ARTICLE pubs.acs.org/bc
Directed Covalent Immobilization of Fluorescently Labeled Cytokines Tobias Recker,† Daniel Haamann,‡ Anne Schmitt,† Andrea K€uster,† Doris Klee,‡ Stefan Barth,§,|| and Gerhard M€uller-Newen*,† †
Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Aachen, Germany ITMC/DWI, RWTH Aachen University, Aachen, Germany § Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, Aachen, Germany Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany
)
‡
ABSTRACT: Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokinereceptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a derivative of O6-alkylguanine-DNA alkyltransferase that couples itself covalently to benzylguanine. Bioactivities of the SNAP-YFP-cytokines were shown to be comparable with the nontagged cytokines. Efficient coupling of SNAP-YFP-cytokines to benzylguanine-modified beads was demonstrated by flow cytometry. The fact that enterokinase treatment released most of the fluorescence from the beads is indicative for directed coupling and only marginal adsorptive binding. Cellular responses to SNAP-YFP-cytokine beads were analyzed in cellular lysates and by confocal microscopy indicating that the directionally immobilized cytokines are fully signaling competent with respect to the activation of ERK and STAT3. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labeled proteins including the convenient and reliable control of coupling efficiency and orientation.
1. INTRODUCTION Cytokines are a large and heterogeneous group of proteins consisting of interleukins, interferons and growth factors.1 These important and versatile mediators of intercellular communication are involved in virtually all physiological processes in the developing and adult organism. Depending on the individual cytokine and the cellular context, these proteins stimulate proliferation or differentiation of cells and may act in a pro- or anti-inflammatory manner on immune responses. Because of these properties, cytokines are promising molecules to modulate the response of the body to biomaterials. Therefore, immobilization of cytokines on the surface of biomaterials to direct the fate of adhered cells, augment wound healing, and control inflammatory responses is an active field of research.24 Cytokines bind specifically to cytokine receptors which consist of two or more subunits. These subunits can be different proteins (heterooligomeric receptors) or two or more copies of the same protein (homooligomeric receptors).5 Shared subunits are known, which are used by different cytokines.6 Gp130 is the shared receptor subunit of the IL-6 family of cytokines.7 Two IL-6 molecules assemble two IL-6 receptor R-subunits (IL-6RR) r 2011 American Chemical Society
and two gp130 molecules leading to the formation of a hexameric receptor complex.8 Other cytokines of this family such as oncostatin M (OSM) and leukemia inhibitory factor (LIF) bind to a heterodimer of gp130 with the OSM receptor (OSMR) or LIF receptor (LIFR), respectively, resulting in a heterotrimeric receptor complex.9 In all cases, large surface areas of the cytokine must be accessible for the interaction with the receptor subunits to allow receptor complex formation. Therefore, random adsorptive or chemical immobilization of cytokines will lead to a large fraction of inactive protein. One could even imagine the generation of partial agonists by inadequate immobilization of cytokines leading to skewed signaling. Signal transduction downstream of gp130 always involves the transcription factor STAT3 and the RAS/RAF/ERK cascade.7 Depending on the cytokine, other pathways may also be activated. The hallmark of STAT3 activation is the phosphorylation of tyrosine residue 705 (Y705) by receptor associated Janus Received: February 8, 2011 Revised: April 27, 2011 Published: May 02, 2011 1210
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Bioconjugate Chemistry kinases (JAKs) and subsequent nuclear accumulation of the transcription factor.10 ERK activation involves phosphorylation of the TEY-motif by the upstream kinase MEK. The kinetics of ERK activation determines the cellular response.11 It is still a matter of debate as to what extent these key signaling events are initiated at the plasma membrane or after internalization of the receptor complex at signaling endosomes.12 This issue is of considerable importance as soon as cytokines are immobilized on surfaces. Cytokines bound to surfaces cannot be internalized and therefore cannot signal from endosomes, raising the possibility that signaling from soluble and immobilized cytokines may differ.13,14 Furthermore, endocytosis of receptor complexes is a way to desensitize cells from further stimulation.15 However, for gp130 it is known that the signaling cascade is downregulated by a classic feedback mechanism: activated STAT3 induces the suppressor of cytokine signaling 3 (SOCS3) protein expression.16 Then, SOCS3 blocks further STAT3 activation at the gp130 receptor complex by mechanisms which are incompletely understood. Therefore, it will be interesting to see as to what extent immobilization of the cytokine interferes with the kinetics of the signaling events. Prerequisite for these investigations is a reliable technique to covalently immobilize cytokines on surfaces in a bioactive form. We used cytokines of the IL-6-family to develop a new strategy for cytokine immobilization on surfaces that guarantees (i) sitedirected covalent coupling through a tag that does not involve the cytokine itself, (ii) straightforward analysis of coupling efficiency by fluorescence detection, and (iii) proof of orientation of coupling by protease cleavage. Cytokines immobilized by this strategy are useful tools for basic research on cytokine signal transduction and the improvement of biomaterials.
2. EXPERIMENTAL PROCEDURES 2.1. Chemicals and Antibodies. PEGylated aminobenzylguanine (BG-PEG-NH2) was provided by New England Biolabs (Ipswich, USA). Magnetic micromer-M PEG-COOH beads with a diameter of 3 μm were produced by Micromod (Rostock, Germany). Primary antibodies were purchased from the following companies: GFP, phosphorylated STAT3, and phosphorylated ERK1&2 (Rockland, Gilbertsville, USA), STAT3 and ERK1&2 (Santa Cruz, Santa Cruz, USA), IL-6, OSM, PEconjugated IL-6 and PE-conjugated OSM (R&D, Minneapolis, USA), and SNAP (Open Biosystems, Huntsville, USA). Secondary antibodies were purchased from DAKO (Glostrup, Denmark). Enterokinase-kit was obtained from EMD Chemicals (Gibbstown, USA). Eukaryotic cell-culture media and antibiotics (Pen Strep) were purchased from Invitrogen (Carlsbad, USA). 2.2. Recombinant Plasmids and Proteins. The expression vector pET-17b from EMD Chemicals was used for the production of YFP-OSM and YFP-IL-6 in E. coli. The constructs consist of a hexahistidine (his)-tag, followed by a enterokinase recognition site (DDDDK), YFP, and the respective mature human cytokine. SNAP-YFP (fusion protein without cytokine) was also produced in E. coli. SNAP-YFP-cytokines were cleaved in E. coli and therefore produced in mammalian cells. The pcDNA5/ FRT/TO vector of the Flp-In system (Invitrogen) was used for the expression of SNAP-YFP-OSM and SNAP-YFP-IL-6 in HEK293 cells. The constructs encode the signal sequence of IL-6 for secretion, followed by a his-tag, a SNAP-tag (New England Biolabs), the enterokinase recognition site, the enhanced yellow fluorescent protein (YFP), and finally the respective mature
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human cytokine. The YFP cDNA originated from the pEYFPN1-vector (Clontech, Mountain View, USA). 2.3. Protein Expression. Flp-In T-Rex HEK293 were stably transfected with expression vectors encoding SNAP-YFP-cytokines using the T-Rex system (Invitrogen). For protein expression, cells were cultured in serum-free DMEM medium in a water-saturated atmosphere containing 5% CO2 at 37 °C. Protein expression was induced by the addition of doxycycline (50 ng/mL). 72 hours after induction, the supernatant was collected and concentrated 20-fold by ultrafiltration using a Vivaspin 20 device (Sartorius Stedim biotech, Aubagne, France). For further characterization, SNAPYFP-cytokines were purified using a Ni-NTA Spin-column according to the instructions of the manufacturer (Qiagen, Hilden, Germany). The different fractions were analyzed by SDSPAGE, followed by Coomassie-staining or Western blotting. SNAP-YFP cytokines were quantified by ELISA using antibodies against the SNAP and YFP-moieties. SNAP-YFP served as a standard. YFP-cytokines and SNAP-YFP were expressed in E. coli (strain BL-21 (DE3) pLysS) by the use of the pET-system.17 Bacteria were grown at 37 °C in LB medium in the presence of 100 mM ZnCl2 to an OD600 of 0.60.8. After the addition of IPTG (0.4 mM) and DTT (1 mM), the culture was incubated at 25 °C for 20 h. Bacteria were sedimented by centrifugation, the pellet shock-frozen and resuspended in lysis-buffer (20 mM phosphate buffer, pH 8.0, containing 500 mM NaCl). Lysozyme (1 mg/mL), benzonase (25 U/mL) and Triton-X100 (1%) were added to complete the lysis of bacteria. Lysates were shaken at 4 °C for 4 h and the recombinant proteins purified as described above. To determine the concentration of SNAP-YFP in the eluate, the absorbance at 514 nm was measured (NanoDrop, NanoDrop products, Wilmington, USA; extinction coefficient of YFP = 83,400 M1 cm1). 2.4. Immobilization of SNAP-YFP-Cytokines. For immobilization, 50 μL of a suspension of micromer-M PEG-COOH beads (3.4 106 beads/μL) was functionalized with 50 μL of BG-PEG-NH2 (2 mg/mL in 10% DMSO and 90% PBS pH 7.4) through EDC/NHS-chemistry:18 At first, beads were shortly washed twice in MES-buffer (25 mM, pH 5.0) and then incubated (30 min, room temperature) in a freshly prepared mixture of 25 μL EDC solution (50 mg/mL) and 25 μL of NHS solution (50 mg/mL) in MES-buffer (25 mM, pH 5.0). After incubation, the supernatant was removed, and the beads were washed again with MES-buffer for 4 min. Then, 50 μL of BG solution was added, and the suspension was incubated for 16 h (4 °C) with slow tilt rotation. After washing four times with 100 μL of 0.05 mM TrisHCl, pH 7.4, 10 μL of the bead suspension (1.6 106 beads/μL) was incubated for 16 h with 50 μL of purified SNAP-YFPcytokine (1 μM) or concentrated supernatants of SNAP-YFPcytokine producing cells. Incubation of beads with YFP-cytokine (no SNAP-tag) and incubation of unmodified beads (no benzylguanine on the surface) with SNAP-YFP-cytokine served as controls. Beads were washed with 100 μL of phosphatebuffer (0.1 M, pH 8.0) containing 2% BSA. After washing, beads were analyzed by flow cytometry or by confocal microscopy. SNAP-YFP-cytokine beads were stored at 80 °C in phosphate buffer (0.1 M, pH 8.0) containing 2% BSA. The concentrations of SNAP-YFP-IL-6 in the coupling buffer before and after coupling were determined with a previously described ELISA19 using a biotinylated GFP antibody (Rockland). 2.5. Analysis of Beads by Flow Cytometry and Confocal Microscopy. Three microliters of a suspension of SNAP-YFPcytokine beads (1.6 105 beads/μL) and the control beads were mixed with 100 μL of BD FACSFlow Sheath Fluid 1211
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Bioconjugate Chemistry (BD Bioscience, Franklin Lakes, USA). Beads were analyzed by flow cytometry (FACS Canto II, BD systems) using the appropriate adjustments for YFP detection. For cytokine detection, 7.5 μL of a suspension of SNAP-YFP-cytokine beads were incubated for 30 min in 25 μL of 0.1 M phosphate buffer (pH 8.0) containing 1 μL of a phycoerythrin (PE)-conjugated cytokine antibody (or without PE-antibody as a control). After incubation, 10 μL of bead suspension was mixed with 100 μL of BD FACSFlow Sheath Fluid and analyzed as described above detecting PE and YFP fluorescence. For confocal microscopy, 2 μL of a suspension of SNAP-YFPcytokine beads (1.6 105 beads/μL) was mixed with 5 μL of Shandon Immu-Mount (Thermo Fisher Scientific Inc., Waltham, USA) on a microscope slide and covered with a coverslip. After a short drying period, probes were analyzed using a Zeiss LSM 510 confocal microscope. 2.6. Stability of Coupling and Enterokinase Cleavage. Fifteen microliters of a suspension of SNAP-YFP-cytokine beads was incubated in 500 μL of serum free medium (DMEM-F:12 including Pen Strep) and incubated for different times at 37 °C. Then, the medium was removed by centrifugation, and beads were washed with 50 μL of 0.1 M phosphate buffer (pH 8.0). Detection of YFP and PE fluorescence by flow cytometry was performed as described above. To 15 μL of a suspension of SNAP-YFP-cytokine beads (1.6 105 beads/μL), 50 μL of enterokinase solution was added. After incubation for 16 h at 4 °C, the beads were prepared for flow cytometry as described above. 2.7. Immobilization on NCO-sP(EO-stat-PO)-Coated Glass Coverslips. Glass coverslips (18 mm diameter, purchased from Menzel (Braunschweig, Germany)) were cleaned by sonication in water, acetone, and 2-propanol for 5 min each, followed by drying in a stream of nitrogen. Afterward, the substrates were transferred to a Unilab glovebox (MBraun, Garching, Germany) and treated with a solution of 0.3 mL of N-[3-(trimethoxysilyl)propyl]ethylendiamine in 50 mL of dry toluene for 2 h, washed several times with dry toluene, and used without further storage. The spin coating solution was prepared by dissolving 10 mg/mL of the NCO-sP(EO-stat-PO) prepolymer (Mn = 12,000 g mol1; PDI = 1.15) in 1 mL of dry THF and adding 9 mL of deionized water. The solutions were used for layer preparation 5 min after mixing. For spin-coating, the samples were placed on the spincoater, covered by the spin coating solution, and then accelerated within 5 s to 2500 rpm for 40 s.20 Afterward, the samples were stored for 10 min and then used for immobilization of BG-PEGNH2 by dip-coating of the samples in a filtered solution of 0.5 mM BG-PEG-NH2 in H2O/DMSO 9:1 for 12 h. Then the samples were washed several times with H2O and dried in a stream of nitrogen. A sticky μSLIDE-insert (IBIDI, Martinsried, Germany) with two chambers was fixed on the coverslip surface. The two chambers were incubated with SNAP-YFPOSM or YFP-OSM (0.1 μM each) in phosphate buffer (0.1 M, pH 8.0) containing 2% BSA for 16 h at 4 °C. Chambers were washed as described in the immobilization section. Fluorescence was detected with a fluorescence scanner using 488 nm laser light for excitation and a 500540 nm bandpass filter for detection. (Typhoon, GE Healthcare, Buckinghamshire, UK). 2.8. Stimulation of Cells. HepG2 cells were seeded on a 24well plate and grown to 70% confluence. After starvation in serum free medium for 2 h, cells were incubated at 37 °C with varying concentrations of recombinant cytokines for different times as
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Figure 1. Scheme of a SNAP-YFP-cytokine and its surface coupling. (A) Schematic representation of the SNAP-YFP-cytokine construct encoded by the expression vector for the production of recombinant protein in mammalian cells. N, N-terminus; S, signal sequence (is cleaved-off during secretion); his, his-tag; SNAP, SNAP-tag; EK, enterokinase recognition site; YFP, yellow fluorescent protein; C, C-terminus. (B) Directed immobilization of a recombinant SNAPYFP-cytokine through the SNAP-tag on a benzylguanine-functionalized bead surface. In this study, PEGylated aminobenzylguanine (BG-PEGNH2) was used. The three ethylene-glycol units that act as a spacer between benzylguanine and the bead are not shown.
indicated in the figure legends. Cellular lysates were analyzed by SDSPAGE and Western blotting using antibodies against phosphorylated STAT3, total STAT3, phosphorylated ERK and total ERK. For stimulation with immobilized cytokines cells were treated as described above but seeded on 24-well plates. Then 15 μL SNAP-YFP-cytokine beads (1.6 105 beads/μL) were added. Benzylguanine-modified beads were used as a control. Cells were incubated for different times. Cellular lysates were analyzed by SDSPAGE and Western blotting as described above. 2.9. Confocal Microscopy of Cells. HEK293 cells transiently transfected with gp130id-CFP 21 and OSMR were seeded on glass coverslips. SNAP-YFP-OSM (1 nM) or SNAP-YFP-OSM beads (1.6 105 beads/μL) were added. Cells were fixed with Shandon Immu-Mount and analyzed by confocal microscopy (Zeiss LSM 510) for CFP and YFP fluorescence. After induction with doxycycline (50 ng/mL) for 72 h, YFP fluorescence of HEK293 cells expressing SNAP-YFP-IL-6 was analyzed in a similar way. Murine embryonic fibroblasts stably transfected with STAT3CFP22 were seeded on coverslips and grown to 40% confluence. Coverslips were transferred to a live-cell imaging chamber, and 15 μL of a suspension of SNAP-YFP-IL-6 beads (1.6 105 beads/μL) was added. After sedimentation of the beads, cells were stimulated by the addition of sIL-6RR (final concentration 0.5 μg/mL). Images of CFP and YFP fluorescence were recorded every 20 min. 1212
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Figure 2. Inducible expression, purification, and activity of SNAP-YFP-cytokines. (A) HEK293 cells stably transfected with an expression vector encoding SNAP-YFP-IL-6 were seeded on coverslips. Protein expression was induced by the addition of doxycycline (50 ng/mL) for 72 h as indicated. Subsequently, cells were fixed and analyzed by confocal microscopy. Scale bars represent 5 μm. (B) Purification of SNAP-YFP-OSM by Ni-chelate affinity chromatography through the his-tag of the fusion protein. Fractions were analyzed by SDSPAGE and subsequent Coomassie-staining of the gel. SN, supernatant from HEK293 cells stably expressing SNAP-YFP-OSM after induction with doxycycline (50 ng/mL) for 72 h; FT, flowthrough of the affinity column; W1 and W2, wash fractions; E, concentrated eluate. (C) Western blot analysis of the fractions described in B with SNAP and OSM antibodies. (D) HepG2 cells were stimulated with OSM (1 nM) or SNAP-YFP-OSM (1 nM) for different times. Cellular lysates were prepared and analyzed for OSM-induced phosphorylation of STAT3 by Western blotting. Detection of ERK served as a loading control. A representative example of three independent experiments is shown.
3. RESULTS 3.1. Expression, Purification, and Activity of SNAP-YFPCytokines. Cytokine constructs were designed that should
permit (i) directed and specific coupling to a functionalized surface, (ii) straightforward detection and quantification of coupling efficiency, (iii) proof of directed coupling versus random coupling or unspecific adsorption, and (iv) purification of the recombinant protein. For this purpose, expression vectors were constructed encoding fusion proteins consisting of an N-terminal hexahistidine (his)-tag (aim iv), followed by a SNAP-tag (aim i), a recognition site for the protease enterokinase (aim iii), a yellow fluorescent protein (YFP, aims ii and iii), and finally the cytokine (Figure 1A). The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase. The SNAP-tag couples itself autocatalytically to benzylguanine derivatives.23 The chemistry of specific covalent coupling of the SNAP-tag to a benzylguanine-functionalized surface is shown in Figure 1B. YFP is a red-shifted variant of the green fluorescent protein (GFP) and can be detected
separately from the cyan fluorescent protein (CFP), a blueshifted variant of GFP.24 Flp-In T-Rex HEK293 cells were stably transfected with SNAPYFP-OSM and SNAP-YFP-IL-6 using the Flp-In T-Rex expression system for targeted recombination and inducible expression of the transgene. Expression of the fluorescent cytokine upon induction with doxycycline is detectable by confocal microscopy (Figure 2A). The recombinant protein is mainly found within the membrane structures of the secretory pathway. The purification of SNAP-YFPOSM from supernatants of stably transfected HEK293 cells by Nichelate affinity chromatography is shown in Figure 2B. SNAP-YFPOSM elutes as a 75 kDa protein. The apparent molecular mass is in good agreement with the estimated molecular mass of the glycosylated fusion protein. In a Western blot, the protein is recognized by both a SNAP antibody and an OSM antibody documenting the integrity of the fusion protein (Figure 2C). SNAP-YFP-OSM and nontagged OSM induce equal STAT3 phosphorylation in HepG2 hepatoma cells (Figure 2D). SNAP-YFP-IL-6 was expressed, purified, and characterized in a similar way as a bioactive cytokine (data not shown). 1213
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Figure 3. Immobilization of SNAP-YFP-cytokines on BG-modified beads. (A) BG-beads were treated for 16 h with SNAP-YFP-cytokines (1 μM) or YFP-cytokines (1 μM) as indicated. Subsequently, beads were analyzed by confocal microscopy using identical settings for laser intensity and detector gain. Scale bars represent 15 μm. (B) Unmodified beads or BG-beads were incubated for 16 h with YFP-IL-6, YFP-OSM, SNAP-YFP-IL-6, or SNAPYFP-OSM (1 μM each). Fluorescence of the beads was analyzed by flow cytometry. Histograms of BG-beads, SNAP-YFP-IL-6 beads, and SNAP-YFPOSM beads are shown with different bead populations in red (monomers), green (dimers), and blue (multimers). Mean fluorescence of the beads as measured by FACS is depicted as bar charts. A representative example of three independent experiments is shown. (C) SNAP-YFP-OSM beads were incubated for different times in cell culture medium at 37 °C. At the indicated time points, fluorescence of the beads was analyzed by flow cytometry. Besides YFP-fluorescence, phycoerythrin (PE) fluorescence of a PE-labeled hOSM antibody was detected to confirm the stability of the recombinant protein on the bead surface.
3.2. Coupling of SNAP-YFP-Cytokines to BenzylguanineModified Surfaces. We used beads as a substrate for surface
immobilization of SNAP-YFP cytokines. We took advantage of the fact that beads can be analyzed by flow cytometry and added as a suspension to cell cultures. The surface of micromer-MCOOH beads (diameter, 3 μm) was functionalized with PEGylated
aminobenzylguanine (BG-PEG-NH2) using NHS/EDC coupling chemistry. The benzylguanine (BG) modified beads were incubated with SNAP-YFP-cytokines or YFP-cytokines and subsequently analyzed by confocal microscopy (Figure 3A). BG-beads treated with SNAP-YFP-cytokines show a strong ringshaped fluorescence, whereas the fluorescence of the YFP-cytokine 1214
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Figure 4. Immobilization of SNAP-YFP-cytokines on a BG-modified, star-PEG-coated surface. (A) Glass coverslips were first aminosilanized and then covered with sP(EO-stat-PO). BG-PEG-NH2 was covalently attached through the reactive NCO-groups (red dots) of sP(EO-stat-PO). (B) The BGmodified sP(EO-stat-PO)-coated surface was incubated with SNAP-YFP-OSM (1 μM) or YFP-OSM (1 μM) for 16 h at 4 °C in two chambers on a glass slide. After washing, the fluorescence of the surface was analyzed by fluorescence scanning.
treated BG-beads is rather weak indicating a specific coupling of SNAP-YFP-cytokines to BG-modified beads. The weak fluorescence of YFP-cytokine treated BG-beads is most probably due to unspecific binding since micromer-M-COOH beads show almost no autofluorescence (not shown). Furthermore, the confocal images confirm that the micromer-M-COOH beads are monodisperse with a diameter of about 3 μm. The modified beads show a tendency to form aggregates. Flow cytometry was used to analyze the relative amounts of immobilized SNAP-YFP-cytokines on BG-modified beads in a quantitative manner (Figure 3B). According to their sizes, three populations of BG-beads can be differentiated (depicted in red, green, and blue in Figure 3B). The red population most probably represents single beads. The green and blue populations represent aggregates of two (green) or three and more beads (blue). The BG-modified beads show almost no fluorescence. After incubation with SNAP-YFP-cytokine, the fluorescence of all populations of BG-beads strongly increases. The representation of several measurements as bar charts shows a strong increase of fluorescence when BG-modified beads were treated with SNAP-YFP-cytokines. A relatively weak fluorescence is measured when nonmodified beads were treated with SNAP-YFP-cytokines or when BG-modified beads were treated with YFP-cytokines lacking the SNAP-tag. These experiments strongly suggest a specific coupling of SNAP-YFPcytokines to BG-modified beads through the SNAP-tag. The concentrations of SNAP-YFP-IL-6 in the coupling buffer before and after coupling to a defined number of beads were determined by ELISA. From the difference of the concentrations, we calculated that about 550.000 molecules of SNAP-YFP-IL-6 are coupled to each bead. On the basis of the known dimensions of YFP, we estimated that a single SNAP-YFP-IL-6 molecule in an extended conformation would occupy 10 nm2. If the beads are regarded as ideal spheres with a diameter of 3 μm, this would result in a surface occupancy of about 20%. To test the stability of coupling, the SNAP-YFP-OSM beads were incubated under cell culture conditions for several hours at 37 °C (Figure 3C). At different times, beads were stained with an OSM specific PE-conjugated antibody. YFP and PE fluorescence was measured by flow cytometry. The almost constant fluorescence of the beads over time in both channels suggests that no SNAP-YFP-OSM is released from the SNAP-YFP-OSM beads and that no cleavage of OSM from the bead surface occurs.
To test the versatility of the approach, SNAP-YFP-OSM was immobilized on a flat glass surface. For this purpose, an aminosilanized glass coverslip was first covered with an isocyanate-terminated, star shaped copolymer containing poly(ethylene oxide) and poly(propylene oxide) (NCO-sP(EOstat-PO) by spin-coating 20 (Figure 4A). The high reactivity of the hydrogel layer shortly after the coating procedure toward amines allowed the covalent attachment of BG-PEG-NH2 by linkage of the amine group to the isocyanate group. The BGmodified surface of the glass slide was incubated with YFP-OSM or SNAP-YFP-OSM. Binding of the YFP-labeled cytokines was analyzed with a fluorescence scanner (Figure 4B). As a consequence of silanization and sP(EO-stat-PO) coating, no unspecific binding of YFP-OSM to the BG-modified surface was detected. The strong fluorescence of the surface area that was incubated with SNAP-YFP-OSM indicates a specific binding mediated by coupling of the SNAP-tag to benzylguanine. 3.3. Proof of Directed Coupling of SNAP-YFP Cytokines to BG-Modified Beads. The enterokinase cleavage site introduced between the SNAP-tag and YFP enabled us to unambiguously prove the directed coupling of SNAP-YFP cytokines to BGmodified beads (Figure 5A). BG-beads were incubated with SNAP-YFP-OSM or SNAP-YFP-IL-6. After the coupling reaction, a fraction of the beads was treated with the protease enterokinase. Subsequently, beads were incubated with PEconjugated OSM or IL-6 antibodies, respectively. YFP and PE fluorescence of the beads were measured by flow cytometry. The results are summarized in Figure 5B. Enterokinase treatment reduces the YFP and PE fluorescence of the SNAP-YFP-cytokine beads by more than 80% indicating that the YFP-cytokine has been cleaved off from the bead surface by the protease. This result clearly shows that the SNAP-YFP-cytokines are bound to the bead surface by directed coupling via the SNAP-tag. Furthermore, the fact that the immobilized cytokines are accessible to the protease indicates that the immobilized cytokines are exposed on the bead surface. Remaining fluorescence of the beads after enterokinase treatment might reflect unspecifically bound protein, incomplete protease cleavage, or both. 3.4. Biological Activities of Immobilized SNAP-YFP-Cytokines. The activities of SNAP-YFP-cytokine beads were analyzed in cell culture. Beads were incubated with medium for 1 h under cell culture conditions. Subsequently, the activities of the supernatants of 1215
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Figure 5. Proof of directed coupling of SNAP-YFP-cytokines. (A) Cleavage of the immobilized SNAP-YFP-cytokine by enterokinase releases the YFPcytokine moiety. his, his-tag; SNAP, SNAP-tag; EK, enterokinase recognition site; YFP, yellow fluorescent protein. (B) SNAP-YFP-cytokine beads were incubated with enterokinase (0.02 U/μL) for 16 h at 4 °C. Beads were incubated with the respective PE-conjugated cytokine antibody and analyzed by flow cytometry f or PE and YFP fluorescence. Mean fluorescence of the beads is depicted in the bar chart. Fluorescence of beads without enterokinase was set to 100%.
the beads and the beads themselves were analyzed. The Western blot in Figure 6A shows that SNAP-YFP-IL-6 beads induce STAT3 phosphorylation in HepG2 cells, whereas the supernatant does not. This experiment shows that IL-6 immobilized through the SNAPtag on BG-beads is biologically active. The observed response does not result from released cytokine because the supernatant of the preincubation of the beads is devoid of any activity. Empty beads show no activity. Interestingly, the time courses of phosphorylation induced by free IL-6 and immobilized IL-6 differ. A similar experiment was performed with free and immobilized SNAP-YFPOSM (Figure 6B). In addition to STAT3, ERK phosphorylation was analyzed. Again the phosphorylation induced by the beads is delayed in comparison to the free cytokine. Supernatants of the SNAP-YFP-OSM beads show only very low activity indicating that
the response mainly results from the immobilized cytokine and not from cytokine released from the beads. 3.5. Interaction of SNAP-YFP-OSM Beads with Cells Expressing gp130-CFP. OSM signals through the cytokine receptors gp130 and OSMR. HEK293 cell were transiently transfected with an expression vector encoding internalization-deficient (id) gp130 fused with the cyan fluorescent protein (gp130id-CFP). The lack of the internalization motif of gp130 increases the cell surface expression of the receptor. Nonlabeled OSMR was cotransfected. These cells were incubated with SNAP-YFPOSM and after fixation analyzed by confocal microscopy (Figure 7A). Surface staining was observed after 30 min that increases during the following 60 min indicating the binding of SNAP-YFP-OSM to the cell surface receptors. 1216
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Figure 6. Biological activities of SNAP-YFP-cytokine beads. (A) SNAP-YFP-IL-6 beads were incubated with medium for 60 min at 37 °C. HepG2 cells were stimulated with the incubation supernatant (SN) or with the SNAP-YFP-IL-6 beads (IL-6-beads). In parallel, cells were stimulated with IL-6 (1 nM), incubated with medium alone () or empty beads (e.b.). Twenty-five minutes after stimulation, cells were lysed. STAT3 phosphorylation was analyzed by Western blotting (pSTAT3). Detection of total STAT3 served as a loading control. (B) In an experiment similar to that described in A, the activity of SNAPYFP-OSM-beads was compared with free SNAP-YFP-OSM. ERK phosphorylation was also analyzed (pERK). Representative examples of three independent experiments are shown.
When the same cells were incubated with SNAP-YFP-OSM beads, enrichment of the beads around a transfected cell was observed (Figure 7B). The image of the CFP channel shows that the cell encompasses the beads to some extent, suggesting an interaction of the cell surface receptors with the SNAP-YFP-OSM beads. 3.6. Effect of SNAP-YFP-Cytokine Beads on the Subcellular Distribution of STAT3-CFP. Tyrosine phosphorylation of STAT3 in response to IL-6 and related cytokines leads to nuclear accumulation of the transcription factor. Murine embryonic fibroblasts (MEFs) lacking endogenous STAT3 (MEFΔ/Δ) were stably transfected with STAT3 fused to the cyan fluorescent protein (STAT3-CFP). Since MEFs do not express the IL-6 receptor R-subunit (IL-6RR), these cells respond to IL-6 only in the presence of soluble IL-6RR (sIL-6RR). Therefore, the timepoint of stimulation is defined by the addition of sIL-6RR. MEFΔ/Δ-STAT3-CFP cells were seeded on coverslips and analyzed under cell culture conditions at the confocal microscope. Upon stimulation with SNAP-YFP-IL-6 beads in the presence of sIL-6RR, nuclear translocation of STAT3 during 60 min of observation is clearly visible (Figure 8A). After 60 min, a z-stack of sections was recorded and displayed as a three-dimensional (3D) image (Figure 8B). The
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Figure 7. Confocal analysis of cell surface-binding of SNAP-YFP-OSM and SNAP-YFP-OSM beads. (A) HEK293 cells were transiently transfected with gp130id-CFP and OSMR. Cells were incubated with SNAPYFP-OSM (1 nM) for the indicated times and subsequently fixed for microscopy. Images were taken by confocal laser-scanning microscopy. A merge of the YFP and CFP channels is shown. (B) The same experiment as that described in A was performed with SNAP-YFPOSM beads. The cells were fixed after 60 min of incubation. Representative examples of three independent experiments are shown.
image demonstrates that all cells showing nuclear accumulation of STAT3-CFP are in contact with SNAP-YFP-IL-6 beads. The confocal images shown in Figure 8C were made from fixed samples. Cells without contact to beads in the neighborhood of stimulated cells do not show any nuclear accumulation of STAT3-CFP. The right image shows that a bead close to cells but without contact does not stimulate the surrounding cells. These observations demonstrate that there is no significant release of free cytokine from the beads. Interestingly, a single beadcell contact is sufficient for stimulation (left image). In a similar experiment, HEK293-STAT3-CFP cells were incubated with SNAP-YFP-OSM beads. The 3D image after 90 min of stimulation shows nuclear accumulation of STAT3 (Figure 8D) and formation of nuclear bodies as described earlier.10 In particular, after rotation of the image by 180° it is clearly visible that all stimulated cells are in close contact with SNAP-YFP-OSM beads. On rare occasions, an accumulation of STAT3-CFP at the cell membrane engulfing a SNAP-YFP-IL-6 bead is visible (Figure 9, arrowhead). In this cell, nuclear accumulation of STAT3-CFP is to some extent inhibited.
4. DISCUSSION In this study, we presented a versatile method for the directed covalent immobilization of proteins on surfaces which includes 1217
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Bioconjugate Chemistry
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Figure 8. Analysis of SNAP-YFP-cytokine beads by confocal live-cell imaging. (A) MEFΔ/Δ-STAT3-CFP cells were incubated with SNAP-YFP-IL-6 beads under cell culture conditions at the confocal microscope. Stimulation of the cells was initiated by the addition of sIL-6RR (0.5 μg/mL), and images were taken at the indicated times. (B) A z-stack of images after 60 min of stimulation from the experiment shown in A is depicted as a 3D representation. (C) Another experiment, as described in A, was performed, and the samples were fixed after 30 min of stimulation. Only cells in contact with SNAPYFPIL-6 beads show nuclear accumulation of STAT3-CFP (left and right image), and contact with a single bead is sufficient (left image). A bead close to cells but without contact is not sufficient for stimulation (right image). (D) HEK293-STAT3-CFP cells were incubated with SNAP-YFP-OSM beads for 90 min. A z-stack of images is depicted as 3D representations. All experiments have been repeated at least 3 times. Representative examples are shown.
the convenient and reliable control of efficiency and orientation of coupling. The method is based on the use of the SNAP-tag for immobilization on benzylguanine-modified surfaces.25 The SNAP-tag is followed by a recognition site for the protease enterokinase, a YFP-tag, and finally the protein of interest. In addition, a his-tag was fused to the N-terminus for purification purposes. The enzyme-like activity of the SNAP-tag has the advantage of performing the coupling reaction under mild conditions without affecting protein structure and function.23 The 1:1 stoichiometry of fluorescent label and protein of interest in the fusion protein allows quantification of the relative amount of immobilized protein even when different proteins of interest are analyzed such as SNAP-YFP-OSM and SNAP-YFP-IL-6 characterized in this study. Directed coupling through a tag versus random chemical coupling of a protein of interest has the great advantage that the site of coupling on the protein is well-defined, and inactivation can be prevented. The tag used for coupling can be arranged
in a way that protein function will not be affected. For the cytokines of the IL-6 family, it is known that the C-terminus is involved in receptor recognition.26 Therefore, the tags were fused to the N-terminus of the protein. Indeed, characterization of the purified tagged fusion proteins revealed no difference in bioactivity compared to that of the nontagged cytokines. For proteins with a functionally important N-terminus, a C-terminal tag with YFP-EK-SNAP-his should be considered. The array of tags also serves as a spacer between surface and cytokine. The resulting exposition and flexibility of the cytokine facilitates receptor complex formation on the cell surface. This is of particular importance for IL-6 where two molecules of cytokine are needed to form a stable receptor complex.8 Nevertheless, restriction of receptor movement can alter the cellular response.27 A general issue in directed protein immobilization on surfaces is the ratio of desired site-specific and covalent coupling versus unspecific adsorptive binding. The latter will lead to a large fraction of inactive or misbehaving proteins. To discriminate 1218
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Bioconjugate Chemistry
Figure 9. Unusual accumulation of STAT3-CFP around a SNAP-YFPIL-6 bead. (A) MEFΔ/Δ-STAT3-CFP cells were incubated with SNAPYFP-IL-6 beads. Stimulation of the cells was initiated by the addition of sIL-6RR (0.5 μg/mL), and confocal images were taken at the indicated times. (B) A z-stack of images after 60 min of stimulation from the experiment shown in A is depicted as a 3D representation. Arrowheads mark the site of STAT3-CFP accumulation.
between these two types of protein binding to surfaces, the enterokinase recognition site was introduced into the fusion protein. Treatment with enterokinase will in the case of directed coupling release the YFP-cytokine moiety of the fusion protein. Thus, a strong loss in fluorescence of the surface upon enterokinase treatment is indicative of directed, covalent coupling. In this study, materials with low protein adsorption were used. Therefore, a large fraction of fluorescence was released by enterokinase treatment clearly demonstrating oriented coupling. Depending on the application, materials with higher protein adsorption may be used. In such a situation, the cleavage assay will be of great value to quantify the amount of site-specifically immobilized protein. Moreover, one can envisage drug delivery systems, in which a protease can be used to release the protein of interest in a controlled manner.28,29 In such a case, labeling with a fluorescent protein will be of great advantage to monitor the release process. After establishing directed coupling, SNAP-YFP-cytokine beads were analyzed with respect to their bioactivity. Immobilized IL-6 and OSM activate STAT3 and ERK indicating that the immobilized cytokines are bioactive. Compared to the free cytokines, STAT3 and ERK phosphorylation in response to the immobilized cytokines is somewhat delayed and prolonged. After the stimulation of cells with beads, the immobilized cytokines may need more time to gain access to receptors than freely diffusing soluble cytokines. A similar delay of signaling was observed with cells seeded on growth-factor-modified surfaces.3 ERK and STAT3 phosphorylation in response to stimulation with both free OSM and SNAP-YFP-OSM beads are transient indicating that feedback inhibition of the signaling pathway works for the free as well as the immobilized cytokine. Taken together, immobilization does not interfere with the basic
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signaling characteristics of the examined IL-6 family cytokines. Because covalently immobilized cytokines cannot be internalized, these findings indicate that internalization of the cytokine is neither necessary for activation of the signaling pathways nor for their attenuation. This conclusion is substantiated by earlier studies showing that feedback inhibition by SOCS3 rather than internalization is required for the transience of IL-6 signaling.30 Thus, adequately immobilized cytokines of the IL-6 family can be used to functionalize surfaces of biomaterials and to investigate signal transduction. Accordingly, it has been shown that immobilized LIF is able to maintain the undifferentiated state of murine embryonic stem cells.31,32 For growth factors that signal through receptor tyrosine kinases, endocytosis of the receptor complex seems to be more important for signaling and downregulation.33 Therefore, immobilization of these kinds of mediators has a stronger impact on the kinetics of the signal.34 Fluorescently labeled and properly immobilized cytokines are a valuable tool to study localized signaling events by confocal microscopy and live cell imaging. In the case of the use of SNAPYFP-cytokine beads for microscopic studies, the fluorescence label fits two purposes: (i) it allows localization of the beads and (ii) provides information of the cytokine load. The latter information is not directly accessible when nonfluorescent proteins are immobilized on fluorescent beads.35 In a first approach, we studied the response of cells transfected with gp130-CFP or STAT3-CFP to stimulation with SNAP-YFP-cytokine beads. Accumulation of receptors was observed at the sites where beads contacted the cells. As a downstream response, nuclear accumulation of STAT3-CFP could be clearly detected. These experiments clearly show that STAT3 finds its way into the nucleus after activation at the plasma membrane and does not require directed transport through the endocytotic pathway. This does not exclude that STAT3 is also activated at signaling endosomes in response to stimulation with free cytokines,36 but it seems that there is no strict requirement of signaling endosomes for STAT3signaling in response to cytokines of the IL-6 family. However, concerning the high cytokine load of the beads some release of bioactive cytokine by, e.g., proteolytic cleavage after receptor binding cannot be entirely excluded. Endocytosis of beads is restricted to nanoparticles with diameters in the submicrometer range because the vesicles formed during the process have a diameter of 100500 nm.37 Therefore, the 3 μm beads we used in this study can serve as a model system to mimic the activities of cytokines immobilized on larger surfaces, e.g., on biomaterials. However, occasionally we observed that the cell “tries” to engulf these particles (Figure 9) leading to mislocalization of signaling proteins such as STAT3. On the basis of these initial observations, it will be interesting to analyze signaling of cytokines immobilized to nanoparticles of different sizes by confocal microscopy.
’ AUTHOR INFORMATION Corresponding Author
*Institut f€ur Biochemie and Molekularbiologie, Universit€atsklinikum RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. Phone: þþ49 241 80 88860. Fax: þþ49 241 80 82428. E-mail:
[email protected].
’ ACKNOWLEDGMENT This research project was supported by grants from the Deutsche Forschungsgemeinschaft (GRK 1035 “Biointerface” 1219
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Bioconjugate Chemistry and SFB 542, projects B12 and Z1). We thank Sabrina Korr for the generation of stable cell lines expressing SNAP-YFP-cytokines.
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