Identification of the hydrophobic strand in the A–B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists

Abstract

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I-III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A-B loop (amino acids 39-42) and in the N-terminal end of LEPR's IGD (amino acids 325-328) that are predicted to participate in site III and to interact with each other in a beta-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39-42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325-328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III.

Development and Characterization of High Affinity Leptins and Leptin Antagonists

“…We previously reported the development of leptin antagonists in four animal species (15,16) by alanine mutagenesis of residues LDF (amino acids 39 -41) or LDFI (amino acids 39 -42). 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).…”

Insensitivity of well-conditioned mature sheep to central administration of a leptin receptor antagonist

“…Treatments were chronic ICV infusion of artificial cerebrospinal fluid (aCSF; 150 mM NaCl, 1.2 mM CaCl 2 , 1 mM MgCl 2 , 2.8 mM KCl), ovine leptin (4 µg/h; treatment designated L) or the combination of ovine leptin (4 µg/h) and its mutant version OLA (40 µg/h; treatment designated L + OLA). OLA and leptin have a similar affinity for the leptin receptor (Niv-Spector et al, 2005). Accordingly, in the case of the L + OLA treatment, the OLA infusion rate was 10 times higher than that of leptin to ensure OLA predominance at the receptor.…”

“…The OLA mutant has the same exact sequence as ovine leptin except for alanine substitution mutations of amino acids 39 to 41. Ovine leptin and OLA were produced in a bacterial expression system, refolded and purified to homogeneity as previously described (Niv-Spector et al, 2005). All proteins were dissolved in aCSF, with each ICV treatment delivered as a 100 µL/h continuous infusion using a syringe pump (model SE 400; Vial Medical, Grenoble, France).…”

“…As the physiological signal read by the CNS in many contexts is falling rather than increasing plasma leptin (Leibel, 2008), we treated well-fed adult sheep with an ovine leptin antagonist mutant (OLA) harboring alanine substitution of amino acid residues 39 to 41 (Niv-Spector et al, 2005). This mutant binds the leptin receptor with an affinity equal to wild type leptin but is totally devoid of agonistic activity.…”

“…OLA binds the leptin receptor with normal affinity but lacks agonistic activity. Accordingly, OLA failed to induce leptin-dependent responses in cell culture when used alone, and more importantly produces dosedependent inhibition of these responses when added simultaneously with leptin (Niv-Spector et al, 2005). However, evidence that OLA can antagonize leptin action in the sheep was lacking, prompting us to first ask whether it could ablate the anorexic effects of ICV leptin.…”