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.

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.

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.

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.

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.

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.

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.

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.

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).