Annals of Clinical & Laboratory Science 37:63-70 (2007)
© 2007 Association of Clinical Scientists
Purification of Biologically Active Human Erythropoietin-Binding Protein and Detection of its Binding Sites
Jong Y. Lee
Department of Medicine, University of Minnesota School of Medicine, Minneapolis, Minnesota
Address correspondence to: Dr. Jong Y. Lee, P.O. Box 14945, Minneapolis, MN 55414, USA; tel 612 408 3125; fax 612 379 2467; e-mail leexx154{at}umn.edu.
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Abstract
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To purify human erythropoietin-binding protein (Epo-bp), the recombinant vector JYL26 was constructed by inserting human Epo-receptor cDNA by PCR into a pGEX-2T plasmid vector with a thrombin cleavage site. EpoRex-th fusion protein, containing glutathione-S-transferase (GST) and Epo-bp, was purified by glutathione-affinity chromatography. Biologically active pure human Epo-bp (MW = 29 kDa) was then purified by Epo-agarose chromatography after cleaving off the GST by thrombin. Purified Epo-bp has a strong binding affinity to 125I-Epo, and unlabeled Epo eliminates the binding (p <0.0001). Trypsin digested Epo-bp to ~20 kDa and completely eliminated the binding. Thus, Epo-bp ligand binding is specific and it may require an intact Epo-bp. Ligand-binding sites were detected using fluorescein-labeling in our new products, Epo-bp and anti-Epo-bp antibody (
Epo-bp), in various blood cell progenitors, including megakaryocytes, erythroblasts, normoblasts, and myeloblasts, while fluorescein-labeled pre-immune Fab-treated cells did not show any binding. Epo, Epo-bp, and their antibodies were measured in serum and plasma specimens by enzyme immunoassay methods developed in our laboratory; Epo in serum and plasma: 25.4 ± 2.17 and 24.2 ± 2.35; Epo-bp in serum and plasma: 24.2 ± 1.84 and 25.0 ± 1.26 mU/ml, respectively. These assays are simple, sensitive, and precise, compared to the conventional Epo radioimmunoassay, and are more environmentally friendly than assays that use radioactive reagents.
Keywords: Epo-receptor expression vector, Epo-binding site, Epo-bp immunoassay
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Introduction
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Erythropoietin (Epo), a glycoprotein hormone of molecular weight 34 kDa produced in mammalian kidney and liver, is the principal regulator of erythropoiesis as a primary inducer and regulator of red cell proliferation and differentiation. This activity is associated with the activation of a number of erythroid-specific genes, including globin and carbonic anhydrase [1,2].
The Epo receptor is a member of the hematopoietic/cytokine/growth factor receptor family, which includes other growth factor receptors such as interleukin (IL)-3, -4, and -6 receptors, the granulocyte macrophage colony-stimulating factor (GM-CSF) receptors, and the prolactin and growth hormone receptors [3]. The entire cytokine receptor family contains conserved 4 cysteines and a tryptophanserine-X-tryptophanserine (WS x WS) motif positioned just outside the transmembrane region near the C-terminal end, which is presumed to be involved in protein-protein interaction [4]. The extracellular segments of the IL-2, -3, -4, -6, and -7, granulocyte colony-stimulating factor (G-CSF) and GM-CSF, and Epo-receptors share a 226 amino acid molecule with conservation of 4 cysteine residues in the N-terminal moiety [4].
The human Epo-receptor cDNA encodes a 508 amino acid polypeptide chain of MW ~55 kDa, and the gene is organized into 8 exons spread over 6 Kb of DNA. Analysis of the coding sequence predicts a 22 amino acid signal peptide, a 226 amino acid extracellular domain, a 23 amino acid membrane-spanning domain, and a 235 amino acid cytoplasmic domain [5]. All human erythroid progenitor cells have been shown to contain Epo receptors, and the binding of Epo continuously declines with an increase in erythroid maturation, until the point at which Epo receptors are not detectable on reticulocytes [6,7].
Epo maintains the cellular viability of the erythroid progenitor cells to allow them to proceed with mitosis and differentiation. Two major responsive erythroid progenitors to Epo are the burst-forming units-erythroid (BFU-E) and the colony-forming units-erythroid (CFU-E). The Epo receptor number correlates very well in response to Epo during normal BFU-E and CFU-E. Epo receptors decline after reaching the peak receptor number at the human and murine CFU-E stage [6,8]. Recovery of the Epo receptors after removal of Epo appears to be dependent on protein synthesis, which suggests a downregulation of the Epo receptors with their degradation, and a subsequent upregulation of receptors with the new synthesis of receptors when Epo is removed [9].
Characterization of the Epo receptor has been difficult due to the miniscule quantities of naturally obtainable Epo-receptor. Thus, the mechanism by which Epo interacts with its receptor and stimulates erythropoiesis is still unknown [10]. Recently, this mechanism has become of great interest in understanding the role of growth factors and their receptors in leukemogenesis; altered hematopoietic growth factors and their receptors may contribute to tumorigenesis [11] and leukemogenesis [12,13].
Functions of Epo may occur beyond hematopoietic and erythropoietic tissues. Epo-receptors exist in the paracrine and autocrine, as well as in the hormonal systems. Thus, the effects of Epo are likely to extend beyond its role in raising hematocrit [14]. Some studies indicate that Epo exists in the human brain, in the neurons of the central nervous system, and in the microglia in cell cultures [15]. Recent studies report multiple protective effects of Epo, including neurotrophic, anti-apoptotic, anti-oxidant, and angiogenic agents [16,17]. All of the studies suggest important roles of the Epo receptor in the system, since Epo and Epo receptor are required for definitive erythropoiesis and maturation of blood progenitors.
The purpose of this study was to develop a method to prepare pure human Epo-binding protein (Epo-bp) that is specific in ligand binding and in binding-site detection.
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Materials and Methods
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Glutathione (GSH)-agarose, pGEX-2T expression vector, and Sephadex G-50 were from Pharmacia (Mechanicsburg, PA). PCR reagents were from Perkin-Elmer Cetus (Norwalk, CT) and Affigel 15 was from BioRad (Richmond, CA). Bacteriophage T4 DNA ligase, restriction enzymes, and isopropylthio-ß-D-galactoside (IPTG) were from BRL Gibco (Gaithersburg, MD). Geneclean II was from Bio 101 (La Jolla, CA). Nitrocellulose was from Schleicher & Schuell (Keene, NH). Chemiluminescence (ECL) reagents and 125I-Epo were from Amersham (Arlington Heights, IL) and unlabeled Epo was a gift of Chugai-Upjohn (Rosemont, IL). Thrombin, trypsin, phenylmethylsulfonylfluoride (PMSF), diisopropylfluorophosphate (DFP), Triton X-100, 2,7-dichlorofluoresein, biotin-amidocaproyl hydroazide, alkaline phosphatase conjugate, disodium p-nitrophenyl phosphate, and o-phenylenediaminedihydrochloride (oPD) were from Sigma Chemical Co., (St. Louis, MO). Biotinylated rabbit anti-sheep antibodies, avidinhorse radish peroxidase, and IgG purification kit were from Pierce Co. (Rockford, IL). Streptavidin peroxidase was from Boehringer Manheim (Indianapolis, IN), and microplates were from Corning Costa (Cambridge, MA). Pure human Epo-bp and sheep anti-Epo-bp antibodies ( Epo-bp), along with Fab- Epo-bp, were prepared in our laboratory. Full-length human erythropoietin receptor (EpoR) cDNA LAP37 was a gift from Dr. Bernard G. Forget, Yale University (New Haven, CT). Oligonucleotides were synthesized by the microchemical facility of the Institute of Human Genetics, University of Minnesota (Minneapolis, MN). All other chemicals were of reagent grade.
Adult Sprague-Dawley (SD) rats were housed in the University animal facilities, fed standard rat chow, and given free access to drinking water. Bone marrow blood cells were used for ligand binding-site investigations. The sheep used for antibody development were housed at the University animal facility. For Epo, Epo-bp, and their antibody measurements, frozen human serum and plasma samples from previous studies were used. All of the study protocols followed the NIH and University of Minnesota guidelines.
Construction of Epo receptor cDNA recombinant vector.
A human Epo receptor recombinant expression vector, pJYL26, was constructed from a PCR product using a full-length human EpoR cDNA and the 5'-sense primer (5'-TTGGATCCGCGCCCCCGCCTAAC-3': BamH1 linker + coding sequence) and the 3'-antisense primer (5'-TGAATTCGGGGTCCAGGTCGCT-3': EcoR 1 linker + coding sequence). Using a Perkin Elmer-Cetus PCR kit, amplification was carried out with a Perkin Elmer Thermal Cycler (Norwalk, CT). The PCR product was confirmed by agarose gel electrophoresis and purified from the gel slice by a Geneclean II method, as described by the manufacturer. The ligation was done with a mixture containing 1 µg/µl each of the PCR product and pGEX-2T and the size was verified to be ~5.5 Kb on 1% agarose gel. The mixture was transformed into E. coli strain JM109. Extracted DNAs from grown colonies having ~5.5 Kb size were selected (colonies #26 and #46) and #26 was named as pJYL26 (Fig. 1
) [18].
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Fig. 1. Schematic representation of pGEX-2T/erythropoietin receptor cDNA PCR insert recombinant vector and Epo-binding protein (Epo-bp purification). Recombinant vector pJYL26 was constructed using the full-length human Epo receptor cDNA with sense primer, 5'-TTGGATCCGCGCCCCCGCCTAAC-3' and antisense primer, 5'-TGAATTCGGGGTCCAGGTCGCT-3'. Following ligation with T4-DNA ligase for the BamH1 and EcoR1 digested PCR and pGEX-2T, the recombinant plasmid was transformed into E. coli strain JM109. After culture for 24 hr at 37°C, colonies were selected to produce fusion proteins by IPTG induction. Fusion protein EpoRex-th induced from the recombinant vector JYL26 contains a protease thrombin cleavage site as shown in this diagram. After cleaving off the foreign protein, GST of the plasmid, the pure human Epo receptor protein, Epo-bp, is isolated by Epo-agarose affinity chromatography.
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Purification of Epo-bp.
Epo receptor recombinant fusion protein (EpoRex-th), containing combined fractions of glutathione-S-transferase (GST) and Epo-bp, was purified by GSH-affinity chromatography. EpoRex-th has a thrombin-specific proteolytic cleavage recognition amino acid sequence of Leu-Val-Pro-Arg-Gly-Ser [19], and Epo-bp was separated from GST following thrombin cleavage. Epo-bp was purified by Epo-agarose affinity chromatography [18].
Binding of Epo to Epo-bp.
For the binding assay, beads bound with 1 µg of Epo-bp in 30 µl of PBS were admixed with 125I-Epo, with or without 200x excess of unlabeled Epo, incubated (1 hr, room temperature), and analyzed in triplicates [18].
Ligand binding site in progenitor cells and detection of Epo and Epo receptor.
We developed Epo-bp in sheep inoculated with Epo-bp every 3–4 wk for 3 mo, and serum antibodies were then collected [20]. The antibodies were further purified for Fab Epo-bp only, which were fluorescein-labeled according to the manufacturer’s directions. These materials were used to detect ligand-binding sites in blood and/or tissue samples. Negative control cells had no antibodies added and positive control cells had Fab from IgG of pre-immune serum. To test for receptor ligand binding sites, rat bone marrow cells were washed in PBS and dispensed in 1–3 x 103 cells per well of round-bottomed tubes and centrifuged into a pellet at 500 x G for 2 min. The supernatant was removed and 100 µl of fluorescein-conjugated Fab antibodies was added. After being mixed well, the mixture was incubated on ice for 30 min. The cells were washed 3 times by adding 400 µl of buffer containing 1% FCS and 0.01% NaN3 in PBS and centrifuged at 500 x G for 2 min to remove supernatant. The cells were resuspended in a total volume of 50 µl of PBS and analyzed under an inverted fluorescence microscope.
A simple enzyme immunoassay (EIA) method, developed in our laboratory, was used to detect and measure levels of Epo, Epo-bp, anti-Epo-antibodies ( Epo), and Epo-bp in untreated human serum or plasma specimens. EIA microplates were coated with 2 µg/well of each Epo or Epo-bp to detect Epo or Epo-bp. To detect circulating Epo and Epo-bp, wells were coated with 200 µl of 1:10 diluted serum or plasma in PBS, pH 7.4. Plates were incubated at room temperature for 30 min or at 4°C overnight. After the plates were coated with antibody or serum, wells were washed 3 times with 200 µl/well PBST (0.05% Tween 20 in PBS). Nonspecific binding sites were blocked with 200 µl/well of 1% BSA in PBST for 30 min at room temperature. Wells were washed 3 times with 200 µl/well PBST. To detect bound antigen, streptavidin peroxidase-labeled Fab Epo (for detecting Epo), Epo (for detecting Epo), Fab Epo-bp (for detecting Epo-bp), and Epo-bp (for detecting Epo-bp) were prepared in our laboratory, and 2 µg/200 µl of the appropriate streptavidin peroxidase-labeled protein was added to each well. The wells were washed 3x with 200 µl PBST. A solution (160 µl) containing 10 mg/ml of o-phenylenediamine · 2HCl (OPD, in citrate buffer (24 mM citrate, and 51 mM Na2HPO4, pH 6.0) was prepared. Before use, H2O2 was added to make 0.1% in the final solution, and 160 µl of the solution was added to each well. The reaction was stopped by adding 40 µl of 5M NaOH, and absorbance at 405 nm was read with a Bio-Tek microplate reader.
Statistics.
Data were analyzed by the two-tailed Student’s t test, the cosinor method, and linear least square rhythmometry [21], as appropriate for the data. Results were expressed as mean ± SE. A p value of <0.05 was considered significant.
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Results
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The recombinant vector pJYL26 cDNA was synthesized using a PCR product encoding human Epo receptor cDNA extracellular domain into pGEX-2T plasmid vector, and a recombinant protein EpoRex-th was purified (Fig. 1
). The EpoRex-th contains a proteolytic-specific site, recognized by thrombin. Thrombin was applied to cleave off GST, and Epo-bp was purified by Epo-agarose affinity chromatography. The recombinant protein EpoRex-th at 55 kDa, Epo-bp at 29 kDa, and GST at 26 kDa were verified on 12.5 % SDS-polyacrylamide gels and Western blotting (Fig. 2).
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Fig. 2. Purified Epo-bp was confirmed by Western blotting. Following thrombin cleavage, EpoRex-th and Epo-bp were subjected to separation electrophoretically and detection immunologically. Primary antibody sheep anti-Epo-bp to Epo-bp was introduced at a 1:2000 concentration and incubated at room temperature for 1 hr with gentle agitation. The first antibody was rinsed off and biotinylated rabbit antiimmunoglobulin-anti-sheep at 1:10,000 was incubated at room temperature for another 1 hr while rocking, and followed by enzyme horse radish peroxidase-avidin, 1:10,000, at room temperature for 45 min. After soaking briefly in ECL reagents, wet blots were exposed immediately on a Kodak X-ray film. As shown in Lane 4, completely cleaved clean Epo-bp was purified, MW = ~29 kDa. Lane 1: Standard molecular weight markers; Lane 2: EpoRex-th; Lane 3: GST; Lane 4: Epo-bp.
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In Fig, 3, the purified Epo-bp binds Epo actively in nmol concentrations (p <0.0001). Specific binding activity of Epo to Epo-bp was greatly increased with increasing Epo concentration, and the binding was almost tripled from 8 nM to 12 nM 125I-Epo. For nonspecific binding, a 200x excess of unlabeled Epo was preincubated before treatment of 125I-Epo. The binding of 125I-Epo to Epo-bp was significantly inhibited in the presence of unlabeled Epo at 8 nM and a higher concentration of 125I-Epo (p <0.0001).
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Fig. 3. Ligand binding of Epo to Epo-bp and effect of concentration on binding to Epo receptor protein. Various concentrations of 125I-Epo were incubated with 1 µg of Epo-bp beads in 60 µl of total volume in PBS, pH 7.4, at room temperature for 1 hr. Nonspecific binding was measured by the same method, except that it was pretreated with 200x excess of unlabeled Epo 1 hr prior to adding labeled Epo. Each sample point was the mean ± SE. Specific binding of Epo to Epo-bp was almost tripled from 8 nM to 12 nM 125I-Epo concentrations, and preincubated Epo-bp with unlabeled Epo eliminated 125I-Epo-specific binding. White bars = 125I-Epo without unlabeled Epo; shaded bars = 125I-Epo + unlabeled Epo preincubation ± SE.
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Trypsin digestion of Epo-bp was done to see if there were smaller Epo-binding proteins other than intact Epo-bp existing in ligand binding (Fig. 4). The final product of Epo receptor protein, as a result of trypsin digestion, was ~20 kDa. However, Epo-bp did not recognize the trypsin digested Epo-bp; thus, the deletion from the full Epo-bp by trypsin digestion completely eliminated the antibody recognition, as verified by Western blotting. This indicates that intact Epo-bp may be required to be effective in Epo-ligand binding.
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Fig. 4. Effect of trypsin concentration (10, 20, 30, 50, 100 µg and 2 mg) per 5 µg of Epo-bp in total volume of 200 ml in PBS, pH 6.7, at 37°C for 3 or 6 hr to find an effective range of trypsin digestion for Epo-bp. This was verified on a 12.5% SDS-PAGE gel. Epo-bp was cleaved effectively at 20 µg or higher concentration of trypsin. The excess of trypsin is evident at 2 mg as a 23.2 kDa protein band. The cleaved Epo-bp is shown as a 20 kDa protein. Lane 1: standard molecular weight markers; lanes 2–7: 10, 20 30, 50, 100 µg and 2 mg, trypsin, respectively, at 37°C for 3 hr; lanes 8–13: the same concentrations at 37°C for 6 hr digestion.
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Figs. 5-1 to 5-3 illustrate the fluorescein-labeled receptor sites of bone marrow progenitor cells where the bound receptor sites are visualized. Fluorescein-labeled pre-immune Fab-treated cells did not show any receptor binding activity; the same was seen on the negative control cells (Fig. 5-1), while receptor sites were labeled in various blood cells (Figs. 5-2 and 5-3). The binding sites were located among the megakaryocytes, erythroblasts and normoblasts, as well as myeloblasts. This is the first report that visualizes the ligand-binding sites in various blood cells and identifies the cell types where the Epo-receptor is located.
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Fig. 5-1 shows control bone marrow cells with or without fluorescein label. A–C: control cells (A and C: 100x; B: 400x) and D–F: pre-immune serum treated cells as positive controls (1000x). Note that the pre-immune serum-treated sample (F) did not show any receptor binding activity, with the same result shown in negative control cells (C).
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Fig. 5-2 shows fluorescein-labeled receptor sites of bone marrow progenitor cells. The receptor was labeled by the following method: Bone marrow cells were washed 3 times in PBS and dispensed in 1–3 x 103 cells per well for the control and samples and centrifuged. Supernatants were removed and 100 µl of purified Fab fractionated fluorescein-conjugated anti-Epo-bp antibodies was added. The mixture was incubated on ice for 30 min. The cells were washed 3 times by adding 400 µl of buffer containing 1% FCS and 0.01% NaN3 in PBS to each sample and centrifuged at 200 x G for 2 min. The packed cells were resuspended in a total volume of 50 µl of PBS and analyzed under an inverted fluorescence microscope. Megakaryocytes = M; basophil erythroblast = asterisk; polychromatophil erythroblast = solid arrowhead; myeloblast = open arrowhead; normoblast = open arrow (magnification: A and B: 400x; C–F: 1000x).
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Fig. 5-3 shows the cells in the same preparation as Fig. 5-2 with a different site set-up. Megakaryocytes = M; basophil erythroblast = asterisk; poly-chromatophil erythroblast = solid arrowhead; normoblast = open arrow (magnification: 1000x).
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The levels of Epo, Epo-bp, and antibodies against Epo and Epo-bp were measured in human serum and plasma samples by use of an enzyme immunoassay (EIA) developed in our laboratories (Fig. 6). Optical density (OD) of each measurement is presented as the mean ± SE of 8–14 individual samples, each analyzed in duplicate. The OD values in Fig. 6 were calculated by subtracting the OD value of the blanks from the OD of each sample. Serum and plasma Epo and Epo-bp OD values were similar: 0.308 ± 0.026 and 0289 ± 0.022 in serum, and 0.289 ± 0.028 and 0.299 ± 0.015 in plasma, respectively. The plasma level of Epo-bp was significantly lower than those of the other 3 antibody categories: 0.058 ± 0.008 serum Epo, 0.052 ± 0.006 serum Epo-bp, 0.054 ± 0.013 plasma Epo, and 0.031 ± 0.004 plasma Epo-bp (p <0.025). The Epo and Epo-bp values were converted with known Epo concentrations prepared as controls in the same plate. The converted values in mU/ml were 25.4 ± 2.17 serum Epo, 24.2 ± 2.35 plasma Epo, 24.2 ± 1.84 serum Epo-bp, 25.0 ± 1.26 plasma Epo-bp. These assay methods are simple, more sensitive, and more precise than the conventional radioimmunoassay. Moreover, the reagents used in our assays are more environmentally friendly than the radioactive or other toxic chemicals used by conventional methods.
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Fig. 6. Graph of optical density of erythropoietin (Epo), Epo-binding protein (Epo-bp), and their antibodies in serum or plasma samples. Error bars represent standard error, SE.
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Discussion
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In 1957, Jacobson et al [22] reported the kidney as a major organ for Epo production. In 1972, Fried [23] discovered the liver as a source of extrarenal Epo production. Since then, many studies have been carried out to understand the biological and physiological aspects of Epo in response to anemia and hypoxia. Purification of Epo from human urine followed [24]. Although its exact role in erythroid differentiation has not yet been elucidated, effects of Epo on DNA and RNA synthesis by marrow erythroid cells have been found [1,25–28].
The expression vector pJYL26 in Fig. 1 was engineered to prepare a pure human Epo-receptor protein (Epo-bp), which is effectively bound to ligand. Since blood cell progenitors respond to Epo by synthesizing hemoglobin, some serine proteases, such as trypsin, act as coordinating agents in a broad range of biological activities and have limited substrate specificity, as required by many physiological functions. Therefore, further experiments were carried out to find a minimum size of Epo-bp in ligand binding. However, tryptic removal of about 30 amino acids from the intact Epo-bp eliminates antibody recognition and causes it no longer to be active in ligand-binding [18]. Lacking a part of the WS X WS motif, with the replacement of 1 of the 2 tryptophan residues in the motif by Gly, or all of the WS X WS, abolished ligand binding to Epo receptor [4]. Thus, this may explain the results of our trypsin digestion experiments.
Our Epobpand its antibodies are characterized by the specific binding of Epo and its antibodies in nmol concentration. With this specificity, the binding sites of blood progenitor cells were detected using Epo-bp and Epo-bp. A major achievement of the current study was the demonstrtion that all human progenitor blood cells have Epo receptors (Figs. 5-1 to 5-3).
The Epo receptor has been cloned [29]. However, we do not yet know what the biophysiological mechanisms of Epo or the second messenger system involved in the interactions of Epo-Epo receptor in ligand binding activities and its subsequent processes are. The cloning of pJYL26 and purification of the pure human Epo-bp may enable us to delineate complete structures of the Epo receptor. In turn, this may allow us to examine the factors involved in ligand binding, as well as the identification of other factors in the regulation of progenitor cell differentiation and proliferating mechanisms. The methods presented in this report will help identify which system, ligand or receptor, is deficient in progenitor cell growth and results in various clinical disorders. Furthermore, Epo-bp and Epo-bp will help in identifying and quantifying Epo, Epo-receptor, and their respective antibodies.
There have been discrepancies in the correlation between the Epo responsiveness of a cell type and the affinities of the receptors to the expression. However, the studies have only involved materials with the recombinant receptors or recombinant proteins [30], and pure human Epo-receptor protein and its antibody have not been available. The invented products may be important in studies of the receptor structure and binding mechanism, as well as providing possible solutions to hematopoietic malignancy and cardiovascular disorders, such as hypertension.
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Conclusion
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Human erythropoietin-binding protein (Epo-bp) was purified from human Epo-receptor cDNA PCR inserted recombinant vector, pJYL26, and anti-Epo-bp antibodies ( Epo-bp) were developed. Binding of Epo to Epo-bp was specific and appeared to require an intact extracellular domain. Our Epo-bp is the first purified human Epo receptor protein that has a specific ligand-binding affinity [18]. The new products may help to elucidate Epo-receptor structures and the mechanisms for the interactions of Epo-Epo receptor ligand binding, as well as progenitor cell differentiation and proliferation. They may also prove useful as clinical tools for differential diagnosis.
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Acknowledgements
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The author thanks Drs. Franz Halberg and Erhard Haus for their material support and their invaluable review and comments. The author also thanks Dr. John Winkelmann for reagents and Mr. John S. Lee for editorial assistance.
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Abstract
Introduction
