P < 0.05). The WT oLEP and WT bLEP interacted with hLBD or chLBD with similar affinity (P > 0.05) and their KD values (LSM) were, respectively, 3.79 and 3.64 (M × 10−9) for hLBD and 2.53 and 2.60 (M × 10−9) for chLBD. In contrast, all LEP muteins exhibited similar, significantly (P < 0.05) reduced, affinity in comparison with WT oLEP. The calculated KD values (LSM) for all muteins examined are summarized in Table 3.

Baf/3 Bioassay

Bioassay of a cell line stably transfected with the long form of human LEP receptor (Baf/3) was used to measure the proliferative activity induced by the WT LEP and their muteins. A representative dose-responsive experiment (1 out of 7) is presented in Figure 3. The relative activity, as calculated by EC50, of various recombinant proteins in each experiment was compared with that of the WT oLEP activity. The proliferative activity did not differ (P > 0.05) among experiments. On the other hand, mutein type had a significant (P < 0.01) effect on Baf/3 proliferative activity. The respective comparative values (WT oLEP = 100) of WT bLEP, ovine R4C, bovine R4C, ovine A59V, and Q62R muteins were (mean ± SEM): 119 ± 30, 179 ± 22, 202 ± 24, 91 ± 22, and 118 ± 22 (Table 4). The WT oLEP, WT bLEP, and the A59V and Q62R oLEP muteins did not differ (P > 0.05) in their ability to induce Baf/3 proliferation. However, the activities of ovine and bovine R4C muteins were similar and significantly greater (P < 0.05) than those of the reference WT LEP.

Figure 3.
Figure 3.

Effect of the reference wild-type ovine leptin (WT oLEP), the reference wild-type bovine leptin (WT bLEP), ovine and bovine R4C muteins, and ovine leptin muteins A59V and Q62R on proliferation of Baf/3 cells stably transected with the long form of human leptin receptor. Results of each experiment were normalized to the maximal response, and absorbance in wells not treated with the reference oLEP was taken as zero. A representative experiment (out of 7 performed for each analog) is shown. The EC50 values for oLEP, bLEP, ovine and bovine R4C, and ovine muteins A59V and Q62R were 15.92, 13.38, 8.88, 7.87, 17.47, and 13.47 pM, respectively. Results are presented as mean ± SEM, but the SEM values were, in most cases, too small to be visible on the graph. For more details, see text.

 
Table 4.

Please see the pdf to view this table.

 

In Vivo Experiment

Three LEP muteins were also tested in vivo and compared with WT oLEP. Feed intake of Suffolk ewes treated with constant ICV infusion of various oLEP recombinant hormones (WT, R4C, A59V, or Q62R) was measured daily. All of the effects, which were statistically significant (P < 0.0001), affected daily feed intake with R2 of the model being 0.92. Average daily feed intake in the pretreatment period was 1.65 ± 0.02 kg/d, and there was no significant (P > 0.05) difference in feed intake among the 4 d of the pretreatment period. After the various LEP muteins ICV administration, daily feed intake was reduced (P < 0.05) to an average of 1.06 ± 0.03 kg, and the effect was gradual; feed intake on the first day of infusion was on average 1.29 ± 0.05 kg, significantly (P < 0.05) less than the average pretreatment daily feed intake values, but significantly (P < 0.05) greater than the ADFI on the second and third days of the infusion: 0.99 ± 0.05 and 0.88 ± 0.05 kg, respectively. In a separate control experiment, infusion of artificial cerebrospinal fluid alone had no effect on feed intake (preinfusion vs. artificial cerebrospinal fluid intake, 1.73 ± 0.02 vs. 1.68 ± 0.01 kg/d).

Infusion of the muteins R4C, A59V, and Q62R significantly (P < 0.05) reduced feed intake to 1.11 ± 0.07, 1.23 ± 0.07, and 1.12 ± 0.07 kg/d, respectively, corresponding to reductions of 32, 25, and 31%. The inhibitory effects of the 3 muteins did not differ (P > 0.05). However, the WT oLEP caused a greater inhibitory effect, reducing feed intake to 0.76 ± 0.07 kg/d, representing a 54% reduction in feed intake. The reduction in feed intake caused by WT oLEP treatment was significantly greater (P < 0.05) than the reduction in feed intake caused by the R4C, A59V, and Q62R muteins.


DISCUSSION

The LEP is a cytokine-like hormone that regulates appetite, energy homeostasis, body composition, reproduction, immunity, and metabolic functions (Ahima and Flier, 2000). Whereas in wild animals, adaptive evolution has been shown to have occurred in pika (Ochotona curzoniae) LEP in response to environmental stress (extreme cold; Yang et al., 2008), in livestock, polymorphism in the LEP gene has been found to be associated with variations in traits of economic importance (van der Lende et al., 2005; Boucher et al., 2006; Singh et al., 2009; Zhou et al., 2009). In sheep, products of the different allele variants in the LEP gene have been shown to differ in their biochemical and biological properties (Reicher et al., 2011). The presence and maintenance of LEP genetic polymorphism in the bovine population suggests that different forms of the protein might have differential functional abilities. To gain insight into this question, recombinant proteins representing products of different alleles can be studied for their in vitro and in vivo biochemical and physiological properties.

To test the biochemical and physiological properties of known variants detected in the bLEP gene, we prepared oLEP muteins incorporating 3 out of the 4 known substitutions (R4C, A59V, and Q62R) of bLEP (the fourth, namely N78S, was detected also in ovine and therefore was investigated previously; Reicher et al., 2011), expressed the proteins in a prokaryotic system and purified them to homogeneity. The percentage of α-helix content was very close to the expected values of LEP, indicating proper refolding.

Examination of the various properties of the 3 muteins relative to the WT LEP proteins did not reveal any fundamental differences. All 3 retained their capacity to bind to LEP receptor and exhibited biological activity both in vitro and in vivo. However, the affinity toward the 2 tested soluble LBD was reduced 29 to 60% compared with the WT oLEP. Those differences originated mainly from the decreased kon values. These results are in agreement with our previous study in which oLEP muteins (Reicher et al., 2011) exhibited reduced affinity to hLBD and chLBD, compared with affinity of the WT oLEP.

On the other hand, the in vitro biological activity of the 3 muteins, as determined in the Baf/3 bioassay, was either unchanged (A59V and Q62R muteins) or doubled in the case of both ovine and bovine R4C muteins. Such a discrepancy between the effect on binding and biological activity has also been demonstrated previously for some other LEP muteins by us (Reicher et al., 2011) and others (Peelman et al., 2004). It may be explained by the fact that the biological activity is dependent not only on the ability of the LEP to bind to its receptor but also on its ability to induce formation of biologically active hexameric complex, as explained further on.

In livestock, the LEP allelic polymorphism has been shown to be involved with variation in feed intake (van der Lende et al., 2005; Wylie, 2010). As LEP muteins were found to differ in their binding affinity and biological activity in vitro, we investigated their impact at the physiological level by characterizing their effect on feed intake after ICV infusion in sheep. The WT oLEP and its 3 muteins (R4C, A59V, and Q62R) inhibited feed intake. Statistical analysis revealed that although there was no significant difference among the inhibitory effects of the 3 muteins, the WT LEP had a significantly stronger inhibitory effect [see Table 4, which summarizes the in vitro relative binding affinity (SPR), the relative biological activity (Baf/3 bioassay), and the in vivo relative feed intake values]. The decrease in affinity toward the receptor paralleled the in vivo results but not the effect on biological activity in vitro. At present, we have no explanation for this discrepancy. It may be related to differences, not in the binding step, but in the subsequent activation of the receptor by formation of the biologically active hexameric (2 leptins:4 receptors) complex. Formation of such a complex (Peelman et al., 2006) and subsequent induction of increased biological activity despite reduced binding affinity may originate from an increased ability to form a hexameric complex due to interaction of site III of LEP with the immunoglobulin-like domain of the neighboring receptor, as we previously suggested (Reicher et al., 2011). Structural changes affecting such interactions may differ between human and ovine LEP receptors, explaining the observed discrepancy between the biological activity in vitro and in vivo. Unfortunately, structural data are not yet available to examine this hypothesis.

The LEP protein circulates in the serum as a free hormone or as a complex with LEP soluble receptor (bound form; Lewandowski et al., 1999). It was found that the proportion of circulating free LEP to bound LEP varies in different physiological conditions; therefore, it was suggested that it influences the biological activity of LEP (Jordan et al., 2005). The R4C alteration might have an effect on the tertiary structure of the protein due to the presence of an extra cysteine, which may destabilize the disulfide bridge found between the 2 cysteines of the LEP (Liefers et al., 2003). In addition, it has been suggested that this variation might disrupt the binding of LEP to its receptor (Buchanan et al., 2002). Our results support the latter notion, as we showed that both ovine and bovine R4C protein variants exhibit reduced affinity to hLBD and chLBD. Furthermore, our results indicate that the reduced affinities of both ovine and bovine R4C muteins are due mainly to a reduction in kon values, rather than to changes in koff values. Disrupted formation of LEP:soluble LEP receptor complex caused by the R4C alteration may lead to a greater proportion of free LEP in the serum. If so, the free LEP, which is not protected from proteolytic degradation, will be cleared more rapidly from circulation (Liefers et al., 2003), and this may explain part of the phenotypic effect, which correlates this mutation to certain traits.

In conclusion, this is the first investigation of known allelic variations detected in the bLEP at the protein level. It serves to reduce the knowledge gap between the association of allelic variation and its associated variation in economically important traits in livestock. We show that 3 of the known LEP variants in cows cause some changes that are directly related to the properties of the respective muteins. However, additional factors, such as expression level, clearance, and tissue-specific effects may affect overall physiological performance.

 

References

Footnotes


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