Exploring the folding landscape of leptin: Insights into threading pathways.

The discovery of new protein topologies with entanglements and loop-crossings have shown the impact of local amino acid arrangement and global three-dimensional structures. This phenomenon plays a crucial role in understanding how protein structure relates to folding and function, affecting the global stability, and biological activity. Protein entanglements encompassing knots and non-trivial topologies add complexity to their folding free energy landscapes. However, the initial native contacts driving the threading event for entangled proteins remains elusive. The Pierced Lasso Topology (PLT) represents an entangled topology where a covalent linker creates a loop in which the polypeptide backbone is threaded through. Compared to true knotted topologies, PLTs are simpler topologies where the covalent-loop persists in all conformations. In this work, the PLT protein leptin, is used to visualize and differentiate the preference for slipknotting over plugging transition pathways along the folding route. We utilize the Energy Landscape Visualization Method (ELViM), a multidimensional projection technique, to visualize and distinguish early threaded conformations that cannot be observed in an in vitro experiment. Critical contacts for the leptin threading mechanisms were identified where the competing pathways are determined by the formation of a hairpin loop in the unfolded basin. Thus, prohibiting the dominant slipknotting pathway. Furthermore, ELViM offers insights into distinct folding pathways associated with slipknotting and plugging providing a novel tool for de novo design and in vitro experiments with residue specific information of threading events in silico.

Topological Reaction Coordinate Captures the Folding Transition State Ensemble in a Pierced Lasso Protein.

Proteins with a pierced lasso topology (PLT) have a covalent loop created by a disulfide bond, and the backbone circles back to thread the loop. This threaded topology has unique features compared to knotted topologies; notably, the topology is controlled by the chemical environment and the covalent loop remains intact even when denatured. In this work, we use the hormone leptin as our model PLT system and study its folding using molecular dynamics simulations that employ a structure-based (Go̅-like) model. We find that the reduced protein has a two-state folding mechanism with a transition state ensemble (TSE) that can be characterized by the reaction coordinate Q, the fraction of native contacts formed. In contrast, the oxidized protein, which must thread part of the polypeptide chain through a covalent loop, has a folding process that is poorly characterized by Q. Instead, we find that a topological coordinate that monitors the residue crossing the loop can identify the TSE of oxidized leptin. By precisely identifying the predicted TSE, one may now reliably calculate theoretical phi-values for the PLT protein, thereby enabling a comparison with experimental measurements. We find the loop-threading constraint leads to noncanonical phi-values that are uniformly small because this PLT protein has a flat energy landscape through the TSE.

A Small Contribution to a Large System: The Leptin Receptor Complex.

Obesity is a classified epidemic, increasing the risk of secondary diseases such as diabetes, inflammation, cardiovascular disease, and cancer. The pleiotropic hormone leptin is the proposed link for the gut-brain axis controlling nutritional status and energy expenditure. Research into leptin signaling provides great promise toward discovering therapeutics for obesity and its related diseases targeting leptin and its cognate leptin receptor (LEP-R). The molecular basis underlying the human leptin receptor complex assembly remains obscure, due to the lack of structural information regarding the biologically active complex. In this work, we investigate the proposed receptor binding sites in human leptin utilizing designed antagonist proteins combined with AlphaFold predictions. Our results show that binding site I has a more intricate role in the active signaling complex than previously described. We hypothesize that the hydrophobic patch in this region engages a third receptor forming a higher-order complex, or a new LEP-R binding site inducing allosteric rearrangement.

Leptin-Activity Modulators and Their Potential Pharmaceutical Applications.

Leptin, a multifunctional hormone primarily, but not exclusively, secreted in adipose tissue, is implicated in a wide range of biological functions that control different processes, such as the regulation of body weight and energy expenditure, reproductive function, immune response, and bone metabolism. In addition, leptin can exert angiogenic and mitogenic actions in peripheral organs. Leptin biological activities are greatly related to its interaction with the leptin receptor. Both leptin excess and leptin deficiency, as well as leptin resistance, are correlated with different human pathologies, such as autoimmune diseases and cancers, making leptin and leptin receptor important drug targets. The development of leptin signaling modulators represents a promising strategy for the treatment of cancers and other leptin-related diseases. In the present manuscript, we provide an update review about leptin-activity modulators, comprising leptin mutants, peptide-based leptin modulators, as well as leptin and leptin receptor specific monoclonal antibodies and nanobodies.

Specific Binding of Leptin to Red Blood Cells Delivers a Pancreatic Hormone and Stimulates ATP Release.

Since its discovery in 1994, leptin continues to have new potential physiological roles uncovered, including a role in the regulation of blood flow. Leptin's role in regulating blood flow is not completely understood. Red blood cell (RBC)-derived ATP is a recognized stimulus of blood flow, and multiple studies suggest that C-peptide, a hormone secreted in equimolar amounts with insulin from the pancreatic ß-cells, can stimulate that release when delivered by albumin and in combination with Zn2+. Here, we report leptin delivers C-peptide and Zn2+ to RBCs in a saturable and specific manner. We labeled leptin with technetium-99 m (99mTc) to perform binding studies while using albumin to block the specific binding of 99mTc-leptin in the presence or absence of C-peptide. Our results suggest that leptin has a saturable and specific binding site on the RBC ((Kd = 1.79 ± 0.46) × 10-7 M) that is statistically equal to the binding affinity in the presence of 20 nM C-peptide ((Kd = 2.05 ± 0.20) × 10-7 M). While the binding affinity between leptin and the RBC did not change with C-peptide, the moles of bound leptin did increase with C-peptide, suggesting a separate binding site on the cell for a leptin/C-peptide complex. The RBC-derived ATP increased in the presence of a leptin/C-peptide/Zn2+ addition, in a concentration-dependent manner. Control RBCs ATP release increased (71 ± 5.6%) in the presence of C-peptide and Zn2+, which increased further to (94 ± 5.6%) in the presence of Zn2+, C-peptide, and leptin.

An immunosensor for the determination of carcinoembryonic antigen by Surface Plasmon Resonance imaging.

Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the "EDC/NHS protocol". The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL-1, and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 µL of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2-16%, a recovery of 101-104% depending on CEA concentration, a detection limit of 0.12 ng mL-1 and a quantification limit of 0.40 ng mL-1. The method is selective (with respect to albumin, leptin, interleukin 6, metalloproteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.

The Pierced Lasso Topology Leptin has a Bolt on Dynamic Domain Composed by the Disordered Loops I and III.

Leptin is an important signaling hormone, mostly known for its role in energy expenditure and satiety. Furthermore, leptin plays a major role in other proteinopathies, such as cancer, marked hyperphagia, impaired immune function, and inflammation. In spite of its biological relevance in human health, there are no NMR resonance assignments of the human protein available, obscuring high-resolution characterization of the soluble protein and/or its conformational dynamics, suggested as being important for receptor interaction and biological activity. Here, we report the nearly complete backbone resonance assignments of human leptin. Chemical shift-based secondary structure prediction confirms that in solution leptin forms a four-helix bundle including a pierced lasso topology. The conformational dynamics, determined on several timescales, show that leptin is monomeric, has a rigid four-helix scaffold, and a dynamic domain, including a transiently formed helix. The dynamic domain is anchored to the helical scaffold by a secondary hydrophobic core, pinning down the long loops of leptin to the protein body, inducing motional restriction without a well-defined secondary or tertiary hydrogen bond stabilized structure. This dynamic region is well suited for and may be involved in functional allosteric dynamics upon receptor binding.

Directional Immobilization of Proteins on Gold Nanoparticles Is Essential for Their Biological Activity: Leptin as a Case Study.

Gold nanomaterials hold great potential for biomedical applications. While this field is evolving rapidly, little attention has been paid to precise nanoparticle design and functionalization. Here, we show that when using proteins as targeting moieties, it is fundamental to immobilize them directionally to preserve their biological activity. Using full-length leptin as a case study, we have developed two alternative conjugation strategies for protein immobilization based on either a site-selective or a nonselective derivatization approach. We show that only nanoparticles with leptin immobilized site-selectively fully retain the ability to interact with the cognate leptin receptor. These results demonstrate the importance of a specified molecular design when preparing nanoparticles labeled with proteins.

Porous graphene-black phosphorus nanocomposite modified electrode for detection of leptin.

Leptin is a vital biomarker of non-alcoholic fatty liver (NAFLD), and its evaluation of the concentration level in vivo is of great significance to NAFLD diagnosis. Therefore, it is pressing to develop a method for rapid and sensitive detection of leptin. This paper describes an environmentally friendly and label-free immunosensor based on porous graphene functionalized black phosphorus (PG-BP) composite to detect of leptin. The PG-BP was synthesized via strong coherent coupling between porous graphene (PG) surface plasmons and anisotropic black phosphorus (BP) localized surface plasmons, which made the electrochemical performance of PG and BP synergistic as well as increased the stability and conductive capability of BP material. The PG-BP modified electrodes was further prepared by gold nanoparticles, cysteamine, and glutaraldehyde in turn. Due to the cross-linking effect of glutaraldehyde, anti-leptin can be firmly fixed. These properties of the platform improved the conductive capability of the immunosensor and enhanced the load capacity of the proteins, thereby, the sensitivity of the immunosensor was significantly increased. Under the optimal conditions, the proposed immunosensor exhibited a wide linear range of 0.150-2500 pg/mL with a low detection limit of 0.036 pg/mL. The leptin immunosensor displayed excellent selectivity and anti-interference ability, which could be used for early screening and diagnosis of clinical NALFD.

Leptin Receptor as a Potential Target to Inhibit Human Testicular Seminoma Growth.

Although in past decades the adipokine leptin and its own receptor have been considered as significant cancer biomarkers, their potential involvement in human testicular seminoma growth and progression remains unexplored. Here, we showed that the expression of leptin and its receptor was significantly higher in human testicular seminoma compared with normal adult testis. Human seminoma cell line TCam-2 also expressed leptin along with the long and short isoforms of leptin receptor, and in response to leptin treatment showed enhanced activation of its downstream effectors. In line with these results, leptin stimulation significantly increased the proliferation and migration of TCam-2 cells. Treatment of TCam-2 cells with the peptide Leu-Asp-Phe-Ile (LDFI), a full leptin-receptor antagonist, completely reversed the leptin-mediated effects on cell growth and motility as well as reduced the expression of several leptin-induced target genes. More importantly, the in vivo xenograft experiments showed that LDFI treatment markedly decreased seminoma tumor growth. Interestingly, LDFI-treated tumors showed reduced levels of the proliferation marker Ki-67 as well as decreased expression of leptin-regulated genes. Taken together, these data identify, for the first time, leptin as a key factor able to affect testicular seminoma behavior, highlighting leptin receptor as a potential target for novel potential treatments in this type of cancer.

Effect of HTST and Holder Pasteurization on the Concentration of Immunoglobulins, Growth Factors, and Hormones in Donor Human Milk.

Donor human milk (DHM) is submitted to Holder pasteurization (HoP) to ensure its microbiological safety in human milk banks but this treatment affects some of its bioactive compounds. The objective of this work was to compare the effects of HoP and high temperature short time (HTST) treatments on some bioactive compounds found in DHM. A total of 24 DHM batches were processed in a continuous HTST system (70, 72, and 75°C for 5-25 s) and by HoP (62.5°C for 30 min). The concentrations of immunoglobulins (Igs) A, G, and M, transforming growth factor-beta 2 (TGF-ß2), adiponectine, ghrelin, and leptin were measured using a multiplex system, whereas the concentration of epidermal growth factor (EGF) was determined by ELISA. In relation to Igs, IgG showed the highest preservation rates (87-101%) after HTST treatments, followed by IgA (54-88%) and IgM (25-73%). Ig retention after any of the HTST treatments was higher than after HoP (p < 0.001). Treatment times required to reduce the concentration of IgM by 90% (D-value) were 130, 88, and 49 s at 70, 72, and 75°C, while the number of degrees Celsius required to change the D-value by one factor of 10 (z-value) was 11.79°C. None of the heat treatments had a significant effect on the concentrations of TGF-ß2, EGF, adiponectin, and ghrelin. In contrast, leptin was detected only in 4 of the samples submitted to HoP, whereas it was present in all samples after the different HTST treatments, with retention rates ranging between 34 and 68%. Globally, the concentration of IgA, IgG, IgM, and leptin in DHM was significantly higher after HTST pasteurization performed in a continuous system designed to be used in human milk banks than after the HoP procedure that is routinely applied at present.

Leptin and leptin receptor genes in tongue sole (Cynoglossus semilaevis): Molecular cloning, tissue distribution and differential regulation of these genes by sex steroids.

Leptin (Lep) is a key factor for the regulation of food intake and energy homeostasis in mammals. To date, a number of studies have provided evidence for the existence of multiple leptin genes in teleosts, but not much information is available in fish regarding the regulation of leptin genes by sex steriods. As a first step, two leptin genes (lepa and lepb) and a leptin receptor (lepr) gene were cloned from the half-smooth tongue sole (Cynoglossus semilaevis), a representative species of the order Pleuronectiformes. The full-length cDNAs of tongue sole lepa and lepb were 1265 bp and 1157 bp in length, encoding for proteins of 160 aa and 158 aa, respectively. The three-dimensional structures modeling of tongue sole LepA and LepB showed strong conservation of tertiary structure with other vertebrates. The full-length cDNA of tongue sole lepr was 4576 bp, encoding a protein of 1133 aa which contained all functionally important domains conserved among vertebrate LepRs. Tissue distribution analysis showed that tongue sole lepa mRNA was highly detectable in the ovary and brain, while lepb mRNA was ubiquitously expressed in various tissues. Notably, the tongue sole lepr mRNA was most abundant in the ovary. Using a primary hepatocyte culture system, we evaluated the effects of sex steroids on lep/lepr gene expression. Both 17ß-estradiol (E2) and testosterone (T) inhibited hepatic lepa and lepr mRNAs without affecting lepb mRNA levels. In addition, T also suppressed growth hormone receptor 1 (ghr1), ghr2, and insulin-like growth factor 2 (igf-2) mRNA levels, and stimulated expression of igf-1 gene. On the other hand, none of these four genes were altered by E2. To the best of our knowledge, this is the first description of a direct and differential regulation of lep/lepr gene expression by sex steroids at the hepatocyte level of a flatfish, supporting that individual leptin peptide may possess different biological roles in teleosts.

[D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, small molecule synthetic peptide leptin mimetics, improve glycemic control in diet-induced obese (DIO) mice.

We have previously shown that following oral delivery in dodecyl maltoside (DDM), [D-Leu-4]-OB3 and its myristic acid conjugate, MA-[D-Leu-4]-OB3, improved energy balance and glucose homeostasis in genetically obese/diabetic mouse models. More recently, we have provided immunohistochemical evidence indicating that these synthetic peptide leptin mimetics cross the blood-brain barrier and concentrate in the area of the arcuate nucleus of the hypothalamus in normal C57BL/6J and Swiss Webster mice, in genetically obese ob/ob mice, and in diet-induced obese (DIO) mice. In the present study, we describe the effects of oral delivery of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on glycemic control in diet-induced (DIO) mice, a non-genetic rodent model of obesity and its associated insulin resistance, which more closely recapitulates common obesity and diabetes in humans. Male C57BL/6J and DIO mice, 17, 20, and 28 weeks of age, were maintained on a low-fat or high-fat diet and given vehicle (DDM) alone or [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in DDM by oral gavage for 12 or 14 days. Body weight gain, food and water intake, fasting blood glucose, oral glucose tolerance, and serum insulin levels were measured. Our data indicate that (1) [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 restore glucose tolerance in male DIO mice maintained on a high-fat diet to levels comparable to those of non-obese C57BL/6J wild-type mice of the same age and sex maintained on a low-fat diet; and (2) the influence of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on glycemic control appears to be independent of their effects on energy balance. These results suggest that [D-Leu-4]-OB3 and/or MA-[D-Leu-4]-OB3 may have application to the management of the majority of cases of common obesity in humans, a state characterized at least in part, by leptin resistance resulting from a defect in leptin transport across the blood-brain barrier. They further suggest that these small molecule synthetic peptide leptin mimetics, through their influence on glycemic control, may prevent the pre-diabetic state associated with most cases of common obesity from escalating into overt type 2 diabetes mellitus.

Light-triggered methylcellulose gold nanoparticle hydrogels for leptin release to inhibit fat stores in adipocytes.

Leptin is released in response to increased triglyceride storage in adipocytes and impacts body weight, but has drawbacks such as poor therapeutic effect and side effects when delivered systemically. Leptin also modifies adipocyte sensitivity to insulin to inhibit lipid accumulation. Here, light-triggered degradation of hydrogels was used to improve accuracy and effectiveness for sustained and controllable release. In our approach, leptin was entrapped within methylcellulose (MC)-based hydrogels, with incorporation of gold nanoparticles (NP). The incorporation of gold NP into MC hydrogels led to a tunable light irradiation response that dictated the hydrogel release rate of leptin. This manuscript demonstrates feasibility in designing tunable thermosensitive hydrogels for loading multimodality therapeutic agents to enhance the bioactivity of leptin for obesity therapy.

Acute effects of intravenous cocaine administration on serum concentrations of ghrelin, amylin, glucagon-like peptide-1, insulin, leptin and peptide YY and relationships with cardiorespiratory and subjective responses.

BACKGROUND: Food intake and use of drugs of abuse like cocaine share common central and peripheral physiological pathways. Appetitive hormones play a major role in regulating food intake; however, little is known about the effects of acute cocaine administration on the blood concentrations of these hormones in cocaine users. METHODS: We evaluated serum concentrations of six appetitive hormones: ghrelin (total and acyl-ghrelin), amylin, glucagon-like peptide-1 (GLP-1), insulin, leptin and peptide YY (PYY), as well as acute cardiorespiratory and subjective responses of 8 experienced cocaine users who received 25mg intravenous (IV) cocaine. RESULTS: Serum concentrations of GLP-1 (p=0.014) and PYY (p=0.036) were significantly decreased one hour following IV cocaine administration; there was a trend towards a decrease for insulin (p=0.055) and amylin (p=0.063) concentrations, while no significant IV cocaine effect was observed for ghrelin (total or acyl-ghrelin) or leptin concentrations (p's≫>0.5). We also observed associations between hormone concentrations acutely affected by IV cocaine (GLP-1, PYY, insulin, amylin) and some cocaine-related cardiorespiratory and subjective responses (e.g., increased heart and respiratory rates; feeling high and anxious). DISCUSSION: These findings show a significant effect of acute IV cocaine administration on some appetitive hormones and suggest potential associations between these hormones and cocaine-related cardiorespiratory and subjective responses. Additional research is needed to further investigate the potential mechanisms underlining these associations.

Pierced Lasso Topology Controls Function in Leptin.

Protein engineering is a powerful tool in drug design and therapeutics, where disulphide bridges are commonly introduced to stabilize proteins. However, these bonds also introduce covalent loops, which are often neglected. These loops may entrap the protein backbone on opposite sides, leading to a "knotted" topology, forming a so-called Pierced Lasso (PL). In this elegant system, the "knot" is held together with a single disulphide bridge where part of the polypeptide chain is threaded through. The size and position of these covalent loops can be manipulated through protein design in vitro, whereas nature uses polymorphism to switch the PL topology. The PL protein leptin shows genetic modification of an N-terminal residue, adding a third cysteine to the same sequence. In an effort to understand the mechanism of threading of these diverse topologies, we designed three loop variants to mimic the polymorphic sequence. This adds elegance to the system under study, as it allows the generation of three possible covalent loops; they are the original wild-type C-terminal loop protein, the fully circularized unthreaded protein, and the N-terminal loop protein, responsible for different lasso topologies. The size of the loop changes the threading mechanism from a slipknotting to a plugging mechanism, with increasing loop size. Interestingly, the ground state of the native protein structure is largely unaffected, but biological assays show that the activity is maximized by properly controlled dynamics in the threaded state. A threaded topology with proper conformational dynamics is important for receptor interaction and activation of the signaling pathways in vivo.

Could tea polyphenols be beneficial for preventing the precocious puberty?

Precocious puberty which impacts children physically and psychologically has become one of the health problem over the world. However, the mechanism and preventive measures of precocious puberty is still not clear. Recent studies suggested that leptin may act as the 'permissive factor' to initiate the puberty by regulating gonadotrophin-releasing hormone secretion. Previous evidence from animal and human studies found that tea polyphenols can reduce serum leptin levels in vivo and inhibit the expression of leptin in adipose tissue. This article focus on whether tea polyphenols could delay the onset of puberty by reducing leptin levels. To verify the possibility of tea polyphenols on preventing precocious puberty, animal experiment can be used. Our hypothesis that tea polyphenols could prevent the precocious puberty may provide important potential way for the prevention and control of children precocious puberty.

Treatment of diet-induced lipodystrophic C57BL/6J mice with long-acting PASylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation.

AIMS/HYPOTHESIS: Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology. METHODS: A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition. RESULTS: In a cohort of female mice, 4 nmol PAS-leptin (applied via four s.c. injections every 3 days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements. CONCLUSIONS/INTERPRETATION: The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.

Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.

Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays.

Leptin Genes in Blunt Snout Bream: Cloning, Phylogeny and Expression Correlated to Gonads Development.

To investigate the leptin related genes expression patterns and their possible function during the gonadal development in fish, the cDNA and genomic sequences of leptin, leptin receptor (leptinR), and leptin receptor overlapping transcript like-1 (leprotl1) were cloned and their expression levels were quantified in the different gonadal development stages of Megalobrama amblycephala. The results showed that the full length cDNA sequences of leptin, leptinR and leprotl1 were 953, 3432 and 1676 bp, coding 168, 1082, and 131 amino acid polypeptides, and the genomic sequences were 1836, 28,528 and 5480 bp, which respectively had 3, 15 and 4 exons, respectively. The phylogenetic analysis revealed that three genes were relatively conserved in fish species. Quantitative real-time PCR results showed that the three genes were ubiquitously expressed in all examined tissues during the different gonadal development stages. The leptin and leptinR took part in the onset of puberty, especially in female M. amblycephala, by increasing the expression levels in brain during the stage I to III of ovary. The expression levels of leptin and leptinR had significant differences between male and female in hypothalamic-pituitary-gonadal (HPG) axis tissues (p < 0.05). The leptinR had the same variation tendency with leptin, but the opposite changes of expression levels were found in leprotl1, which may resist the expression of leptinR for inhibiting the function of leptin in target organ. These findings revealed details about the possible role of these genes in regulating gonadal maturation in fish species.