Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

Abstract

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.

Development and Characterization of High Affinity Leptins and Leptin Antagonists

“…5) are structurally similar and face the same orientation, and they are most likely involved in interacting with CHR2. Mutations of those residues, such as D9S, T12Q, K15S, T16N, R20N, Q75S, N82S, D85S and L86A, L86S, L86N, and L86Q, significantly lowered the affinity of leptin for CHR2 and affected both binding and leptin receptor signaling (4,5). To date, no report regarding the putative role of Asp-23 has been published; nevertheless, our data (see Table 3) strongly indicate that replacement of Asp-23 by any non-negatively charged amino acid is sufficient to increase affinity for hLBD (CHR2) and subsequent biological activity.…”

“…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).…”

“…The following kits were purchased: GeneMorph kit, QuikChange mutagenesis kit and XL- 4 and supplemented with 5% (w/v) bovine serum albumin (BSA) and 0.05% (w/v) azide, lysis buffer for luciferase activity (25 mM Tris phosphate, 2 mM DTT, 2 mM CDTA, 10% (w/v) glycerol, 1% w/v Triton X-100, pH 7.8), TN buffer for gel filtration experiments (25 mM Tris-HCl, pH 8 or 9, containing 300 mM NaCl).…”

“…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). Pegylation of the antagonist resulted in further improvement of the in vivo antagonistic activity via inhibition of the transport of endogenous leptins through the brain-blood barrier (17).…”

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

“…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.…”