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BiaCore analysis of leptin–leptin receptor interaction: evidence for 1:1 stoichiometry
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 327 (2004) 271–277 www.elsevier.com/locate/yabio
BiaCore analysis of leptin–leptin receptor interaction: evidence for 1:1 stoichiometry P. Mistrík,a,1 F. Moreau,b and J.M. Allena,b,¤,2 a
Division of Biochemistry and Molecular Biology, IBLS, Wolfson Building, Level 2, University of Glasgow, Glasgow G12 8QQ, UK b PWzer Global Research and Development, 3–9 Rue de la Loge, 94265 Fresnes Cedex, France Received 21 November 2003
Abstract Leptin is a hormonal protein involved in energy homeostatis that acts to inhibit food intake, to stimulate energy expenditure, and to inXuence insulin secretion, lipolysis, and sugar transport. Its action is mediated by a speciWc 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 IgG1 were captured on to the sensor via protein G. Kinetic data Wtted 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 highaYnity interaction. The KD was 0.23 § 0.08 nM, with ka D (1.9 § 0.4) £ 106 M¡1 s¡1 and kd D (4.4 § 0.6)£10¡4 s¡1 for human leptin with its cognate receptor. Similar results were observed for the aYnity of diVerent species of leptin binding to mouse leptin receptor. 2004 Elsevier Inc. All rights reserved. Keywords: Surface plasmon resonance; Dimerization; Cytokine; Receptor activation
Leptin (Ob),3 a 16-kDa protein isolated in 1994, seemed to represent an eagerly expected molecular component which could be exploited in the development of novel therapeutics for weight control [1]. Leptin was originally thought to be expressed exclusively in the adipose tissue and in accord with the lipostatic theory [2] to form a feedback loop with the hypothalamus in the control of the appetite. It is now recognized that leptin is a ¤
Corresponding author. Fax: +44-207-074-4746. E-mail address: [email protected] (J. Allen). 1 Present address: Department of Pharmacy, Center for Drug Research, University of Munich, 81377 Munich, Germany. 2 Present address: Inpharmatica, 60 Charlotte Street, London W1T 2NU, UK. 3 Abbreviations used: Ob, leptin; ObR, leptin receptor; ObR-Fc, leptin receptor chimera; RU, resonance unit; Rmax, analyte binding capacity; R1, ligand response; Epo, erythropoietin; EpoR, erythropoietin receptor; CHR, cytokine-binding homology regions; NHS, N-hydroxysuccinimide; EDC, N-ethyl-N0-(3-diethylaminopropyl)carbodiimide. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.01.022
component of a highly complex molecular network whose physiological manifestations extend beyond the initial lipostatic concept. Leptin is produced in several organs and participates in a broad range of metabolic processes [3–7]. Interestingly, the crystal structure of a mutant human leptin (leptin-E100) [8] revealed that leptin bears striking structural similarity to some cytokine molecules, such as growth hormone, leukemia inhibitory factor, and granulocyte-stimulating hormone. This suggests that leptin may also possess immunomodulatory and antiinXammatory eVects [9]. The high complexity of leptin action is mirrored by the complex pattern of expression of the leptin receptor (ObR) [10]. High levels were detected in lung and kidney, in addition to the hypothalamus and choroid plexus. Lower levels were found in heart, spleen, liver, skeletal muscle, and testes [10–13]. Therefore, leptin represents an interesting target for studying the molecular interactions between immune, neuronal, digestive, and reproductive systems.
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We have used the BiaCore to evaluate the interaction between leptin and receptor. The nature of this ligand– receptor interaction is controversial. ObR as a member of the cytokine receptor superfamily should be activated by ligand-induced dimerization [14,15]. However, recent studies [16–18] have suggested that ObR does not change its oligomerization state during its interaction with a ligand; rather, it exists as a dimer in both ligandfree and ligand-bound states. We used human or mouse chimeric receptors and measured the interactions with human, mouse, and rat leptin. We have evaluated the kinetics and stoichiometry of the interaction in all possible combinations. Kinetic data Wtted 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.
Materials and methods Surface plasmon resonance BiaCore biosensor BIAcore 2000, CM5 researchgrade biosensor chips, N-hydroxysuccinimide (NHS), N-ethyl-N0-(3-diethylaminopropyl)carbodiimide (EDC), ethanolamine-HCl, and HBS-EP buVer were obtained from BiaCore AB (Uppsala, Sweden). Protein G was purchased from Sigma (Poole, UK). Recombinant human and mouse leptin receptor-Fc chimeras and recombinant human, mouse, and rat leptin were obtained from R&D Systems Europe, Ltd. (Abingdon, UK). The leptin receptor-Fc chimera exists as a disulWde-linked homodimeric protein with the monomeric molecular weights of 190–210 kDa for the human receptor chimera and 150–170 kDa for the mouse receptor chimera as determined by SDS–PAGE. The molecular weight of leptin molecules is 16 kDa. To prepare the biosensor assay, protein G was immobilized on to the four channels of a research-grade carboxymethyl dextran chip (CM5) using an amine coupling procedure described previously [19]. The surface was activated with NHS/EDC for 5 min. Protein G was injected at a concentration of 200 g/ml in sodium acetate buVer (10 mM, pH 4.0) until 2000 resonance units (RU) of protein were coupled. Remaining activated groups were then blocked with a 5-min injection of 1 M ethanolamine (pH 8.5). For kinetic experiments the recombinant human or mouse leptin receptor-Fc chimera was captured on to the protein G surface. Typically, 15 l of a 0.5-g/ml solution was injected to immobilize 100–300 RU of the receptor chimera. After an 1800-s equilibration time, a
100-l injection of leptin or HBS-EP buVer was performed. A range of leptin concentrations (0.1–30 nM) was used. The dissociation phase was then monitored for 900 s, followed by two 5-l injections of 10 mM glycine, pH 2.0, to regenerate a fully active protein G surface. All kinetic experiments were performed at 25 °C in a buVer containing 10 mM Hepes, pH 7.4, 150 mM sodium chloride, and 0.005% Tween 20, at a Xow rate of 20 l/min. In control experiments, protein G was used instead of the leptin receptor-Fc chimera to evaluate leptin nonspeciWc binding. Generation of good-quality data for analysis was achieved classically as follows. Leptin, in concentration range 0.1–30 nM, or buVer alone was injected on to the active (ObR-Fc) or control (protein G) surfaces. To remove contributions of nonspeciWc binding and refractive index, the control responses were subtracted from those obtained from the active surface. A minor baseline draft resulting from a slow dissociation of the complex between ObR-Fc and protein G was eliminated by subtracting data of a bulk injection (0 nM Ob) from data corrected for nonspeciWc binding. The kinetics parameters were derived from obtained data using BIA evaluation software 3.1 (Biacore AB). Association and dissociation binding data were Wtted simultaneously to a one-to-one, single-site binding model using nonlinear least squares analysis model [20]. Stoichiometry The BiaCore instrument measures the mass of molecules bound to the sensor surface. Thus the stoichiometry of the surface molecular complex can be determined using the equation:
where Rmax, analyte binding capacity, can be extrapolated from experimental data and ligand response (Rl) is given directly from a sensorgram recorded during ligand immobilization.
Results To study the interaction between the leptin and its speciWc receptor, we used human and mouse receptor chimeras (ObR-Fc), which consist of two receptor extracellular domains fused together via the Fc region of IgG1. The following assay was used to evaluate their aYnity for human, mouse, and rat leptin (Fig. 1). The ObR-Fc was captured on to the chip surface via the protein G, a high-aYnity receptor for the Fc portion of IgG, which was covalently bound to the dextran matrix of the chip. In this conWguration, a homogenous receptor
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273
Fig. 1. Schematic of the biosensor assay. The ObR-Fc molecule was captured on to the chip surface via protein G, which was covalently bound to the dextran matrix of the chip. In this conWguration, a homogenous receptor surface is formed and its interaction with Ob can be monitored.
surface is formed and its interaction with Ob can be monitored. Typical sensorgrams, representing diVerent leptin molecules (human, mouse, and rat) binding to the receptor chimera (human) are shown in Fig. 2. The association phase of the ligand–receptor interaction is monitored in real time during the Wrst 300 s of each sensorgram (t D 0–300 s) when the ligand was continuously injected on to the active chip surface. The dissociation phase of the ligand–receptor complex is monitored during the following 900 s (t D 300–1200 s) after the injection of the ligand was Wnished (see also Materials and methods). Mass transport BiaCore evaluation of reaction kinetics is in general aVected by a phenomenon of mass transport. To evaluate whether the reaction is kinetic or mass transport controlled, 180 RU of the hObR-Fc was immobilized and the Xow rate of the hOb (5 nM) injection was changed from 15 to 100 l/min. The binding rate was monitored for an initial 100 s. A typical result is shown in Fig. 3. Because no increase in the initial binding rate was observed, mass transport was considered negligible and the interaction fully kinetic controlled. Data Wtting Association and dissociation phases of all corrected sensorgrams (0.1–30 nM) were simultaneously Wtted to a 1:1 binding model. The rate constants ka and kd were determined as global Wtting parameters [20]. The analyte binding capacity Rmax was Wtted locally for each sensorgram to reXect possible minor diVerences of ObR-Fc coating level on the protein G surface (Table 1). Kinetic
Fig. 2. Data Wtting. Association and dissociation phases of all corrected sensorgrams (0.1–30 nM) were simultaneously Wtted to a 1:1 binding model. The rate constants ka and kd were determined as global Wtting parameters. The analyte binding capacity Rmax was Wtted locally for each sensorgram to reXect possible minor diVerences due to ObR-Fc dissociation from the protein G surface. A, B, and C represent typical sensorgrams obtained with human, mouse, and rat leptin, respectively (human ObR-Fc was used).
constants kinetics for the binding of human, rat, and mouse leptin to both the mouse leptin receptor chimera and the human leptin receptor chimera are presented in Table 2. The values shown represent average values from three separate measurements. For both sets of experiments, the Ob/ObR-Fc complex has very fast association and extremely slow dissociation phases (Fig 2). Its high stability is striking (kd » 3 and 10 £ 10¡4 s¡1 for human and mouse ObR-Fc, respectively). Such slow
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P. Mistrík et al. / Analytical Biochemistry 327 (2004) 271–277 Table 3 Stoichiometry of the Ob–ObR interaction Ob species
Fig. 3. Evaluation of mass transport. 180 RU of the hObR-Fc was immobilized onto the chip. The Xow rate of 5 nM hOb injection was changed from 15 to 100 l/min. The binding rate was monitored for an initial 100 s. Because no increase was observed, mass transport was considered negligible and reaction fully kinetic controlled.
Table 1 Determination of the analyte binding capacity, Rmax Conc [nM]
Rmax Rl D 124.0
Rl D 124.5
Rl D 123.0
30 10 5 3 1 0.1
9.0 8.6 9.2 9.6 10.8 8.7
8.9 8.7 8.6 8.8 9.7
8.4 8.8 8.8 8.8 9.7 7.6
Average Rmax
9.3 § 0.8
9.0 § 0.5
8.6 § 0.7
It was determined from the experimental data as a local Wtting parameter for a 1:1 binding model. The Rmax value obtained for each sensorgram is depicted following the immobilization of about 120 RU of hOBR-Fc. The average value for each experiment was calculated. The Rmax and Rl values are presented in resonance units.
Table 2 Kinetic constants for the Ob–ObR interaction ka [106 M¡1 s¡1]
kd [10¡4 s¡1]
KD [10¡10 s¡1]
Mouse ObR-Fc was immobilizeda Human 2.3 § 0.1 Mouse 1.5 § 0.2 Rat 2.8 § 0.3
11.6 § 0.1 7§1 12 § 1
5.0 § 0.6 5§1 4.3 § 0.8
Human ObR-Fc was immobilizedb Human 1.9 § 0.4 Mouse 1.1 § 0.2 Rat 2.9 § 0.1
4.4 § 0.6 1.3 § 0.6 2.8 § 0.2
2.3 § 0.8 1.2 § 0.8 1.0 § 0.1
Ob species
The association and dissociation rate constants ka and kd were determined as global Wtting parameters for a 1:1 binding model. a The kinetics of the interaction with human, rat, and mouse leptin were analyzed. The equilibrium dissociation constant KD was determined as kd/ka. Numbers represent averaged values from three independent measurements on the same mObR-Fc surface (R1 D 300 RU). b The kinetics of the interaction with human, rat, and mouse leptin were analyzed. The equilibrium dissociation constant KD was determined as kd/ka. Numbers represent averaged values from three independent measurements on the same hObR-Fc surface (R1 D 300 RU).
Stoichiometry when Rl D 120 RU
Stoichiometry when Rl D 300 RU
Mouse ObR-Fc immobilized Human 1.0 Mouse 0.9 Rat 1.0
0.9 1.0 1.0
Human ObR-Fc immobilized Human 0.9 Mouse — Rat —
1.1 1.1 1.1
Derived from Eq. 1. The analyte binding capacity Rmax and the ligand response Rl were determined from a 1:1 binding model or obtained directly from a sensorgram, respectively. Numbers represent averaged values from three experiments.
dissociation could, in principle, signiWcantly aVect data Wtting by reducing the accuracy of the kd determination. To improve this determination, we increased the dissociation time to such an extent that standard errors on kd as reported from three independent experiments are acceptable. Stoichiometry As the BiaCore instrument measures the mass of molecules bound to the sensor surface, the stoichiometry of the surface molecular complex can be evaluated. The stoichiometry of ObR-Fc/Ob interaction was determined from Eq. (1). Two diVerent amounts of the ObRFc chimeras were immobilized (120 and 300 RU). Because Rmax was determined as local Wtting parameter, its average value was used (Table 1). Stoichiometry was calculated using molecular weights of monomeric molecules (see Materials and methods). The derived stoichiometry, therefore, represents the number of Ob molecules bound to one of the two ObR extracellular domains of the ObR-Fc molecule. The obtained values are summarized in Table 3. The observed stoichiometry is consistently observed to be around 0.99, i.e., describing a 1:1 stoichiometry. Thus, for both receptors, one extracellular ObR domain of ObR-Fc molecule interacts with one Ob molecule (one ObR-Fc molecule interacts with two Ob molecules).
Discussion Leptin plays a complex role in homeostasis. Leptin conducts this task by coordinating activity of the neuronal, reproductive, immune, and digestive systems. A crucial role in this is played by the leptin receptor, which speciWcally mediates leptin action in target tissues. ObR is a membrane protein with extracellular, transmembrane, and intracellular domains [10]. Its extracellular
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domain bears the Ob binding site while the intracellular domain triggers the response to ligand binding. Here we report a BiaCore analysis of the leptin/leptin receptor interaction. Our aim was to clarify the mechanism of leptin binding to the receptor as this issue is at the moment still highly controversial. Based upon its primary sequence analysis, the leptin receptor has been classiWed as a member of the cytokine receptor superfamily [10]. It is generally accepted that ligand binding activates cytokine receptors by inducing a change in their oligomerization state. The most common mechanism is ligand-induced dimerization of monomeric receptor [14]. However, the leptin receptor has recently been shown to exist as a dimer, not a monomer, in both its ligand-free and its activated states [16–18]. This observation has created diYculties with the proposed activation mechanism for this receptor. We decided to investigate leptin binding to its receptor by using chimeric ObR receptor which consists of two receptor extracellular domains fused together via Fc region of IgG1 (ObR-Fc). The chimeric receptor used provides a close mutual localization of the receptor extracellular domains on the chip surface. Although the relative orientation of the extracellular domains cannot be assumed to be identical to that on the cell surface, the chimera serves as a useful system to mimic the situation where the leptin receptor adopts a very ambiguous stoichiometrical state, existing as a mixture of monomers and dimers [18]. We evaluated the reaction kinetics using human, mouse, and rat leptin in combination with human or mouse chimeric receptors (ObR-Fc). Association and dissociation phases of all sensorgrams (0.1–30 nM leptin) for all leptin–leptin receptor combinations Wtted well to a 1:1 binding model (Fig. 3). All three leptin molecules bind both human and mouse chimeric receptors (ObRFc) with very similar aYnity (KD[0.4 nM). The association rate to either receptor is very similar for the diVerent
275
species of leptin. The minor diVerences in aYnity result from a variance in the dissociation rate constant (kd[3 and 10 £ 10¡4 s¡1 for human and mouse ObR-Fc, respectively). The similarity in the kinetics for these interactions likely reXects the conservation in the sequences across species. Thus, the leptin sequence is very highly conserved across species, there being 84% sequence identity shared across all three species (Fig. 4). However, the extracellular domains of mouse and human receptors are less well conserved, having 68% identity. Apart from the reaction kinetics, the stoichiometry of interaction can also be determined with the BiaCore instrument. The observed stoichiometry was measured for both the human and the mouse receptors using two diVerent surface densities (120 and 300 RU). The derived stoichiometry was the same for both densities, around 0.99, describing a 1:1 stoichiometry for both the human and the mouse receptors. This indicates that one ObR extracellular domain of ObR-Fc molecule interacts with one Ob molecule (i.e., one ObR-Fc molecule interacts with two Ob molecules). Our data suggest that the leptin receptor is not activated classically by ligand-induced dimerization. The data Wtting to the simplest 1/1 kinetic model and the 1:1 reaction stoichiometry at the ligand saturation indicate that ligand binds to the dimerized ObR-Fc receptor with two independent binding sites. This Wnding implies that leptin binding to its receptor does not follow the general model of the cytokine receptor activation which presumes that the ligand induces dimerization of monomeric receptor (with growth hormone/growth hormone receptor as prototype [21]). The same conclusion has been drawn recently from results obtained with alternative techniques, gel Wltration chromatography [16] or Xuorescence resonance energy transfer [18]. The notion that leptin binds to the dimerized ObR-Fc receptor with two independent binding sites is supported
Fig. 4. Sequence alignment of human, mouse, and rat Ob proteins (hOb, mOb, and rOb, respectively). Shading indicates the degree of similarity of sequences at a given position. Black shading is used for residues that are exactly the same as the symbol in the consensus sequence (not shown). Medium gray shading is used for symbols whose comparison value is greater than or equal to the average nonidentical comparison value in the scoring matrix. Light-gray shading is used for symbols whose comparison scores are less than this value but are greater than or equal to 1. All other sequence symbols are shown with white backgrounds. Made with the Pileup and PrettyBox programes from the GCG package (Genetics Computer Group).
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also by a possible geometry of the receptor–ligand complex derived from information on the functional motifs in the ObR extracellular domain. The leptin receptor extracellular domain possesses two cytokine-binding homology regions, a possible ligand binding site predicted from the primary sequence [22]. However, only the second cytokine-binding homology region has been found to bind the ligand [23]. Thus, the Fc-linked receptor could be predicted to accommodate the two leptin molecules as proposed by our BiaCore data. An unexpected reaction mechanism of Ob/ObR-Fc described above could account also for a very high aYnity of human leptin for its Fc-linked receptor (KD). This was found to be 0.23 § 0.08 nM (Table 2). This high KD value reXects a very strong ligand binding to the Fclinked receptor. Of interest, the KD determined by BiaCore previously for monomeric receptor was found to be 10.5 nM [24]. This indicates that the Fc-linked dimeric receptor has consistently »25-fold higher aYnity for ligand than its monomeric counterpart. This diVerence is not negligible. It may be the consequence of the oligomerization state of the Fc-receptor. It is possible that receptor dimerization induces a particular structural modiWcation of the receptor, modifying its ligand recognition sites and hence improving their aYnity for the ligand. Both ligand sites would in this case still remain functional. The improved aYnity would therefore not be a consequence of ligand bivalent binding, as usually observed. This could suggest that receptor dimerization, a feature for the native receptor on the cell surface [18], stabilizes the receptor–ligand complex. However, it must be also considered that the monomeric receptor used previously and the Fc-receptor chimera employed here were expressed in diVerent systems and, hence, may diVer in their levels of glycosylation. The monomeric receptor was expressed in Baculovirus while the Fc-receptor chimera was expressed in mammalian cells (mouse myeloma cell line, NSO). This could contribute to the observed diVerent aYnities. In conclusion, our data provide important detailed insight into the kinetics and stoichiometry of the leptin binding to its cognate receptor. Presented data support the notion that the extracellular domain of the receptor exists as a homodimer, even in the absence of ligand, and that its dimerization increases the ligand aYnity. Furthermore, the stoichiometry of the interaction was conWrmed to be 1:1. This challenges the classical model of the ligand-induced dimerization as the activation mechanism of leptin receptor and argues for an investigation of an alternative mechanism. Acknowledgments Pavel Mistrík was funded by a Wellcome Trust Prize 4-year PhD Studentship.
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BiaCore analysis of leptin–leptin receptor interaction: evidence for 1:1 stoichiometry P. Mistrík,a,1 F. Moreau,b and J.M. Allena,b,¤,2 a
Division of Biochemistry and Molecular Biology, IBLS, Wolfson Building, Level 2, University of Glasgow, Glasgow G12 8QQ, UK b PWzer Global Research and Development, 3–9 Rue de la Loge, 94265 Fresnes Cedex, France Received 21 November 2003
Abstract Leptin is a hormonal protein involved in energy homeostatis that acts to inhibit food intake, to stimulate energy expenditure, and to inXuence insulin secretion, lipolysis, and sugar transport. Its action is mediated by a speciWc 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 IgG1 were captured on to the sensor via protein G. Kinetic data Wtted 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 highaYnity interaction. The KD was 0.23 § 0.08 nM, with ka D (1.9 § 0.4) £ 106 M¡1 s¡1 and kd D (4.4 § 0.6)£10¡4 s¡1 for human leptin with its cognate receptor. Similar results were observed for the aYnity of diVerent species of leptin binding to mouse leptin receptor. 2004 Elsevier Inc. All rights reserved. Keywords: Surface plasmon resonance; Dimerization; Cytokine; Receptor activation
Leptin (Ob),3 a 16-kDa protein isolated in 1994, seemed to represent an eagerly expected molecular component which could be exploited in the development of novel therapeutics for weight control [1]. Leptin was originally thought to be expressed exclusively in the adipose tissue and in accord with the lipostatic theory [2] to form a feedback loop with the hypothalamus in the control of the appetite. It is now recognized that leptin is a ¤
Corresponding author. Fax: +44-207-074-4746. E-mail address: [email protected] (J. Allen). 1 Present address: Department of Pharmacy, Center for Drug Research, University of Munich, 81377 Munich, Germany. 2 Present address: Inpharmatica, 60 Charlotte Street, London W1T 2NU, UK. 3 Abbreviations used: Ob, leptin; ObR, leptin receptor; ObR-Fc, leptin receptor chimera; RU, resonance unit; Rmax, analyte binding capacity; R1, ligand response; Epo, erythropoietin; EpoR, erythropoietin receptor; CHR, cytokine-binding homology regions; NHS, N-hydroxysuccinimide; EDC, N-ethyl-N0-(3-diethylaminopropyl)carbodiimide. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.01.022
component of a highly complex molecular network whose physiological manifestations extend beyond the initial lipostatic concept. Leptin is produced in several organs and participates in a broad range of metabolic processes [3–7]. Interestingly, the crystal structure of a mutant human leptin (leptin-E100) [8] revealed that leptin bears striking structural similarity to some cytokine molecules, such as growth hormone, leukemia inhibitory factor, and granulocyte-stimulating hormone. This suggests that leptin may also possess immunomodulatory and antiinXammatory eVects [9]. The high complexity of leptin action is mirrored by the complex pattern of expression of the leptin receptor (ObR) [10]. High levels were detected in lung and kidney, in addition to the hypothalamus and choroid plexus. Lower levels were found in heart, spleen, liver, skeletal muscle, and testes [10–13]. Therefore, leptin represents an interesting target for studying the molecular interactions between immune, neuronal, digestive, and reproductive systems.
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We have used the BiaCore to evaluate the interaction between leptin and receptor. The nature of this ligand– receptor interaction is controversial. ObR as a member of the cytokine receptor superfamily should be activated by ligand-induced dimerization [14,15]. However, recent studies [16–18] have suggested that ObR does not change its oligomerization state during its interaction with a ligand; rather, it exists as a dimer in both ligandfree and ligand-bound states. We used human or mouse chimeric receptors and measured the interactions with human, mouse, and rat leptin. We have evaluated the kinetics and stoichiometry of the interaction in all possible combinations. Kinetic data Wtted 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.
Materials and methods Surface plasmon resonance BiaCore biosensor BIAcore 2000, CM5 researchgrade biosensor chips, N-hydroxysuccinimide (NHS), N-ethyl-N0-(3-diethylaminopropyl)carbodiimide (EDC), ethanolamine-HCl, and HBS-EP buVer were obtained from BiaCore AB (Uppsala, Sweden). Protein G was purchased from Sigma (Poole, UK). Recombinant human and mouse leptin receptor-Fc chimeras and recombinant human, mouse, and rat leptin were obtained from R&D Systems Europe, Ltd. (Abingdon, UK). The leptin receptor-Fc chimera exists as a disulWde-linked homodimeric protein with the monomeric molecular weights of 190–210 kDa for the human receptor chimera and 150–170 kDa for the mouse receptor chimera as determined by SDS–PAGE. The molecular weight of leptin molecules is 16 kDa. To prepare the biosensor assay, protein G was immobilized on to the four channels of a research-grade carboxymethyl dextran chip (CM5) using an amine coupling procedure described previously [19]. The surface was activated with NHS/EDC for 5 min. Protein G was injected at a concentration of 200 g/ml in sodium acetate buVer (10 mM, pH 4.0) until 2000 resonance units (RU) of protein were coupled. Remaining activated groups were then blocked with a 5-min injection of 1 M ethanolamine (pH 8.5). For kinetic experiments the recombinant human or mouse leptin receptor-Fc chimera was captured on to the protein G surface. Typically, 15 l of a 0.5-g/ml solution was injected to immobilize 100–300 RU of the receptor chimera. After an 1800-s equilibration time, a
100-l injection of leptin or HBS-EP buVer was performed. A range of leptin concentrations (0.1–30 nM) was used. The dissociation phase was then monitored for 900 s, followed by two 5-l injections of 10 mM glycine, pH 2.0, to regenerate a fully active protein G surface. All kinetic experiments were performed at 25 °C in a buVer containing 10 mM Hepes, pH 7.4, 150 mM sodium chloride, and 0.005% Tween 20, at a Xow rate of 20 l/min. In control experiments, protein G was used instead of the leptin receptor-Fc chimera to evaluate leptin nonspeciWc binding. Generation of good-quality data for analysis was achieved classically as follows. Leptin, in concentration range 0.1–30 nM, or buVer alone was injected on to the active (ObR-Fc) or control (protein G) surfaces. To remove contributions of nonspeciWc binding and refractive index, the control responses were subtracted from those obtained from the active surface. A minor baseline draft resulting from a slow dissociation of the complex between ObR-Fc and protein G was eliminated by subtracting data of a bulk injection (0 nM Ob) from data corrected for nonspeciWc binding. The kinetics parameters were derived from obtained data using BIA evaluation software 3.1 (Biacore AB). Association and dissociation binding data were Wtted simultaneously to a one-to-one, single-site binding model using nonlinear least squares analysis model [20]. Stoichiometry The BiaCore instrument measures the mass of molecules bound to the sensor surface. Thus the stoichiometry of the surface molecular complex can be determined using the equation:
where Rmax, analyte binding capacity, can be extrapolated from experimental data and ligand response (Rl) is given directly from a sensorgram recorded during ligand immobilization.
Results To study the interaction between the leptin and its speciWc receptor, we used human and mouse receptor chimeras (ObR-Fc), which consist of two receptor extracellular domains fused together via the Fc region of IgG1. The following assay was used to evaluate their aYnity for human, mouse, and rat leptin (Fig. 1). The ObR-Fc was captured on to the chip surface via the protein G, a high-aYnity receptor for the Fc portion of IgG, which was covalently bound to the dextran matrix of the chip. In this conWguration, a homogenous receptor
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Fig. 1. Schematic of the biosensor assay. The ObR-Fc molecule was captured on to the chip surface via protein G, which was covalently bound to the dextran matrix of the chip. In this conWguration, a homogenous receptor surface is formed and its interaction with Ob can be monitored.
surface is formed and its interaction with Ob can be monitored. Typical sensorgrams, representing diVerent leptin molecules (human, mouse, and rat) binding to the receptor chimera (human) are shown in Fig. 2. The association phase of the ligand–receptor interaction is monitored in real time during the Wrst 300 s of each sensorgram (t D 0–300 s) when the ligand was continuously injected on to the active chip surface. The dissociation phase of the ligand–receptor complex is monitored during the following 900 s (t D 300–1200 s) after the injection of the ligand was Wnished (see also Materials and methods). Mass transport BiaCore evaluation of reaction kinetics is in general aVected by a phenomenon of mass transport. To evaluate whether the reaction is kinetic or mass transport controlled, 180 RU of the hObR-Fc was immobilized and the Xow rate of the hOb (5 nM) injection was changed from 15 to 100 l/min. The binding rate was monitored for an initial 100 s. A typical result is shown in Fig. 3. Because no increase in the initial binding rate was observed, mass transport was considered negligible and the interaction fully kinetic controlled. Data Wtting Association and dissociation phases of all corrected sensorgrams (0.1–30 nM) were simultaneously Wtted to a 1:1 binding model. The rate constants ka and kd were determined as global Wtting parameters [20]. The analyte binding capacity Rmax was Wtted locally for each sensorgram to reXect possible minor diVerences of ObR-Fc coating level on the protein G surface (Table 1). Kinetic
Fig. 2. Data Wtting. Association and dissociation phases of all corrected sensorgrams (0.1–30 nM) were simultaneously Wtted to a 1:1 binding model. The rate constants ka and kd were determined as global Wtting parameters. The analyte binding capacity Rmax was Wtted locally for each sensorgram to reXect possible minor diVerences due to ObR-Fc dissociation from the protein G surface. A, B, and C represent typical sensorgrams obtained with human, mouse, and rat leptin, respectively (human ObR-Fc was used).
constants kinetics for the binding of human, rat, and mouse leptin to both the mouse leptin receptor chimera and the human leptin receptor chimera are presented in Table 2. The values shown represent average values from three separate measurements. For both sets of experiments, the Ob/ObR-Fc complex has very fast association and extremely slow dissociation phases (Fig 2). Its high stability is striking (kd » 3 and 10 £ 10¡4 s¡1 for human and mouse ObR-Fc, respectively). Such slow
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P. Mistrík et al. / Analytical Biochemistry 327 (2004) 271–277 Table 3 Stoichiometry of the Ob–ObR interaction Ob species
Fig. 3. Evaluation of mass transport. 180 RU of the hObR-Fc was immobilized onto the chip. The Xow rate of 5 nM hOb injection was changed from 15 to 100 l/min. The binding rate was monitored for an initial 100 s. Because no increase was observed, mass transport was considered negligible and reaction fully kinetic controlled.
Table 1 Determination of the analyte binding capacity, Rmax Conc [nM]
Rmax Rl D 124.0
Rl D 124.5
Rl D 123.0
30 10 5 3 1 0.1
9.0 8.6 9.2 9.6 10.8 8.7
8.9 8.7 8.6 8.8 9.7
8.4 8.8 8.8 8.8 9.7 7.6
Average Rmax
9.3 § 0.8
9.0 § 0.5
8.6 § 0.7
It was determined from the experimental data as a local Wtting parameter for a 1:1 binding model. The Rmax value obtained for each sensorgram is depicted following the immobilization of about 120 RU of hOBR-Fc. The average value for each experiment was calculated. The Rmax and Rl values are presented in resonance units.
Table 2 Kinetic constants for the Ob–ObR interaction ka [106 M¡1 s¡1]
kd [10¡4 s¡1]
KD [10¡10 s¡1]
Mouse ObR-Fc was immobilizeda Human 2.3 § 0.1 Mouse 1.5 § 0.2 Rat 2.8 § 0.3
11.6 § 0.1 7§1 12 § 1
5.0 § 0.6 5§1 4.3 § 0.8
Human ObR-Fc was immobilizedb Human 1.9 § 0.4 Mouse 1.1 § 0.2 Rat 2.9 § 0.1
4.4 § 0.6 1.3 § 0.6 2.8 § 0.2
2.3 § 0.8 1.2 § 0.8 1.0 § 0.1
Ob species
The association and dissociation rate constants ka and kd were determined as global Wtting parameters for a 1:1 binding model. a The kinetics of the interaction with human, rat, and mouse leptin were analyzed. The equilibrium dissociation constant KD was determined as kd/ka. Numbers represent averaged values from three independent measurements on the same mObR-Fc surface (R1 D 300 RU). b The kinetics of the interaction with human, rat, and mouse leptin were analyzed. The equilibrium dissociation constant KD was determined as kd/ka. Numbers represent averaged values from three independent measurements on the same hObR-Fc surface (R1 D 300 RU).
Stoichiometry when Rl D 120 RU
Stoichiometry when Rl D 300 RU
Mouse ObR-Fc immobilized Human 1.0 Mouse 0.9 Rat 1.0
0.9 1.0 1.0
Human ObR-Fc immobilized Human 0.9 Mouse — Rat —
1.1 1.1 1.1
Derived from Eq. 1. The analyte binding capacity Rmax and the ligand response Rl were determined from a 1:1 binding model or obtained directly from a sensorgram, respectively. Numbers represent averaged values from three experiments.
dissociation could, in principle, signiWcantly aVect data Wtting by reducing the accuracy of the kd determination. To improve this determination, we increased the dissociation time to such an extent that standard errors on kd as reported from three independent experiments are acceptable. Stoichiometry As the BiaCore instrument measures the mass of molecules bound to the sensor surface, the stoichiometry of the surface molecular complex can be evaluated. The stoichiometry of ObR-Fc/Ob interaction was determined from Eq. (1). Two diVerent amounts of the ObRFc chimeras were immobilized (120 and 300 RU). Because Rmax was determined as local Wtting parameter, its average value was used (Table 1). Stoichiometry was calculated using molecular weights of monomeric molecules (see Materials and methods). The derived stoichiometry, therefore, represents the number of Ob molecules bound to one of the two ObR extracellular domains of the ObR-Fc molecule. The obtained values are summarized in Table 3. The observed stoichiometry is consistently observed to be around 0.99, i.e., describing a 1:1 stoichiometry. Thus, for both receptors, one extracellular ObR domain of ObR-Fc molecule interacts with one Ob molecule (one ObR-Fc molecule interacts with two Ob molecules).
Discussion Leptin plays a complex role in homeostasis. Leptin conducts this task by coordinating activity of the neuronal, reproductive, immune, and digestive systems. A crucial role in this is played by the leptin receptor, which speciWcally mediates leptin action in target tissues. ObR is a membrane protein with extracellular, transmembrane, and intracellular domains [10]. Its extracellular
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domain bears the Ob binding site while the intracellular domain triggers the response to ligand binding. Here we report a BiaCore analysis of the leptin/leptin receptor interaction. Our aim was to clarify the mechanism of leptin binding to the receptor as this issue is at the moment still highly controversial. Based upon its primary sequence analysis, the leptin receptor has been classiWed as a member of the cytokine receptor superfamily [10]. It is generally accepted that ligand binding activates cytokine receptors by inducing a change in their oligomerization state. The most common mechanism is ligand-induced dimerization of monomeric receptor [14]. However, the leptin receptor has recently been shown to exist as a dimer, not a monomer, in both its ligand-free and its activated states [16–18]. This observation has created diYculties with the proposed activation mechanism for this receptor. We decided to investigate leptin binding to its receptor by using chimeric ObR receptor which consists of two receptor extracellular domains fused together via Fc region of IgG1 (ObR-Fc). The chimeric receptor used provides a close mutual localization of the receptor extracellular domains on the chip surface. Although the relative orientation of the extracellular domains cannot be assumed to be identical to that on the cell surface, the chimera serves as a useful system to mimic the situation where the leptin receptor adopts a very ambiguous stoichiometrical state, existing as a mixture of monomers and dimers [18]. We evaluated the reaction kinetics using human, mouse, and rat leptin in combination with human or mouse chimeric receptors (ObR-Fc). Association and dissociation phases of all sensorgrams (0.1–30 nM leptin) for all leptin–leptin receptor combinations Wtted well to a 1:1 binding model (Fig. 3). All three leptin molecules bind both human and mouse chimeric receptors (ObRFc) with very similar aYnity (KD[0.4 nM). The association rate to either receptor is very similar for the diVerent
275
species of leptin. The minor diVerences in aYnity result from a variance in the dissociation rate constant (kd[3 and 10 £ 10¡4 s¡1 for human and mouse ObR-Fc, respectively). The similarity in the kinetics for these interactions likely reXects the conservation in the sequences across species. Thus, the leptin sequence is very highly conserved across species, there being 84% sequence identity shared across all three species (Fig. 4). However, the extracellular domains of mouse and human receptors are less well conserved, having 68% identity. Apart from the reaction kinetics, the stoichiometry of interaction can also be determined with the BiaCore instrument. The observed stoichiometry was measured for both the human and the mouse receptors using two diVerent surface densities (120 and 300 RU). The derived stoichiometry was the same for both densities, around 0.99, describing a 1:1 stoichiometry for both the human and the mouse receptors. This indicates that one ObR extracellular domain of ObR-Fc molecule interacts with one Ob molecule (i.e., one ObR-Fc molecule interacts with two Ob molecules). Our data suggest that the leptin receptor is not activated classically by ligand-induced dimerization. The data Wtting to the simplest 1/1 kinetic model and the 1:1 reaction stoichiometry at the ligand saturation indicate that ligand binds to the dimerized ObR-Fc receptor with two independent binding sites. This Wnding implies that leptin binding to its receptor does not follow the general model of the cytokine receptor activation which presumes that the ligand induces dimerization of monomeric receptor (with growth hormone/growth hormone receptor as prototype [21]). The same conclusion has been drawn recently from results obtained with alternative techniques, gel Wltration chromatography [16] or Xuorescence resonance energy transfer [18]. The notion that leptin binds to the dimerized ObR-Fc receptor with two independent binding sites is supported
Fig. 4. Sequence alignment of human, mouse, and rat Ob proteins (hOb, mOb, and rOb, respectively). Shading indicates the degree of similarity of sequences at a given position. Black shading is used for residues that are exactly the same as the symbol in the consensus sequence (not shown). Medium gray shading is used for symbols whose comparison value is greater than or equal to the average nonidentical comparison value in the scoring matrix. Light-gray shading is used for symbols whose comparison scores are less than this value but are greater than or equal to 1. All other sequence symbols are shown with white backgrounds. Made with the Pileup and PrettyBox programes from the GCG package (Genetics Computer Group).
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also by a possible geometry of the receptor–ligand complex derived from information on the functional motifs in the ObR extracellular domain. The leptin receptor extracellular domain possesses two cytokine-binding homology regions, a possible ligand binding site predicted from the primary sequence [22]. However, only the second cytokine-binding homology region has been found to bind the ligand [23]. Thus, the Fc-linked receptor could be predicted to accommodate the two leptin molecules as proposed by our BiaCore data. An unexpected reaction mechanism of Ob/ObR-Fc described above could account also for a very high aYnity of human leptin for its Fc-linked receptor (KD). This was found to be 0.23 § 0.08 nM (Table 2). This high KD value reXects a very strong ligand binding to the Fclinked receptor. Of interest, the KD determined by BiaCore previously for monomeric receptor was found to be 10.5 nM [24]. This indicates that the Fc-linked dimeric receptor has consistently »25-fold higher aYnity for ligand than its monomeric counterpart. This diVerence is not negligible. It may be the consequence of the oligomerization state of the Fc-receptor. It is possible that receptor dimerization induces a particular structural modiWcation of the receptor, modifying its ligand recognition sites and hence improving their aYnity for the ligand. Both ligand sites would in this case still remain functional. The improved aYnity would therefore not be a consequence of ligand bivalent binding, as usually observed. This could suggest that receptor dimerization, a feature for the native receptor on the cell surface [18], stabilizes the receptor–ligand complex. However, it must be also considered that the monomeric receptor used previously and the Fc-receptor chimera employed here were expressed in diVerent systems and, hence, may diVer in their levels of glycosylation. The monomeric receptor was expressed in Baculovirus while the Fc-receptor chimera was expressed in mammalian cells (mouse myeloma cell line, NSO). This could contribute to the observed diVerent aYnities. In conclusion, our data provide important detailed insight into the kinetics and stoichiometry of the leptin binding to its cognate receptor. Presented data support the notion that the extracellular domain of the receptor exists as a homodimer, even in the absence of ligand, and that its dimerization increases the ligand aYnity. Furthermore, the stoichiometry of the interaction was conWrmed to be 1:1. This challenges the classical model of the ligand-induced dimerization as the activation mechanism of leptin receptor and argues for an investigation of an alternative mechanism. Acknowledgments Pavel Mistrík was funded by a Wellcome Trust Prize 4-year PhD Studentship.
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