Subcloning, Expression, Purification, and Characterization of Recombinant Human Leptin-binding Domain

Sandowski, Yael; Raver, N; Gussakovsky, Eugene; Shochat, S.; Dym, Orly; Livnah, Oded; Rubinstein, Menachem; Krishna, Radha; Gertler, Arieh

doi:10.1074/jbc.m207556200

Cited by 59 publications

(48 citation statements)

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“…6C). Disulfide-dependent dimer- ization in the absence of leptin has also been demonstrated for bacterially expressed CRH2 protein (31). Our co-precipitation data furthermore indicate that this CRH2-dimerization occurs intracellularly (Fig.…”

Section: Discussionsupporting

confidence: 62%

Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains

Zabeau

Defeau

Iserentant

et al. 2005

Journal of Biological Chemistry

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The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligandinduced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membranebound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.The 16-kDa cytokine-like hormone leptin has been identified as one of the key players in the control of body weight. The product of the ob gene is produced and secreted mainly by adipocytes (1, 2), and plasma protein levels positively correlate with body fat energy stores. Leptin reduces food intake and stimulates energy expenditure by activating its receptor in specific hypothalamic nuclei (3-5). Spontaneous mutations that lead to a functional defect in either leptin (6) or its receptor (7-9) result in a complex syndrome that includes morbid obesity, hypothermia, infertility, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. Besides serving a weight-regulating function, leptin also plays a role in other processes including metabolism, reproduction, hematopoiesis, and immunity (10 -13).The leptin receptor (LR), 1 the product of the db gene (14), is a member of the class I cytokine receptor family. The extracellular (EC) domain is composed of two so-called cytokine receptor homology (CRH) domains, a membrane distal CRH1 and a membrane proximal CRH2. Both domains are separated by an immunoglobulin-like (Ig) domain and are followed by two fibronectin type III (FNIII) domains proximal to the membrane. The CRH2 domain is necessary and sufficient for leptin binding (15). Despite the lack of any binding affinity for the ligand, the two FNIII and the Ig domains are needed for receptor activation (15, 16). Because of alternative splicing and ectodomain shedding, the LR can exist as six isoforms: a LR long ...

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Section: Discussionsupporting

confidence: 62%

Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains

Zabeau

Defeau

Iserentant

et al. 2005

Journal of Biological Chemistry

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“…Binding site II, which is a major binding site, is found on the surface of helix A and C and binds to the CHR2 (cytokine homology region II) subdomain of the leptin receptor. This subdomain of human leptin receptor was subcloned in our laboratory and expressed as a soluble recombinant protein termed leptin binding domain, capable of forming high affinity 1:1 complex with human or other mammalian leptins (21). This protein, which is also termed CHR2, was used in this study as a hook to fish out the high affinity leptin mutants expressed on the surface of yeast cells.…”

Section: Discussionmentioning

confidence: 99%

“…Materials-Recombinant soluble human leptin binding domain (hLBD) (21), human leptin triple antagonist, and mouse leptin were prepared in our laboratory as described previously (15,16). Synthetic mouse leptin WT cDNA optimized for expression in Escherichia coli was synthesized by Entelechon Co. (Rensberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Development and Characterization of High Affinity Leptins and Leptin Antagonists

Shpilman

Niv-Spector

Katz

et al. 2011

Journal of Biological Chemistry

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Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/ D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/ F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.Leptin, a 16-kDa protein, is a central regulator of body weight (1), as well as a pleiotropic hormone whose involvement in many physiological processes has been well established (2). The three-dimensional structure of leptin was elucidated shortly after its discovery (3), but so far no structural information on its complex with leptin receptor has been published. Although several theoretical models of such a complex have been proposed (4 -6), lack of a valid crystallographic structure hampers reliable structural interpretation of any new leptin mutations. In the last decade, leptin has also been documented as a major regulator of the innate and adaptive immune response and a modulator of the onset and progression of autoimmunity in several animal models of disease (7), including rheumatoid arthritis, experimental autoimmune encephalomyelitis (4), and immune-mediated colitis (8). Work published from our and others' laboratories has also shown that leptin enhances thioacetamide-induced liver fibrosis (9) and liver inflammation in several mouse models (10). In addition, leptin acts as a mitogenic agent in many tissues and is suggested to promote cancer cell growth. In fact...

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“…Glycine at this base position is highly conserved across genera. Structural analysis of the human LEPR extracellular domain suggests that this amino acid change likely could affect the nearby ligand binding domain (44). RT-PCR showed that this mutation did not prevent transcription of a full-length (LEPR-Rb) transcript in the hypothalamus.…”

Section: Db-5jmentioning

confidence: 99%

Mouse Models and the Genetics of Diabetes

Leiter

Lee

2005

Diabetes

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In humans, both type 1 and type 2 diabetes exemplify genetically heterogeneous complex diseases in which epigenetic factors contribute to underlying genetic susceptibility. Extended human pedigrees often show inheritance of both diabetes types. A common pathophysiological denominator in both disease forms is pancreatic ␤-cell exposure to proinflammatory cytokines. Hence, it is intuitive that systemically expressed genes regulating ␤-cell ability to withstand chronic diabetogenic stress may represent a component of shared susceptibility to both major disease forms. In this review, the authors assemble evidence from genetic experiments using animal models developing clearly distinct diabetes syndromes to inquire whether some degree of overlap in genes contributing susceptibility can be demonstrated. The conclusion is that although overlap exists in the pathophysiological insults leading to ␤-cell destruction in the currently studied rodent models, the genetic bases seem quite distinct. Diabetes 54 (Suppl. 2):S151-S158, 2005 T he complex of genes in the HLA locus (IDDM1) contributing the major component of human susceptibility to autoimmune type 1 diabetes clearly provides a starting point for comparison for overlap with major susceptibility contributors to classic type 2 diabetes. Indications of humoral immunity against pancreatic ␤-cell autoantigens in 10 -30% of patients deemed clinically to have type 2 diabetes led to the concept that these patients exhibit latent autoimmune diabetes in adults (LADA), or "type 1.5" diabetes (1). Many LADA patients exhibit decreased frequency of the highest risk HLA class I and class II alleles and increased frequency of HLA alleles conferring strong protection against juvenile-onset type 1 diabetes (1). Although most type 2 diabetes cases clearly are not autoimmune in causation, and thus, specific HLA alleles are not identified as major type 2 diabetes susceptibility contributors, the LADA cases raise the possibility that a subset of non-HLA susceptibility may be shared. Under such circumstances, the absence of the high-risk HLA alleles results in a more slowly progressive disease that only presents in adulthood when pathophysiological stresses on ␤-cells, including obesity and insulin resistance shared in common with patients with classic type 2 diabetes, become prevalent.Polymorphisms in the upstream regulatory region of the insulin gene currently represent the strongest non-HLA locus linked to human type 1 diabetes susceptibility (IDDM2) (2). The pathogenic mechanism has not been associated with insulin processing, secretion, or action, but rather with the ability to express intrathymically and elicit T-cell tolerance to this major type 1 diabetes autoantigen (3,4). Alleles associated with resistance to type 1 diabetes, on the other hand, have been associated with polycystic ovarian syndrome (5), an insulin resistance syndrome that often leads to type 2 diabetes. Because type 2 diabetes often culminates in insulin deficiency attributed to ␤-cell failure associated with chron...

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Subcloning, Expression, Purification, and Characterization of Recombinant Human Leptin-binding Domain

Cited by 59 publications

References 60 publications

Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains

Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains

Development and Characterization of High Affinity Leptins and Leptin Antagonists

Mouse Models and the Genetics of Diabetes

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