Abstract
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.
Highlights
Using mouse leptin antagonist (MLA),4 we demonstrated that inhibition of leptin signaling is beneficial in several mouse models of fibrosis and inflammation (9, 10)
In-frame surface expression of the full-length Aga2p ϩ leptin fusion protein was established by flow cytometric c-Myc labeling, whereas affinity to leptin receptor was confirmed by binding to a fluorescently labeled human leptin binding domain (hLBD) (Fig. 1B)
We report the discovery of the Asp-23 residue as a site that modulates leptin binding to its receptor, and we show that mutagenesis of this residue is associated with a dramatic increase in leptin binding affinity
Summary
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). Yeast cells transformed with pCT302/mouse leptin WT were grown overnight at 30 °C with shaking in 3 ml of selective glucose medium SD-CAA. Einat (ARO, Israel), consists of HEK-293 cells stably transfected with three constructs as follows: phOB-Rb (long form of human leptin receptor), pAH32 (luciferase reporter construct), and pgkPuro (expression vector containing the puromycin resistance gene) at a ratio of 4:4:1 as described previously (33). The cells were incubated in serum-free medium in the presence or absence of various concentrations (0.2–12.8 g/ml) of MLA or SMLA and one concentration of WT mouse leptin (0.1 g/ml) for 20 min in 0.5 ml in 24-well plates Following these treatments, cells were harvested in 75 l of ice-cold lysis buffer. Animals were maintained under 12-h light/dark cycles, in accordance with regulations of the institutional animal and care authority of the Tel Aviv Sourasky Medical Center
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
