ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 6, pp. 653–657. © Pleiades Publishing, Ltd., 2017. Original Russian Text © A.A. Mozhaev, T.N. Erokhina, O.V. Serova, I.E. Deyev, A.G. Petrenko, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 6, pp. 631–636.
Production and Immunochemical Characterization of Monoclonal Antibody to IRR Ectodomain A. A. Mozhaev, T. N. Erokhina, O. V. Serova, I. E. Deyev, and A. G. Petrenko1 Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia Received December 9, 2016; in final form, June 1, 2017
Abstract⎯The insulin receptor-related receptor (IRR) is the only known metabotropic sensor of extracellular alkaline medium involved in the regulation of the acid–base balance in the body. IRR is expressed in certain cell populations of the kidney, stomach, and pancreas that can come into contact with the extracellular fluids with alkaline pH. To study IRR structure and function, we obtained a stable hybridoma cell-line-producing antibody to the extracellular portion of the receptor. The monoclonal antibody isolated from ascitic fluids showed a positive reaction with the antigen in the ELISA test. The minimum working concentration of antibodies was 12.5 ng/mL. The ability of the antibodies to specifically recognize the purified ectodomain of IRR and the fulllength receptor was confirmed by western blot, immunoprecipitation, and immunocytochemistry. Keywords: monoclonal antibodies, insulin receptor-related receptor DOI: 10.1134/S1068162017060097
INTRODUCTION The insulin receptor-related receptor (IRR) belongs to the family of insulin receptors. In addition to IRR, the family comprises insulin receptor and insulin-like growth factor receptors. The three receptors are highly homologous and, supposedly, have closely related functionality [1]. Despite the long history of studies over 30 years on their structure and action, many details of their functioning remain unclear. Today, the structure of the insulin receptor extracellular domain has been determined; also, the extracellular domain of the insulin receptor in complex with insulin has been crystallized [1, 2]. The insulin receptor extracellular domain was stabilized in a complex with four Fab fragments of monoclonal antibodies, which eventually resulted in a crystal for X-ray crystallography analysis [1]. Nevertheless, until now it has not been clear which conformational changes occur in the receptors upon their activation—four different models are considered today [3]. Further, the mechanism of autophosphorylation of the intracellular parts is not clear. Previously, we have demonstrated that IRR is a sensor of extracellular alkaline medium and it is involved in the regulation of the acid–base balance in the body [4]. Mice with the IRR gene knockout exhibited impaired behavior [5]. IRR is activated upon alkalization of the extracellular medium (pH > 7.9) with the 50%-effect point at approximately pH 8.0, and is saturated at pH over 9.0. The receptor is expressed in certain cell populations of the kidney, 1 Corresponding author: e-mail:
[email protected].
stomach, and spleen that are in contact with extracellular alkaline fluids [6]. Fragments involved in pH sensing by the receptor were determined using chimeric constructs and site-directed mutagenesis [7–10]. Activation of the IRR was demonstrated to trigger phosphorylation of signaling intracellular proteins insulin receptor substrate 1 (IRS-1) and protein kinase B (Akt); it is also able to induce cytoskeletal rearrangements in cells [4, 11]. Study of the IRR structure, understanding the activation by alkali, and elucidation of its physiological importance in the body are of great interest and can lead to an explanation of the activation of homologous receptor tyrosine kinases of the insulin receptor family. The aim of the work was to produce and characterize monoclonal antibodies (mAb) against extracellular part (ectodomain) of IRR to study its structure and functions. An undoubted advantage of mAbs is the highly reproducible results since, in contrast to polyclonal antibodies, mAbs are characterized by stable immunochemical properties. RESULTS AND DISCUSSION The recombinant ectodomain of the IRR receptor expressed in eukaryotic cells was used as an antigen to produce mAbs. The choice is explained by the fact that until now no high-affinity polyclonal antibodies to the extracellular part of the receptor, which would have been used for experiments in vivo or in intact cell cultures, could be produced. Earlier, we demonstrated that the IRR ectodomain is responsible for the pH
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Fig. 1. Titration curve of immobilized IRR with 4D5 mAb.
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100 Fig. 2. Western blot of 4D5 mAbs with denatured IRR. Insulin receptor, IR, and insulin-like growth factor 1 receptor, IGF1R. The arrow indicates alpha-subunit of the receptor. On the left, marker protein weight (kDa) is indicated.
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Fig. 3. Immunoprecipitation of IRR and IR using 4D5 mAbs. Arrows indicate beta-subunits of IRR and IR. On the left, marker protein weight (kDa) is indicated. Lysates of non-transfected HEK293 cells, C; cells expressing the IRR receptor, IRR; cells expressing the IR receptor, IR. IP designates samples obtained during immunoprecipitation using the 4D5 mAbs and anti-myc tag antibodies (4D5 and a-myc, respectively) from cells expressing the IRR (PI IRR) and IR (IP IR) receptors.
sensing. Therefore, it should be possible to use mAbs against the ectodomain in functional tests. To produce the recombinant ectodomain of the IRR receptor with a myc tag, we used Glutamine Synthetase Xceed Gene Expression system in CHO-K1 cells (Lonza) allowing for higher recombinant protein yield compared to common transfection. The target protein was secreted into a cultural serum-free medium, where it was isolated from using a combination of ion exchange and size exclusion chromatography, as described in the Experimental. The isolated protein was identified with western blotting using the previously obtained rabbit polyclonal anti-IRR antibodies [4]. Purity of the protein was evaluated using PAGE with Coomassie staining. The protein yield was 0.5 mg/L cultural fluid; protein purity was over 90%. Structural identity of the protein was additionally confirmed by mass spectrometry. Anti-IRR ectodomain mAbs were obtained in mice according to a classical method described in detail in the Experimental. According to PAGE, purity of mAbs isolated from ascitic fluid was above 90%. The yield was 6 mg/mL ascites. A stable hybridoma 4D5 line producing specific mAbs against the extracellular part of the IRR receptor tyrosine kinase was obtained. 4D5 mAbs isolated from ascitic fluid demonstrated a positive reaction to antigen in the indirect solid-phase ELISA. Minimal concentration of mAbs necessary for a signal exceeding twice the background was approximately 12.5 ng/mL. The titration curve is presented in Fig. 1. The values evidence the high affinity of interaction of 4D5 mAbs with the antigen. Then, 4D5 mAbs were assayed in western blotting (Fig. 2). Samples of HEK293 cells transfected with IRR receptor, as well as cells transfected with insulin receptor and insulin-like growth factor receptor, were subjected to SDS-PAGE and transferred onto nitrocellulose. Samples of nontransfected cells were used as control. Staining of the blot using 4D5 mAbs revealed their specific interaction with the alpha-subunit of the IRR, but not the other receptors. To verify antibody binding with native protein, immunoprecipitation and immunocytochemistry experiments were conducted. Cells transfected with IRR were lysed with a nonionic detergent-containing buffer; then, antibodies were added and incubated with protein G conjugated Sepharose. The adsorbed proteins were eluted and analyzed by western blotting using polyclonal anti-IRR beta-subunit antibodies. Staining revealed interaction of monoclonal antibodies with native full-length IRR (Fig. 3). Transfected cells were also fixed and directly incubated with monoclonal antibodies and then stained with secondary fluorescent antibodies. Specific staining of IRR transfected cells was observed (Fig. 4). Earlier, monoclonal antibodies produced against the chimeric protein of the IRR and insulin receptor were described. However, their interaction with full-
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Fig. 4. Immunocytochemistry. 4D5 mAb-stained HEK293 cells (a) transfected and (b) not transfected with IRR. Red filter and 570-nm emission were used. (c) HEK293 cells in transmitted light. Scale bar is 10 μm.
size native IRR was not studied [12]. We provide evidence that 4D5 mAbs recognize the full-length IRR, but not the related receptors, and are able to bind specifically and with high affinity with its native and denatured forms. An undoubted advantage of the mAbs is the high reproducibility of study results, since in contrast to polyclonal antibodies, they have stable immunochemical properties. Further, we plan to verify the reactivity of mAbs in immunohistochemical analysis of mouse tissue sections obtained from wildtype and IRR knock out animals, as well as study their effect on the receptor activation and elucidate the region of specific binding with the antigen. EXPERIMENTAL The following reagents were used in the work: Dulbecco’s modified Eagle medium (DMEM, Gibco, United States), selective medium supplemented with 100× hypoxanthine, aminopterin, and thymidine (DMEM-HAT, Gibco), Freund’s complete adjuvant (MP Biomedicals), PEG1500 (Sigma) solution, fetal bovine serum (Gibco), dimethyl sulfoxide, G protein conjugated Sepharose (Amersham Biosciences), 96well ELISA plates (Costar, United States), 24- and 96well cell culture plates (TPP, Switzerland), horseradish peroxidase-conjugated rabbit anti-mouse IgG (DAKO, Denmark), bovine serum albumin (BSA, Sigma), SuperSignal West Pico (Thermo Fischer, United States) luminescent substrate, nitrocellulose membrane, Glycergel Mounting Medium (DAKO), Sp2/0 murine myeloma cells, goat antimouse CY3 antibodies (Jackson ImmunoResearch Laboratories), Tween 20, polyacrylamide (Amresco), phosphatebuffered saline, pH 7.4 (PBS), alkaline phosphatase substrate buffer, Triton X-100, formaldehyde, and 50 mM Tris-HCl, pH 8.0, 150 mM HCl, 0.05% Tween 20 (TBST). Protein expression and purification. According to the manufacturer’s protocol, CHO-K1 cell line which constitutively expressed IRR ectodomain with C-terminal myc tag excreted in the extracellular medium was produced using the Glutamine Synthetase Xceed Gene Expression (Lonza, Switzerland) system. The RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
producer line was cultured in DMEM supplemented with 10% FBS (Gibco), 100 μg/mL penicillin, 100 μg/mL streptomycin, 1× GS supplement (Sigma), 40 μM MSX (Sigma) in T175 (Eppendorf) cell culture flasks till the monolayer was formed. Then, cultural fluid was substituted with a serum-free medium HybriS-1-CHO (PanEko) supplemented with alanyl-glutamine. Cells were incubated at 37°C, 5% CO2. After 2–3 days, cultural fluid was collected and a fresh portion of serum-free medium was added. Protein secreted into the medium was isolated from cultural fluid by ion exchange chromatography on a DEAE-Toyopearl 650M (TSOH, Japan) resin in a C16/20 (GE, United States) column using linear NaCl gradient (from 0 to 1 M, total volume of 40 mL) in 20 mM Tris-HCl, pH 8.0. Fractions of 2 mL were analyzed with PAGE and western blotting using polyclonal anti-IRR antibodies [4]. Fractions containing the target protein were joined, concentrated using the Amicon Ultra-4 (Millipore, Germany) filter, and applied onto a Superdex 200 Increase 10/300 GL (GE) high-performance size exclusion chromatography column equilibrated with 50 mM Na2HPO4, 150 mM NaCl, pH 7.0, at a flow rate of 0.5 mL/min. Fractions of 2 mL were collected and analyzed with PAGE. Isolated protein was identified with a MALDITOF mass spectrometry analysis. Final yield of the protein after all purification procedures was 0.2 mg/L of initial cultural fluid. Immunization of BALB/c mice was performed three times in two-week intervals. For this purpose, 0.2 mL of emulsion containing equal volumes of Freund’s complete adjuvant and 100 μg purified IRR dissolved in PBS was administered to mice via intraperitoneal injection. Then, the same amount of antigen was administered to mice twice, with two-week intervals, in Freund’s incomplete adjuvant. One week after the last immunization, blood was collected from the tail vein to determine the antibody titer in blood serum. Three days prior to fusion of immune lymphocytes with murine myeloma cells, 100 μg purified IRR ectodomain were administered to mice intraperitoneVol. 43
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ally in PBS without an adjuvant (buster immunization). Establishment of the mAb-producing hybridoma. Splenocytes of immune mice were hybridized with Sp2/0 myeloma cells in 45% polyethylene glycol solution supplemented with 10% dimethyl sulfoxide according to Kohler and Milstein [13]. Cells were spread over the 24-well plates. Selective DMEM-HAT medium supplemented with 15% fetal bovine serum was used for hybridoma cultivation in the presence of murine peritoneal macrophages as feeder cells [14]. Cloning and recloning of hybridoma was performed with the limiting dilution method. Positive clones were grown as ascites in BALB/c mice. Hybridomas were stored in liquid nitrogen containers in cryoprotector medium prepared of fetal bovine serum (85%) and dimethyl sulfoxide (15%). Monoclonal antibodies were isolated from ascitic fluid by precipitation with ammonium sulfate followed by affinity chromatography on a Fast Flow G protein-conjugated Sepharose (Sigma). Indirect solid-phase enzyme-linked immunoassay. IRR ectodomain at the concentration of 5 μg/mL was introduced into ELISA plates in PBS and left to adsorb overnight at +4°С. Non-specific binding was blocked with 5% BSA solution in PBS for 1 h. Then, study samples containing antibodies were introduced into wells and incubated for 1 h at room temperature. After incubation, the plates were washed with PBS supplemented with 0.1% Tween 20; then, the horseradish peroxidase-conjugated rabbit antimouse IgG antibodies were added and were incubated for 1 h. Sodium 4-nitrophenylphosphate (1 mg/mL) solution in substrate buffer (100 mM Tris-HCl, pH 9.5, 5 mM MgCl2, 100 mM NaCl) was used as a substrate. Optical absorbance at 405 nm was measured with Labsystem Multiskan (United States) spectrophotometer. PAGE and western blotting. Electrophoresis in 8% PAA gel in the presence of sodium dodecyl sulfate followed by western blotting was performed according to a standard protocol as described in [15, 16]. Proteins were transferred onto a nitrocellulose membrane during 1.5 h at 250 mA. Nonspecific protein sorption was prevented with membrane incubation in 1% BSA solution in TBST for 1 h. Then, the membrane was incubated with primary antibodies. To identify the isolated protein, rabbit polyclonal anti-IRR antibodies were used. The antibodies were obtained against the N-terminal fragment of murine IRR ectodomain (amino acid residues 539–686) [4]. To verify the reactivity, 4D5 mAbs in TBST were added at 1 : 3000 dilution and incubated for 1 h. Then, primary antibodies were washed off and secondary horseradish peroxidase-conjugated rabbit antimouse IgG antibodies were added. Bands were visualized by treatment with a SuperSignal West Pico luminescent substrate on a Fusion Solo (Vilber Lourmat) equipment.
HEK293 cells were grown in DMEM containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine under standard conditions (37°С, 5% CO2). Cells were transfected with a pcDNA3.1 plasmid encoding IRR with an HA tag at the C-terminus [4] using the Unifectin-56 (UnifectGroup). After transfection, cells were grown for two days and then used for western blotting and immunocytochemistry. Immunoprecipitation. HEK293 cells transfected with plasmids encoding IRR and IR [4] were washed with PBS and lysed in a 20 mM Tris-HCl buffer, pH 8, containing 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged at 15000 g for 30 min at +4°C. To supernatant, 5 μg 4D5 mAbs were added. Lysates were incubated with antibodies for 2 h at +4°C; then, 20 μL G protein-conjugated Sepharose pretreated with lysing buffer was added to each sample and incubated overnight at +4°C. The adsorbent was washed three times with a lysing buffer. Elution of bound proteins was performed in the PAGE sample buffer. Eluates were analyzed with western blotting using anti-IRR C-terminus antibodies. Immunocytochemistry. Transfected HEK293 cells were fixed in 4% formaldehyde solution in PBS for 20 min at room temperature. After permeabilization with 0.1% Triton X-100 solution and washing with PBS, cells were incubated with 1% BSA for 1 h and with primary antibodies for 1 h at room temperature. Then, samples were washed thrice with PBS and incubated with secondary goat antimouse CY3 antibodies in 1 : 3000 dilution for 1 h at room temperature. After washing, cells were fixed on slides with the Glycergel Mounting Medium. The samples thus prepared were studied using the Olympus IX70 microscope equipped with an immersion lens with 60-fold magnification. ACKNOWLEDGMENTS The work was supported by the Russian Science Foundation (project no. 14-14-01195). REFERENCES 1. McKern, N.M., Lawrence, M.C., Streltsov, V.A., Lou, M.Z., Adams, T.E., Lovrecz, G.O., Elleman, T.C., Richards, K.M., Bentley, J.D., Pilling, P.A., Hoyne, P.A., Cartledge, K.A., Pham, T.M., Lewis, J.L., Sankovich, S.E., Stoichevska, V., Silva, E.D., Robinson, C.P., Frenkel, M.J., Sparrow, L.G., Fernley, R.T., Epa, V.C., and Ward, C.W., Nature, 2006, vol. 443, pp. 218–221. 2. Menting, J.G., Whittaker, J., Margetts, M.B., Whittaker, L.J., Kong, G.K., Smith, B.J., Watson, C.J., Zakova, L., Kletvikova, E., Jiracek, J., et al., Nature, 2013, vol. 493, pp. 241–245. 3. De Meyts, P., The Insulin Receptor and Its Signal Transduction Network, in Endotext, De Groot, L.J., et al., Eds., South Dartmouth (MA), 2016. 4. Deyev, I.E., Sohet, F., Vassilenko, K.P., Serova, O.V., Popova, N.V., Zozulya, S.A., Burova, E.B., Houillier, P.,
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8. Deyev, I.E., Chachina, N.A., Shayahmetova, D.M., Serova, O.V., and Petrenko, A.G., Biochimie, 2015, vol. 111, pp. 1–9.
10. Deyev, I.E., Popova, N.V., and Petrenko, A.G., Acta Naturae, 2015, vol. 7, pp. 80–86. 11. Deyev, I.E., Popova, N.V., Serova, O.V., Zhenilo, S.V., Regoli, M., Bertelli, E., and Petrenko, A.G., Biochimie, 2017, vol. 138, pp. 62–69. 12. Kovacina, K.S. and Roth, R.A., J. Biol. Chem., 1995, vol. 270, pp. 1881–1887. 13. Kohler, G. and Milstein, C., Nature, 1975, vol. 256, pp. 495–497. 14. Erokhina, T.N., Zinovkin, R.A., Vitushkina, M.V., Jelkmann, W., and Agranovsky, A.A., J. Gen. Virol., 2000, vol. 81, pp. 597–603. 15. Deyev, I.E. and Petrenko, A.G., Biochimie, 2010, vol. 92, pp. 418–422. 16. Krasnoperov, V., Deyev, I.E., Serova, O.V., Xu, C., Lu, Y., Buryanovsky, L., Gabibov, A.G., Neubert, T.A., and Petrenko, A.G., Biochemistry, 2009, vol. 48, pp. 3230– 3238.
9. Popova, N.V., Deyev, I.E., and Petrenko, A.G., Dokl. Biochem. Biophys., 2013, vol. 450, pp. 160–163.
Translated by N. Onishchenko
Rzhevsky, D.I., Berchatova, A.A., Murashev, A.N., Chugunov, A.O., Efremov, R.G., Nikol’sky, N.N., Bertelli, E., Eladari, D., and Petrenko, A.G., Cell Metab., 2011, vol. 13, pp. 679–689. 5. Zubkov, E.A., Chachina N.A., Shayakhmetova D.M., Mozhaev A.A., Chekhonin V.P., Petrenko A.G, Zh. Vyssh. Nerv. Deyat., 2017, vol. 67, pp. 106–112. 6. Petrenko, A.G., Zozulya, S.A., Deyev, I.E., and Eladari, D., Biochim. Biophys. Acta, 2013, vol. 1834, pp. 2170–2175. 7. Deyev, I.E., Mitrofanova, A.V., Zhevlenev, E.S., Radionov, N., Berchatova, A.A., Popova, N.V., Serova, O.V., and Petrenko, A.G., J. Biol. Chem., 2013, vol. 288, pp. 33884–33893.
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