Biochimica et Biophysica Acta 1633 (2003) 90 – 95 www.bba-direct.com
Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer Jesper Svartz a,*, Robert Blomgran b, Sven Hammarstro¨m a, Mats So¨derstro¨m a a
Division of Cell Biology, Department of Biomedicine and Surgery, Linko¨ping University, SE-581 85 Linko¨ping, Sweden b Graduate School in Biomedical Research, Linko¨ping University, SE-581 85 Linko¨ping, Sweden Received 18 March 2003; received in revised form 3 June 2003; accepted 6 June 2003
Abstract Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC4 and its metabolites LTD4 and LTE4 stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC4 is formed by addition of glutathione to LTA4, catalyzed by the integral membrane protein, LTC4 synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homooligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/ LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114 – 150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57 – 88. The functional significance of LTCS homo-oligomer formation is currently being investigated. D 2003 Elsevier B.V. All rights reserved. Keywords: Eicosanoid; Fusion protein; Green fluorescent protein; Human embryonic kidney cell; Oligomerization
1. Introduction Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid with important functions in allergy and inflammation [1]. Cysteinyl LTs have been recovered from the airways of asthmatic individuals at rest and following allergen challenge or isocapnic hyperventilation [2 –4]. The inflammatory response in asthma is characterized by partial degranulation of mast cells and eosinophils [5] as well as release of preformed granule enzymes and newly generated lipid mediators such as platelet-activating factor and LTC4, LTD4 and LTE4. The latter compounds, (which are the biologically active constituents of slow
Abbreviations: BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney; LT, leukotriene; LTCS, leukotriene C4 synthase; Rluc, Renilla luciferase * Corresponding author. Tel.: +46-13-223917; fax: +46-13-224149. E-mail address:
[email protected] (J. Svartz). 1388-1981/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1388-1981(03)00091-X
reacting substance of anaphylaxis, SRS-A), stimulate bronchoconstriction, airway mucous formation and edema formation in nearly every vascular bed investigated [6]. The formation of LTC4 takes place on the nuclear envelope and is initiated after activation of cytosolic phospholipase A2 (cPLA2). This enzyme releases arachidonic acid from membrane phospholipids. Arachidonic acid is converted to LTA4 by 5-lipoxygenase (5-LO) in the presence of five-lipoxygenase activating protein (FLAP). 5-Lipoxygenase and cPLA2 translocate to the nuclear envelope [7 –9] upon cell activation. LTC4 synthase (LTCS), a 17-kDa membrane protein, is also found at the nuclear envelope [10,11], where it conjugates LTA4 with reduced glutathione to form LTC4. LTC4 is exported from the cell and metabolized to LTD4 and LTE4. LTCS is expressed, for example, in eosinophils [12], mast cells [13] and monocytes/macrophages [14]. It also occurs in platelets [15]. Earlier experiments have suggested formation of LTCS dimers in cell-free systems [16] and it has been suggested that artificial mutant/wild-type heterodimer formation res-
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tores catalytic activity of inactive mutants [17]. This report provides evidence that LTCS forms homo-oligomers in living cells using bioluminescence resonance energy transfer (BRET) and in cell free-assays using glutathione S-transferase (GST) pull-down techniques. Putative oligomerization domains were suggested based on BRET experiments with truncated variants of LTCS.
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2. Materials and methods
23 – 115, 57 – 88, 57 –115, 87 –115), respectively, were amplified by PCR with pGFP2C2/LTCS as template. The PCR products were ligated in frame with GFP into the EcoRI/ EcoRV sites of pGFP2C2. Four N-terminally truncated forms (amino acids 23 – 150, 57– 150, 87 –150, 114 –150) were amplified by PCR using the same template. The PCR products were ligated in frame with GFP into the EcoRI/ BamHI sites of pGFP2C2. The primers used are listed in Table 1. All plasmids were purified using cesium chloride density gradient centrifugation.
2.1. Materials
2.4. Transfections
Dulbecco’s modified Eagle’s medium supplemented with 4500 mg/l glucose and pyridoxine HCl, fetal calf serum, antibiotics, restriction enzymes, T4 DNA polymerase and competent Escherichia coli DH5a were obtained from Invitrogen (Life Technologies). Deep Vent DNA polymerase and T4 DNA ligase were from New England Biolabs. Monoclonal anti-green fluorescent protein (GFP) antibody (B-2) was from Santa Cruz Biotechnology. Renilla luciferase fusion protein plasmid vector (pRlucC3), green fluorescent protein fusion protein plasmid vector (pGFP2C2) and DeepBlueCk (the coelenterazine substrate for luciferase used in BRET 2) were from BioSignalPackard (Montreal, Canada). Mounting medium (SlowFade Antifade Kit) was from Molecular Probes. Enhanced chemiluminescence kit (ECLplus), 35S-labeled methionine and glutathione Sepharose 4B and pGEX fusion protein vector were from Amersham Pharmacia Biotech. SDS-PAGE molecular weight standard (broad range) was purchased from BioRad. Rabbit reticulocyte lysate-coupled transcription/translation TNTR kit was from Promega. All other chemicals used were from Sigma. White shallow 96-well plates were from Packard Biosciences (Pangbourne, UK). Mouse LTCS cDNA was kindly provided by Dr. Bing K. Lam, Harvard Medical School.
One day before transfection, 2.5 105 HEK 293 cells were seeded per well in six-well dishes. Cells were transfected using a calcium phosphate precipitation method [19]. After 16 h, the cells were washed with PBS and fresh medium was added. The cells were then incubated for an additional 24– 28 h before being analyzed.
2.2. Cell culture Human embryonic kidney (HEK 293) cells, a kind gift from Dr. Johan Stenflo, Lund University, Sweden, were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum, penicillin 100 units/ml and streptomycin 100 Ag/ml. Cell cultures were split 1:5 when reaching confluency. 2.3. Recombinant plasmids Human LTCS cDNA [18] was subcloned into the EcoRI/ BamHI sites of pRlucC3 and pGFP2C2. Correct reading frames were verified by DNA sequencing. Human LTCS cDNA was also subcloned into the EcoRI/XhoI sites of the glutathione S-transferase fusion protein vector pGEX. cDNA encoding four C-terminally truncated forms of LTCS (amino acids 1– 24, 1 – 58, 1 –88, 1 – 115) and six forms truncated from both the C and N termini (amino acids 23 –58, 23 –88,
2.5. BRET2 assays HEK 293 cells co-transfected with 0.5 Ag of pRluc/ LTCS and 2 Ag pGFP/LTCS construct or pGFP, respectively, per well were detached by treatment with 1 mM EDTA (37 jC for 20 min), washed twice and resuspended in BRET buffer (PBS supplemented with 1 g/l glucose, 0.1 g/l MgCl2 and 0.1 g/l CaCl2). Each cell suspension was divided among three wells on a 96-well dish (white shallow plate). Just before analysis, 5 Al of DeepBlueCk dissolved in BRET buffer was added to a final concentration of 5 AM. BRET readings were recorded in dual luminescence mode using a Victor2 1420 Multilabel Counter (Wallac, Perkin Elmer). Transmission filters (Packard instruments) 410 nm (bandwidth 80 nm) and 515 nm (bandwidth 30 nm) were used for Rluc and GFP emission, respectively. Energy transfer was defined as BRET ratio (Emission515 Emission410 Cf)/ Emission410 (Cf = Emission515/Emission410 calculated for Rluc/LTCS expressed alone in the same set of experiment). Co-transfections were also performed with 0.25 Ag pRluc/ LTCS, 1 Ag pGFP/LTCS plus 0, 3, or 10 Ag of the expression vector pcDNA3/LTCS. Empty pcDNA3 vector was added to
Table 1 PCR primers used Primers used forwGFP forw23 forw57 forw87 forw114 revGFP rev24 rev58 rev88 rev115
5VTCCTGCTGGAGTTCGTGACC3V 5VGCCGAATTCTCCCTGCAGGTGATCTC3V 5VCAGGTGGAATTCAGCGAGTACTTCC3V 5VCCTGGAATTCCTGTTCGCGCGCC3V 5VGCGAGCGAATTCGCCCTCTGGC3V 5VAAACCTCTACAAATGTGGTATGGC3V 5VCTACAGGGAGAAGTAGGCTTG3V 5VGAATTACTCGCTGCAGTTCACCTG3V 5VCGCTAGAACAGGTAGACCAGGC3V 5VCCACCAGCAGTCAGAGGGC3V
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give the same amount of DNA in all transfection experiments. In some experiments, the transfected cells were incubated with LTA4 methyl ester (final concentration 50 AM) for 15 min at room temperature before adding the coelenterazine. 2.6. Western blotting Aliquots of the cell suspensions used for BRET measurements were analyzed by Western blotting. Cells were collected by centrifugation and boiled in 100 Al SDS sample buffer for 3 min. Twenty microliters of each sample was loaded on SDS-polyacrylamide gels (10% (w/v) acrylamide), proteins were separated by electrophoresis and electrotransferred to nitrocellulose membrane (100 V for 1 h, 20% aqueous (v/v) methanol, 25 mM Tris, 192 mM glycine). The membrane was blocked with 3% (w/v) bovine albumin fraction V, incubated for 16 h at + 4 jC with primary antibody (anti-GFP) diluted 1:200 in 1% BSA. The membrane was washed three times with PBS containing 0.05% Tween-20 and then incubated with secondary antibody (HRP conjugated goat-anti-mouse) diluted 1:10,000 with 1% BSA. After four washings, the membrane was incubated with ECL-plus reagent for 1 min and chemiluminescence was recorded with a Fuji 1000CH LAS camera.
containing buffer (8 mM Tris – HCl, pH 7.4, 0.12 M KCl, 8% (w/v) glycerol, 4 mM DTT, 0.5% CHAPS), and 35S-labeled LTCS (approximately 100,000 cpm per sample) was added. After incubation at 4 jC for 1.5 h with nutation, the beads were washed five times in NETN buffer and boiled in SDS sample cocktail. The samples were separated by SDS-polyacrylamide gel electrophoresis (15% (w/v) acrylamide). The gel was stained with Coomassie brilliant blue, dried, and exposed to autoradiography film (Kodak X-omat) for 2 weeks at room temperature.
3. Results 3.1. Detection of LTCS oligomerization in transfected HEK 293 cells using BRET technology HEK 293 cells were co-transfected with plasmids encoding Rluc/LTCS and either GFP/LTCS fusion protein or nonchimeric GFP. High BRET ratios resulting from non-radiant energy transfer from luciferase to GFP, were obtained after addition of the luciferase substrate DeepBlueCk to cells coexpressing the fusion proteins Rluc/LTCS and GFP/LTCS (Fig. 1). Background BRET ratios in control cells co-expressing Rluc/LTCS and GFP were much lower. Co-expression of non-chimeric LTCS (Fig. 1) attenuated the BRET signal in
2.7. Fluorescence microscopy Transfected HEK 293 cells (1 Ag GFP plasmid per well) grown on cover slips in six-well dishes were washed with PBS and fixed in 3% (w/v) paraformaldehyde for 20 min. The cells were washed twice with PBS and mounted on slides. GFP fluorescence was imaged using a Nikon Eclipse E800 upright fluorescence microscope. Photographs were taken with a Nikon FDX35 camera equipped with a Nikon U-III multipoint sensor system. 2.8. GST pull-down assay 35
S-Methionine-labeled LTCS was synthesized using Promega’s TNTR kit. GST and GST/LTCS fusion proteins were expressed in 500 ml cultures of E. coli Y1090 transformed with pGEX and pGEX/LTCS plasmid vectors, respectively. After treatment with 1 mM IPTG at 25 jC for 3 h, the cells were harvested by centrifugation, resuspended in 10 ml of a buffer containing of 50 mM Tris – HCl, pH 7.4, 1.5 mM EDTA, 10% (w/v) glycerol, 0.4 M NaCl, and lysed by ultrasound. Triton X-100 was added dropwise, while mixing, to a final concentration of 3% (w/v) and the solubilized lysates were centrifuged in a Beckman SW-41 rotor using an L8-70 M ultracentrifuge (30 min, 25,000 rpm, 4 jC). The supernatants were mixed with 200 Al of glutathione Sepharose suspension and incubated at 4 jC for 16 h with continuous nutation. The Sepharose beads were washed five times in NETN buffer (50 mM Tris – HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Igepal). Aliquots were diluted in CHAPS-
Fig. 1. LTCS homo-oligomerization detected by BRET. HEK 293 cells were seeded on cover slips in six-well dishes and transfected the next day with 0.25 Ag pRlucLTCS, 1 Ag pGFPLTCS and 0, 3, or 10 Ag of pcDNA3/ LTCS. The amount of transfected DNA was equalized with pcDNA3 vector. After 44 h, cells were detached with EDTA, washed and resuspended in PBS containing calcium and magnesium ions and glucose. BRET2 assays were performed in 96-well plates by light-emission acquisition with 410 and 515 nm transmission filters immediately after addition of the luciferase substrate DeepBlueCk. BRET ratio=[Emission515 Emission410 Cf]/ Emission410 (Cf = Emission515/Emission410 for Rluc/LTCS expressed alone in the same experiments). Data expressed as mean values F standard deviation (n = 3). For further details, see Materials and methods.
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Fig. 2. Fusion protein constructs used. Shaded areas indicate LTCS membrane spanning regions as determined from hydrophobicity plots.
cells expressing Rluc/LTCS and GFP/LTCS: at a 10-fold DNA excess, pcDNA3LTCS reduced the BRET signal by about 50%. These data demonstrated that homo-oligomers of LTCS fusion proteins were formed in living cells. Incubation with LTA4 methyl ester (50 AM, 15 min) did not affect the BRET ratios (data not shown), suggesting that LTA4 substrate binding does not influence oligomer formation.
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Fig. 4. Western blot analysis of GFP/LTCS and GFP/LTCS deletion mutants. Aliquots of cell suspensions used for BRET measurements were boiled in SDS sample buffer and loaded onto SDS-polyacrylamide gels (10% (w/v) acrylamide). After electrophoresis and transfer onto nitrocellulose membranes, detection was performed using anti-GFP antibody and HRPconjugated secondary antibody as described in Materials and methods. The figure shows a representative gel from one of the experiments.
signals (Fig. 3) were observed for two of the C-terminally truncated forms, [LTCS(1 –88) and LTCS(1 –115)], but not for the shorter LTCS(1 – 58) and LTCS(1 – 24). Four Nterminally truncated variants [LTCS(23 –150), LTCS(57–
3.2. Mapping of oligomerization domains A number of GFP/LTCS deletion mutant constructs (Fig. 2) were prepared to map oligomerization domains. BRET
Fig. 3. Mapping of LTCS oligomerization domains. HEK 293 cells were seeded on cover slips in six-well dishes and transfected the following day with pRlucLTCS and different GFP/LTCS vector constructs. Numbers below the x-axis indicate the LTCS fusion part amino acid numbers expressed as full-length LTCS numbers. Mean values and standard deviations were calculated from three independent experiments. For further details, see Materials and methods.
Fig. 5. Subcellular localization of GFP and GFP/LTCS fusion proteins. Transfected HEK 293 cells (1 Ag GFP plasmid per well) grown on cover glasses were fixed in 3% (w/v) paraformaldehyde and mounted on slides. GFP fluorescence was imaged using a Nikon Eclipse E800 upright fluorescence microscope. Photographs were taken with Nikon FDX35 camera equipped with a Nikon U-III multipoint sensor system. For additional details, see Materials and methods.
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showed no staining (Fig. 6). These results demonstrate that LTCS formed homo-oligomers in cell-free assays.
4. Discussion
Fig. 6. GST pull-down assay demonstrates LTCS homo-oligomerization in cell-free assays. The figure shows an autoradiogram from a typical GST pull-down experiment. GST/LTCS fusion protein or GST alone were expressed in E. coli and immobilized on glutathione Sepharose. 35Smethionine-labeled LTCS was incubated with the immobilized proteins. Samples were boiled in sample buffer before analyses by SDS-PAGE. Lane 1: 10% of the input of 35S-labeled LTCS. Lane 2: molecular weight marker (non radioactive). Lane 3: GST. Lane 4: GST/LTCS. For additional details, see Materials and methods.
150), LTCS(87 –150), and LTCS(114– 150)] gave BRET signals comparable to or somewhat higher than those obtained with full-length enzyme (Fig. 3). When using LTCS truncated from both the N terminus and the C terminus, intermediate BRET signals were observed, except for LTCS(23 –115) which gave a signal comparable to fulllength LTCS (Fig. 3).1 Aliquots of the cells used for BRET measurements were also analyzed by Western blotting (Fig. 4). The results verified protein expression for all constructs, and showed that the fusion proteins had the correct size. 3.3. Subcellular localization Fluorescence microscopy of HEK 293 cells transiently transfected with GFP/LTCS showed GFP fluorescence around the nucleus, indicating localization to the nuclear membrane/ER (cf. Ref. [10]). In contrast, non-chimeric GFP was evenly distributed throughout the cells (Fig. 5). The subcellular distribution of the truncated forms of GFP/LTCS (Fig. 2) was sufficiently similar to that of full-length LTCS (Fig. 5 shows images of two non-interacting mutants) to exclude the possibility that different localization would preclude interaction to occur. The transfection efficiency was around 30% in most experiments. 3.4. GST pull-down assay GST/LTCS fusion proteins expressed in E. coli were immobilized on GSH Sepharose and incubated with 35Smethionine-labeled LTCS. Immobilized proteins were detached and analyzed by SDS-PAGE. Autoradiographic staining corresponding to 35S-labeled LTCS (17 kDa) was seen in lanes containing 10% input and GST/LTCS fusion protein, respectively. In contrast, lanes containing non-chimeric GST
1
The BRET ratios recorded in Fig. 1 are lower than those in Fig. 3 because the amount of recombinant BRET vectors used was lower in the former case in order not to overload DNA in the transfections.
BRET is a naturally occurring phenomenon caused by nonradiant energy transfer from a luminescent donor to a fluorescent acceptor protein: Luminescence arising during catalytic decarboxylation of coelenterazine by Renilla luciferase is transferred to green fluorescent protein [20] when these proteins are in close proximity. The BRET phenomenon depends strictly on molecular proximity between energy donor and acceptor. The BRET technique has been used to study interaction between circadian clock proteins in E. coli [21], dimerization of the h2-adrenergic receptor [22], homooligomerization of the thyrotropin-releasing hormone receptor [23] and oligomer formation of type A cholecystokinin receptor [24]. BRET was also proposed as a way to monitor the activation state of the insulin receptor [25] and as a method for high throughput screening [26]. A variant of BRET (BRET2) using a modified luciferase substrate (DeepBlueCk) and a GFP variant (GFP2) was used here to study LTCS homo-oligomerization. LTCS and FLAP are located on the nuclear envelope and peripheral endoplasmic reticulum in transiently transfected CV-1 cells [10] and non-transfected RBL cells [11]. 5Lipoxygenase translocates to the same location upon cell activation [7,9]. We designed truncated variants of LTCS starting or ending near the three predicted membrane regions [16]. The corresponding cDNAs were subcloned into the pGFP2 vector to permit detection of the subcellular localization of the truncated forms. Western blot analyses confirmed that the different fusion proteins were expressed and had the expected molecular weights. BRET signals are influenced by the ratio of GFP to luciferase plasmids transfected and an optional ratio of GFP to Rluc plasmid of 4:1 was used throughout this study, cf. Ref. [23]. Gel filtration data have suggested formation of LTCS dimers in cell-free systems [16]. The high BRET ratios obtained for cells co-expressing Rluc/LTCS and GFP/LTCS, compared to control cells expressing Rluc/LTCS and GFP, plus the fact that non-chimeric LTCS attenuated the BRET signal, clearly demonstrated that LTCS homo-oligomers were formed in living cells. The present results identified a C-terminal domain sufficient for LTCS oligomerization (amino acids 114 – 150). In addition, a centrally located oligomerization domain appeared to be present between amino acids 57 and 88, as judged by the BRET data. About half of the C-terminal interaction domain is located in membrane spanning region III (amino acids 114 – 135), whereas the entire central interaction domain is located in membrane spanning region II (amino acids 60 –89). Homooligomerization of LTCS was shown to occur also in cell-free assays by GST pull-down. Artificial mutant/wild-type LTCS
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heterodimer formation restored catalytic activity to the mutant, inactive monomer [17]. Therefore, LTCS homo-oligomerization likely contributes to the structural and catalytic stability of LTCS.
Acknowledgements This work was supported by grants from the Swedish Medical Research Council (31X-05914 and 31BI-14751) and the Swedish Foundation for Strategic Research.
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