Mapping of Binding Site III in the Leptin Receptor and Modeling of a Hexameric Leptin·Leptin Receptor Complex

Peelman, Frank; Iserentant, Hannes; Smet, A. De; Vandekerckhove, Joël; Zabeau, Lennart; Tavernier, Jan

doi:10.1074/jbc.m512622200

Cited by 77 publications

(79 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…To test the generality of the antibody-CDR fusion approach, we next attempted to fuse human leptin, also a member of the four-helix-bundle hormone family, into the Herceptin CDR3 regions of the heavy and light chains. Previous studies have indicated that residues at the N terminus of helix D of hLeptin are essential for binding to and activating hLeptin receptor (38,39). These residues are on the opposite face of hLeptin relative to the free N and C termini.…”

Section: Resultsmentioning

confidence: 99%

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

Liu

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

On the basis of the 3D structure of a bovine antibody with a wellfolded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20-to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.antibody | protein engineering | growth hormone | leptin | pharmacology M any endocrine hormones are used therapeutically (1); however, they generally suffer from short circulating halflives (2, 3) and therefore require high doses and frequent injections to achieve efficacious exposures. For example, human growth hormone (hGH) is a 22-kDa four-helix-bundle protein produced by the pituitary gland, and its recombinant form has long been used for the treatment of growth hormone deficiency (GHD). Due to the short in vivo half-life of hGH, which is on the timescale of minutes, current GHD therapy requires daily injection (4), resulting in lower patient compliance (5). Studies have shown that a continuous infusion of recombinant (r)hGH has a similar efficacy and safety profile as daily rhGH therapy (6), and thus there is considerable interest in the development of long-acting forms of hGH. The first and only approved long-acting hGH, Nutropin Depot, a sustained-release formulation based on a biodegradable polymer, was withdrawn due to manufacturing difficulties. Various forms of long-acting GH have since been generated: those currently in clinical development include LB03002 (microparticle suspension), NNC126-0083 (PEGylation), NNC0195-0092 (reversible albumin-binding GH derivative), MOD-4023 (CTP modification), ACP-001 (prodrug strategy), Albutropin (albumin fusion), and VRS-317 (XTEN fusion), and have been extensively reviewed elsewhere (5). However, challenges associated with heterogeneity, stability, immunogenicity, toxicity, and/or compromised potency could potentially limit their clinical utility (3, 7-10).Another short-lived hormone in which there is considerable clinical interest is human leptin, a 16-kDa four-helix-bundle protein that plays a central role in the homeostasis of body weight. Recombinant leptin has been approved for the treatment of leptindeficient lipodystrophy patients (11), and ...

show abstract

Section: Resultsmentioning

confidence: 99%

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

Liu

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The extracellular region contains two cytokine receptor homology domains, i.e. cytokine receptor homology domain 1 (CRH1) and cytokine receptor homology domain 2 (CRH2) [9,110,115,116]. Based on previously studies, the CRH2 domain is essential for the interaction of leptin and its receptor [9,115,117,118], and it bind to the site II in leptin [9,110,[115][116][117][118].…”

Section: Leptin and Leptin Receptor Co-evolutionmentioning

confidence: 99%

“…The leptin receptor is a large single-transmembrane-domain receptor of the class I cytokine receptor family and is coded by the diabetes (db) gene [10,[108][109][110][111]. Homozygous mutant db/db mice produce a syndrome that is phenotypically identical to the ob/ob mouse [108,109,112,113].…”

Section: Leptin and Leptin Receptor Co-evolutionmentioning

confidence: 99%

Natural selection and adaptive evolution of leptin

Guo

Zhang

2013

Chin. Sci. Bull.

View full text Add to dashboard Cite

Leptin is an adiposity-secreted hormone that is pivotal in regulating feeding behavior, energy metabolism and body mass. The study of leptin has been of crucial importance for public health and pharmaceutical intervention given its role in obesity. Generally, leptin is highly conserved due to its functional importance. However, episodes of rapid sequence evolution and positive selection have been observed in some mammalian species, indicating that the leptin functions in these animals may have undergone adaptive modification to their environments. In this article, we review the adaptive evolution of leptin and its potential functional consequences. This review is expected to guide future research of molecular evolution and functional assays of this gene, and also to provide a theoretical foundation for the use of leptin in therapeutic applications. natural selection, adaptive evolution, leptin, leptin receptor Citation:Zou G, Zhang Y P, Yu L. Natural selection and adaptive evolution of leptin. Chin Sci Bull, 2013Bull, , 58: 21042112, doi: 10.1007 The relationship between the adaptations of an organism to different ecological niches with genetic changes (e.g. the change of genes) has been one of the essential subjects in the study of life sciences. Accordingly, the mechanism underlying how organisms adapt to different living environments at molecular level has fascinated evolutionary biologists [1][2][3][4][5][6][7][8][9]. Metabolism is a series of biochemical reactions, allowing organisms to exchange materials and energy with the environment. These processes control growth, reproduction and behavior of organisms. Although the mechanisms of increased energy utilization are likely to be complex, one important component involves the activation of adipose tissue [10].Leptin, encoded by the obese gene, is a hormone that mainly secreted by adipose tissue. It is important in regulating feeding behavior, energy metabolism and body mass [11][12][13][14][15][16]. The level of plasma leptin is generally regarded as a signal to direct the central nervous system (CNS) to regulate food intake and energy expenditure, which is an important pathway of metabolism to maintain constancy of the adipose mass [12,14,17,18]. The mutation of ob gene results in a myriad of disturbance of metabolism [12,19]. Mice that are homozygous (ob/ob mouse) for mutations in this gene exhibit a profound obesity resulting from defects in energy expenditure, food intake and nutrient partitioning [12,20,21]. In humans, mutation of this gene results in a profound obesity and type II diabetes [12,[20][21][22][23]. Therefore, due to its critical role in obesity, the study of leptin will shed insight into molecular mechanism of energy homeostasis, future pharmaceutical intervention and public health [9,10,24].The leptin gene, spanning over 4.5-kb, consists of three exons and two introns [9,25]. The coding exons (exons 2 and 3) are 501 bp in length in total. Leptin contains an amino-terminal signal peptide (21 residues) and a mature peptide (146 residues),...

show abstract

“…While the CRH2 module serves as the primary ligand binding site [1], the Ig-like and FNIII domains have also been implicated in complex formation and receptor activation. Binding of the cytokine to the extracellular portion of the leptin receptor is thought to induce a conformational change that promotes transphosphorylation of constitutively bound tyrosine janus kinase 2 (JAK2) on the intracellular site of the receptor.The mechanism of activation of L-R as well as the exact stoichiometry of the complex is controversial [2,3]. Additionally, the relative large molecular size (∼170kDa) and flexibility of the receptor make its structural determination with techniques like NMR and x-ray chrystallography exceedingly challenging.…”

mentioning

confidence: 99%

“…The mechanism of activation of L-R as well as the exact stoichiometry of the complex is controversial [2,3]. Additionally, the relative large molecular size (∼170kDa) and flexibility of the receptor make its structural determination with techniques like NMR and x-ray chrystallography exceedingly challenging.…”

mentioning

confidence: 99%

Structural Characterization of the Activated Leptin Receptor Complex

2010

View full text Add to dashboard Cite

Leptin is a hormone, secreted from adipocytes in levels proportional to body fat, and binds to the leptin receptor (L-R) in the hypothalamus, thereby regulating body energy homeostasis. Leptin signaling reduces food intake while it increases energy expenditure, and mutations in either the hormone or its receptor result in marked obesity in both mice and humans. Beyond this role, leptin action is also heavily implicated in hyperinsulinaemia and type 2 diabetes, tumorigenesis, angiogenesis, bone formation, sexual maturation, immunity and wound healing.The L-R is a transmembrane protein with an extracellular domain organization that is similar to other members of the class I cytokine receptor family. The ectodomain is characterized by two cytokine receptor homology region (CRH1 and CRH2) separated by an Ig-like domain, and also includes two fibronectin III (FNIII) membrane proximal domains. While the CRH2 module serves as the primary ligand binding site [1], the Ig-like and FNIII domains have also been implicated in complex formation and receptor activation. Binding of the cytokine to the extracellular portion of the leptin receptor is thought to induce a conformational change that promotes transphosphorylation of constitutively bound tyrosine janus kinase 2 (JAK2) on the intracellular site of the receptor.The mechanism of activation of L-R as well as the exact stoichiometry of the complex is controversial [2,3]. Additionally, the relative large molecular size (∼170kDa) and flexibility of the receptor make its structural determination with techniques like NMR and x-ray chrystallography exceedingly challenging. We employ single-particle electron microscopy to structurally characterize the extracellular portion of the receptor alone and in complex with leptin.Our results show that the extracellular portion of L-R is a monomer in solution, but forms a dimer after incubation with leptin in a 2:2 stoichiometric ratio. 2D EM averages and modeling of the L/L-R complex suggest that leptin employs conserved epitope II to engage the CRH2 of one L-R, while epitope III engages the Ig-like domain of a second L-R. In addition, the FNIII domains of the 2 L-R chains appear to interact at their C-terminal tips, by a homotypic interaction not observed in the nonliganded receptor. We postulate that the close proximity and resulting orientation of the membrane proximal FNIII domains is promoting JAK2 transphosphorylation on the intracellular side of the receptor.

show abstract

Mapping of Binding Site III in the Leptin Receptor and Modeling of a Hexameric Leptin·Leptin Receptor Complex

Cited by 77 publications

References 45 publications

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

Natural selection and adaptive evolution of leptin

Structural Characterization of the Activated Leptin Receptor Complex

Contact Info

Product

Resources

About