Development and characterization of high affinity leptins and leptin antagonists.

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.

Large-scale preparation and characterization of non-pegylated and pegylated superactive ovine leptin antagonist.

Superactive ovine leptin antagonist (SOLA) was prepared by rational mutagenesis of the ovine leptin antagonist L39A/D40A/F41A mutant prepared previously in our lab by mutating wild type leptin to D23L/L39A/D40A/F41A. SOLA was expressed in Escherichia coli as insoluble inclusion bodies, refolded and purified to homogeneity (as evidenced by SDS-PAGE and analytical gel filtration) by ion-exchange chromatography. The purified protein was mono-pegylated at its N terminus by 20-kDa linear pegylation reagent. The D23L mutation resulted in ca. 5- to 6-fold increased affinity toward soluble human leptin binding domain and 6- to 8-fold increased inhibitory activity in two different in vitro bioassays. This increase was similar, though not identical, to our previous results with superactive mouse and human leptin antagonists. Pegylation decreased overall activity by 5- to 8-fold, but as shown previously for superactive mouse leptin antagonist, the prolonged half life in the circulation will likely result in higher activity in vivo. As amino acids 6-31 (VQDDTKTLIKTIVTRINDISHTQSVS), making up a main part of the first α-helix, are identical in human, mouse, rat, ovine, bovine and pig leptins, we anticipate that D23L mutations of the respective leptins will result in similar increases in affinity and consequent activity of other leptin antagonists.

Leptin signaling and apoptotic effects in human prostate cancer cell lines.

BACKGROUND: Prostate cancer (PCa) progression is often associated with transactivation of the androgen receptor (AR) by endogenous hormones/growth factors. One such factor affecting growth, proliferation, and apoptostis (pro-/anti-) in various cancers is the adipokine leptin. This research studied leptin-induced signaling and apoptosis in androgen sensitive (LNCaP, PC3/AR) and insensitive (PC3, DU145) PCa cell lines. METHODS: Signaling was studied by immunoblotting in cells overexpressing leptin receptors (LRb), Janus kinase 2 (JAK2), and kinase negative-HER2-YFP cDNAs. Apoptosis was measured by immunoblotting of apoptotic proteins and by Hoechst staining of condensed DNA. RESULTS: Leptin rapidly induced activation of JAK2, STAT3, and MAPK (ERK1/2) signaling cascades; it may also induce HER2 transactivation via leptin-induced phospho-JAK2. Leptin was then shown to exert clear pro-apoptotic effects, increasing levels of caspase 3, cleavage of its substrate, poly (ADP-ribose) polymerase (PARP) to cleaved PARP(89) , levels of CK 18, a cytoskeletal protein formed during apoptosis, and DNA condensation. Kinase inhibitors indicated that leptin-induced apoptosis is probably mediated by balanced activation of JAK2/STAT3, p38 MAPK, and PKC pathways in PCa cells. A human leptin mutein LRb antagonist, L39A/D40A/F41A, fully inhibited leptin-induced phosphorylation of JAK2, ERK1/2, and Akt/PKB, and partially abrogated effects on apoptotic proteins. In LNCaP and PC3/AR cells, leptin increased AR protein levels in correlation with raised apoptotic markers. Thus, AR may mediate, at least partly, the leptin-induced apoptotic response. CONCLUSIONS: Leptin can clearly induce apoptosis in human PCa cell lines. These findings could lead to development of new leptin agonists with enhanced pro-apoptotic effects and targeted for use in human PCa.

The obese phenotype-inducing N82K mutation in human leptin disrupts receptor-binding and biological activity.

A novel homozygous mutation of the leptin gene was recently reported in an Egyptian child and his sister with severe early onset obesity. This mutation results from the substitution of asparagine (AAC) by lysine (AAA) at codon 103 of a non-mature (signal peptide-containing) leptin and corresponds to the N82K mutation in the mature protein. The patient had very low serum leptin levels, raising the question of whether the obese phenotype resulted from low leptin levels or from its lower intrinsic activity. To answer this question, we characterized the functional consequences of the N82K mutation. Wild-type (WT) human leptin was mutated accordingly, expressed in Escherichia coli at high yield, purified to homogeneity as a monomer and compared to WT human leptin prepared by the same methodology. Circular dichroism analysis of the mutated leptin indicated proper refolding and a secondary structure identical to that of the WT human leptin. In contrast to WT human leptin, the N82K mutant did not form a detectable complex with human leptin-binding domain (hLBD) and its binding capacity to hLBD assessed in a nonradioactive receptor-binding assay was at least 500-fold lower than that of WT human leptin. The biological activity of the N82K mutant, tested in two cell bioassays, was reduced by more than three orders of magnitude relative to WT human leptin. Therefore, though the present report does not explain the reason for the low circulating leptin levels it definitely documents that the reported obese phenotype originates not only from low serum leptin levels but also from the N82K mutant's almost total lack of intrinsic leptin activity.

Preparation of leptin antagonists by site-directed mutagenesis of human, ovine, rat, and mouse leptin's site III: implications on blocking undesired leptin action in vivo.

Six muteins of human, ovine, rat, and mouse leptins mutated to Ala in amino acids 39-41 or 39-42 were prepared by site-directed mutagenesis of the putative site III, which does not affect binding but is necessary for receptor activation, then expressed, solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose or combination of anion-exchange chromatography followed by gel filtration. The overall yields were 400-800 mg from 5 L of fermentation. All proteins were >98% pure as evidenced by SDS-PAGE and contained at least 95% monomers as documented by gel-filtration chromatography under nondenaturing conditions. Circular dichroism analysis revealed that all six muteins have identical secondary structure characteristic of nonmutated leptins, namely 52-63% of alpha helix content. All muteins formed a 1:1 complex with chicken leptin binding domain, (chLBD) and bound chLBD or membrane-embedded leptin receptor with affinity identical to WT leptins. Muteins were devoid of any biological activity in several bioassays but were potent competitive antagonists. Some muteins were pegylated using 40 kDa PEG. Although pegylation decreased the in vitro activity, increasing circulation half-life can recompensate this deficit, so pegylated antagonists are expected to be more potent in vivo.

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.

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.

Preparation and characterization of mouse IL-22 and its four single-amino-acid muteins that act as IL-22 receptor-1 antagonists.

Recombinant mouse interleukin 22 (mIL-22) and its variants encoding four muteins (Y51A, N54A, R55A and E117A) were expressed in Escherichia coli, refolded and purified to homogeneity as monomeric proteins by one-step ion-exchange chromatography. The binding of IL-22 and its four muteins to immobilized mIL-22 receptor α1 extracellular domain (mIL-22 Rα1-ECD) exhibited similar affinity, indicating that the single-amino-acid mutations do not affect its binding properties. Similarly, no differences were found in binding to IL-22 binding protein expressed on the surface of yeast cells, although the affinity of all five proteins to the binding protein was higher than that to IL-22 Rα1-ECD. In an in vitro bioassay, recombinant mIL-22 stimulated signal transducer and activator of transcription-3 phosphorylation in HepG2 cells, whereas the four muteins were completely (Y51A) or almost completely (N54A, R55A and E117A) devoid of this agonistic activity. Furthermore, the agonistic activity of mIL-22 could be inhibited in a dose-dependent manner by the four muteins with almost identical efficiency. mIL-22 and its Y51A mutein were pegylated by methoxy polyethylene glycol-propionylaldehyde-20 kDa, yielding a mixture of mono (75-80%) and double (20-25%) pegylated proteins. The pegylated proteins showed lower affinity (50 and 25%) toward immobilized mIL-22 Rα1-ECD than their non-pegylated analogs. Wild-type pegylated IL-22 exhibited 5- to 10-fold lower activity in the HepG2 bioassay than its non-pegylated counterpart. Preparation of recombinant mIL-22 antagonists provides new tools for the study of IL-22 activity and of eventual therapeutic means for attenuating its negative effects.

Pegylated leptin antagonist is a potent orexigenic agent: preparation and mechanism of activity.

Leptin, a pleiotropic adipokine, is a central regulator of appetite and weight and a key immunomodulatory protein. Although inborn leptin deficiency causes weight gain, it is unclear whether induced leptin deficiency in adult wild-type animals would be orexigenic. Previous work with a potent competitive leptin antagonist did not induce a true metabolic state of leptin deficiency in mice because of a short circulating half-life. In this study, we increased the half-life of the leptin antagonist by pegylation, which resulted in significantly increased bioavailability and retaining of antagonistic activity. Mice administered the pegylated antagonist showed a rapid and dramatic increase in food intake with weight gain. Resulting fat was confined to the mesenteric region with no accumulation in the liver. Serum cholesterol, triglyceride, and hepatic aminotransferases remained unaffected. Weight changes were reversible on cessation of leptin antagonist treatment. The mechanism of severe central leptin deficiency was found to be primarily caused by blockade of transport of circulating leptin across the blood-brain barrier with antagonisms at the arcuate nucleus playing a more minor role. Altogether we introduce a novel compound that induces central and peripheral leptin deficiency. This compound should be useful in exploring the involvement of leptin in metabolic and immune processes and could serve as a therapeutic for the treatment of cachexia.

Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists.

Expression plasmids encoding mouse and rat leptins and their L39A/D40A/F41A muteins were prepared. The proteins were expressed in Escherichia coli, refolded and purified to homogeneity, yielding electrophoretically pure, over 98% monomeric protein. Circular dichroism (CD) analysis revealed that the mutations hardly affect the leptins' secondary structure, and they were similar to previously reported CD spectra for human leptin. Both mouse and rat leptins were biologically active in promoting proliferation in BAF/3 cells stably transfected with the long form of human leptin receptor. The mutations did not change the binding properties to BAF/3 cells as compared, respectively, to non-mutated mouse, rat or human leptins, or their ability to form 1:1 complexes with the leptin-binding domain of chicken leptin receptor. In contrast, their biological activity, tested in a BAF/3 proliferation assay, was abolished and both became potent antagonists. As the LDF (amino acids 39-41) sequence is preserved in all known leptins, the present results substantiate the hypothesis that this sequence plays a pivotal role in leptins' site III and that interaction of leptin with its receptors resembles the corresponding interactions of interleukin-6 and granulocyte colony-stimulating factor their receptors.