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.     

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.     

Avian IgY binds to a monocyte receptor with IgG-like kinetics despite an IgE-like structure

An ancestor of avian IgY was the evolutionary precursor of mammalian IgG and IgE, and present day chicken IgY performs the function of human IgG despite having the domain structure of human IgE. The kinetics of IgY binding to its receptor on a chicken monocyte cell line, MQ-NCSU, were measured, the first time that the binding of a non-mammalian antibody to a non-mammalian cell has been investigated (k(+1) = 1.14 +/- 0.46 x 10(5) mol(-1)sec(-1), k(-1) = 2.30 +/- 0.14 x 10(-3) s(-1), and K(a) = 4.95 x 10(7) m(-1)). This is a lower affinity than that recorded for mammalian IgE-high affinity receptor interactions (Ka approximately 10(10) m(-1)) but is within the range of mammalian IgG-high affinity receptor interactions (human: Ka approximately 10(8)-10(9) m(-1) mouse: Ka approximately 10(7)-10(8) m(-1). IgE has an extra pair of immunoglobulin domains when compared with IgG. Their presence reduces the dissociation rate of IgE from its receptor 20-fold, thus contributing to the high affinity of IgE. To assess the effect of the equivalent domains on the kinetics of IgY binding, IgY-Fc fragments with and without this domain were cloned and expressed in mammalian cells. In contrast to IgE, their presence in IgY has little effect on the association rate and no effect on dissociation. Whatever the function of this extra domain pair in avian IgY, it has persisted for at least 310 million years and has been co-opted in mammalian IgE to generate a uniquely slow dissociation rate and high affinity.     

Structure of the human obesity receptor leptin-binding domain reveals the mechanism of leptin antagonism by a monoclonal antibody

Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of?a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling.     

Preparation of recombinant bovine, porcine, and porcine W4R/R5K leptins and comparison of their activity and immunoreactivity with ovine, chicken, and human leptins

Recombinant ovine Ala-leptin (GenBank Accession No. U84247, of ovine leptin), previously prepared in our laboratory in prokaryotic expression plasmid pMON3401, was mutated using a mutagenesis kit to prepare plasmids encoding for bovine (GenBank Accession No. U50365) and porcine (GenBank Accession No. U59894) leptins and for porcine leptin analogue W4R/R5K. Escherichia coli cells transformed with these plasmids overexpressed large amounts of these proteins upon induction with nalidixic acid. The expressed proteins, found in inclusion bodies, were refolded and purified to homogeneity using subsequently anion- and cation-exchange chromatography. All three purified proteins showed a single band of the expected molecular mass of 16 kDa in SDS-PAGE in the presence of reducing agent and were composed of 90-100% monomers. Proper refolding was evidenced by comparing their CD spectra to those of previously prepared chicken and ovine leptins and to commercially available human leptin. The amino acid content of the purified proteins closely resembled the predicted composition. The biological activity of bovine leptin, porcine leptin, and porcine leptin analogue W4R/R5K was evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor. All three proteins, as well as ovine and chicken leptins, but not human leptin, exhibited a very high degree of cross-immunoreactivity against antiserum raised against ovine leptin in rabbits. In contrast, none or very low cross-immunoreactivity was observed against antiserum raised against ovine leptin in goats.     

Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins

The chicken leptin sequence, in contrast to mammalian leptins, contains an unpaired Cys at position 3 of the original cDNA (AF012727). The presence of an extra Cys may confer a different structure and affect the leptin's biological activity. To address this, we studied the effects of wild-type and mutated (C4S) chicken leptins in vitro and in vivo and compared them with mammalian leptin prepared from ovine leptin cDNA. The prokaryotic expression vector pMON, encoding full-size A(-1) chicken leptin (AF012727), was mutated using a mutagenesis kit, yielding the C4S analog. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin C4S upon induction with nalidixic acid. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding three electrophoretically pure fractions, eluted from the column by 100, 125, and 150 mM NaCl, respectively. All three fractions showed a single band of the expected molecular mass (16 kDa) and were composed of >95% monomeric protein. Proper refolding was evidenced by comparing the circular dichroism spectrum of the analog with spectra of nonmutated chicken and ovine leptins. The biological activity of the C4S analog was evidenced by its ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct similar to its nonmutated counterpart, indicating that Cys4 plays no role in leptin activity. The in vitro activity of both wild-type and mutated chicken leptins was approximately 10-fold lower than that of ovine leptin. After intravenous or intraperitoneal injections, C4S analog and the nonmutated chicken and ovine leptins all lowered the food intake of starved 9-day-old broiler or 5-wk-old layer male chickens by 11-34%. Monitoring food behavior revealed that the attenuated food intake resulted not from a decreased number of approaches to the feeders but from a decrease in the average time spent eating during each approach.     

BiaCore analysis of leptin-leptin receptor interaction: evidence for 1:1 stoichiometry

Leptin is a hormonal protein involved in energy homeostatis that acts to inhibit food intake, to stimulate energy expenditure, and to influence insulin secretion, lipolysis, and sugar transport. Its action is mediated by a specific receptor whose activation is highly controversial. As a member of the cytokine receptor superfamily, it has been predicted to be activated by ligand-induced dimerization. However, recent evidence has indicated that this receptor exists as a dimer in both ligand-free and ligand-bound states. Here, the BiaCore has been used to measure the kinetics and stoichiometry of the interaction between the leptin and its receptor. Human or mouse receptor chimeras comprising two receptor extracellular domains fused to the Fc region of IgG(1) were captured on to the sensor via protein G. Kinetic data fitted to the simplest 1/1 model. The observed stoichiometry at ligand saturation was 1:1. Analyzing the binding mode and the reaction stoichiometry allowed us to conclude that the leptin receptor dimerization is not induced by ligand binding. This contradicts the common paradigm of cytokine receptor activation. Furthermore, data demonstrated a high-affinity interaction. The KD was 0.23+/-0.08 nM, with ka = (1.9 +/- 0.4) x 10(6) M(-1)s(-1) and kd = (4.4 +/- 0.6) x 10(-4) s(-1) for human leptin with its cognate receptor. Similar results were observed for the affinity of different species of leptin binding to mouse leptin receptor.     

The kinetics of mebendazole binding to Haemonchus contortus tubulin.

The kinetics of the binding of mebendazole (MBZ) to tubulin from the third-stage (L3) larvae of the parasitic nematode, Haemonchus contortus, have been characterized. In partially purified preparations, the association of [3H]MBZ to nematode tubulin was rapid, k1 = (2.6 +/- 0.3) x 10(5) M-1 min-1, but dissociation was slow, k-1 = (1.58 +/- 0.02) x 10(-3) min-1. The affinity constant (K(a)) for the interaction, determined by the ratio k1/k-1, was (1.6 +/- 0.2) x 10(8) M-1. Similar results were obtained with crude cytosolic fractions. In equilibrium studies, performed with partially purified nematode tubulin under similar conditions, a K(a) of (5.3 +/- 1.6) x 10(6) M-1 was obtained. The best estimate for the K(a) of the MBZ-nematode tubulin interaction is considered to be the 'kinetic' value determined from the ratio of rate constants. The slow dissociation of MBZ from nematode tubulin, which contrasts with the rapid dissociation of MBZ from mammalian tubulin, supports the hypothesis that the selective toxicity of the benzimidazole anthelmintics results from a difference between the affinities of mammalian and nematode tubulins for these drugs.     

Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence

A putative growth hormone receptor from detergent-solubilized rabbit liver membranes and the growth hormone binding protein from rabbit serum have been purified 59,000- and 400,000-fold, respectively, primarily by affinity chromatography. Both purified proteins exhibit high affinity binding for human growth hormone; K alpha = 9-30 x 10(9) M-1 for the liver receptor and K alpha = 6 x 10(9) M-1 for the binding protein. The apparent molecular weight of the liver receptor is 130,000 by reduced sodium dodecyl sulfate gel electrophoresis, while that of the binding protein is 51,000. Both contain N-linked carbohydrate. The amino-terminal sequences of the liver growth hormone receptor and the serum binding protein were found to be the same, indicating that the binding protein corresponds to the extracellular domain of the liver receptor. Ubiquitin was found covalently linked to the liver receptor but not to the serum binding protein. The amino acid sequences of several peptides from the liver receptor were also determined after tryptic and V8 protease digestion.     

Antiferritin VL homodimer binds human spleen ferritin with high specificity

The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody.     

Mechanism of formation of the complex between transferrin and bismuth, and interaction with transferrin receptor 1

The kinetics and thermodynamics of Bi(III) exchange between bismuth mononitrilotriacetate (BiL) and human serum transferrin as well as those of the interaction between bismuth-loaded transferrin and transferrin receptor 1 (TFR) were investigated at pH 7.4-8.9. Bismuth is rapidly exchanged between BiL and the C-site of human serum apotransferrin in interaction with bicarbonate to yield an intermediate complex with an effective equilibrium constant K(1) of 6 +/- 4, a direct second-order rate constant k(1) of (2.45 +/- 0.20) x 10(5) M(-1) s(-1), and a reverse second-order rate constant k(-1) of (1.5 +/- 0.5) x 10(6) M(-1) s(-1). The intermediate complex loses a single proton with a proton dissociation constant K(1a) of 2.4 +/- 1 nM to yield a first kinetic product. This product then undergoes a modification in its conformation followed by two proton losses with a first-order rate constant k(2) = 25 +/- 1.5 s(-1) to produce a second kinetic intermediate, which in turn undergoes a last modification in the conformation to yield the bismuth-saturated transferrin in its final state. This last process rate-controls Bi(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau(3)(-1) of (3 +/- 1) x 10(-2) s(-1). The mechanism of bismuth uptake differs from that of iron and probably does not involve the same transition in conformation from open to closed upon iron uptake. The interaction of bismuth-loaded transferrin with TFR occurs in a single very fast kinetic step with a dissociation constant K(d) of 4 +/- 0.4 microM, a second-order rate constant k(d) of (2.2 +/- 1.5) x 10(8) M(-1) s(-1), and a first-order rate constant k(-d) of 900 +/- 400 s(-1). This mechanism is different from that observed with the ferric holotransferrin and implies that the interaction between TFR and bismuth-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of bismuth incorporation by the transferrin receptor-mediated iron acquisition pathway is discussed.     

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.     

Reversible DIDS binding to band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.     

Opioid binding properties of the purified kappa receptor from human placenta

A glycoprotein with a molecular weight of 63,000 has been purified, in an active form, from human placental villus tissue membranes. The binding properties of this glycoprotein to opioid alkaloids and peptides indicates that it is the kappa opiate receptor of human placenta. The receptor binds the tritiated ligands etorphine, bremazocine, ethylketocyclazocine and naloxone specifically and reversibly with Kd values of 3.3, 4.4, 5.1 and 7.0nM, respectively. The binding of 3H-Bremazocine to the purified receptor is inhibited by the following compounds with the corresponding Ki values EKC, 1.3 x 10(-8)M; Dynorphin 1-8, 3.03 x 10(-7); U50,488H, 4.48 x 10(-9); U69-593,2.28 x 10(-8), morphine, 4.05 x 10(-6) DADLE, 6.47 x 10(-6) and naloxone, 2.64 x 10(-8). The purified receptor binds 8 nmole of 3H-Etorphine and 1.7 nmole 3H-BZC per mg protein. The theoretical binding capacity of a protein of this molecular weight is 15.8. Although the iodinated purified receptor appears by autoradiography as one band on SDS-PAGE, yet homogeneity of the preparation is not claimed.     

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.     

Filter binding assay for the geldanamycin-heat shock protein 90 interaction

A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.