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Pharmacological Characterization of a Recombinant, Fluorescent Somatostatin Receptor Agonist Varun K. A. Sreenivasan,† Oleg A. Stremovskiy,§ Timothy A. Kelf,† Marika Heblinski,‡ Ann K. Goodchild,‡ Mark Connor,‡ Sergey M. Deyev,§ and Andrei V. Zvyagin*,† †
MQ Biofocus Research Centre Australian School of Advanced Medicine Macquarie University, Australia 2109 § Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Russia ‡
bS Supporting Information ABSTRACT: Somatostatin (SST) is a peptide neurotransmitter/hormone found in several mammalian tissue types. Apart from its natural importance, labeled SST/analogues are utilized in clinical applications such as targeting/ diagnosis of neuroendocrine tumors. We report on the development and characterization of a novel, recombinant, fluorescent somatostatin analogue that has potential to elucidate somatostatin-activated cell signaling. SST was genetically fused with a monomeric-red fluorescent protein (mRFP) as the fluorescent label. The attachment of SST to mRFP had no detectable effect on its fluorescent properties. This analogue’s potency to activate the endogenous and transfected somatostatin receptors was characterized using assays of membrane potential and Ca2+ mobilization and immunocytochemistry. SSTmRFP was found to be an effective somatostatin receptor agonist, able to trigger the membrane hyperpolarization, mobilization of the intracellular Ca2+ and receptorligand internalization in cells expressing somatostatin receptors. This complex represents a novel optical reporter due to its red emission spectral band suitable for in vivo imaging and tracking of the somatostatin receptor signaling pathways, affording higher resolution and sensitivity than those of the state-of-the-art radiolabeling bioassays.
’ INTRODUCTION Somatostatin-14 (SST) is a tetradecapeptide widely distributed in mammalian tissues participating in several crucial functions from regulating insulin release to modifying the central neural control of blood pressure and breathing.13 SST, when bilaterally microinjected into discrete regions of the ventral medulla oblongata, evokes profound sympathoinhibition, leading to a precipitous fall in blood pressure. However, SST administered to the neighboring sites evokes a cessation of breathing, or apnea, but does not affect blood pressure.4 SST functions via G-protein coupled receptors of 6 subtypes: sst1, sst2A, sst2B, sst3, sst4, and sst5 (in general, sst) which are found differentially distributed throughout the body and brain.5 Of all these subtypes, the most abundant in human tissues and neuroendocrine tumors, hence the most studied, is sst2A.6 The physiological effects evoked following sst activation are mediated through several intracellular signaling pathways including adenylyl cyclase inhibition, K+ channel opening, inhibition of voltage gated Ca2+ channels, and in some cases activation of phospholipase C or phospholipase A2 leading to opening of intracellular Ca2+ stores.3,79 Although the former three mechanisms are found to be pertussis toxin (PTX) sensitive, the latter is mostly found to occur following activation of sst in cells heterologously r 2011 American Chemical Society
expressing the receptor and is only partially PTX sensitive and has been suggested to be mediated via Gi/o and Gq/G11 proteins.1,7,10 SST also acts via other PTX insensitive pathways which could result in stimulation of Na+-H+ exchange and inhibition of cell growth.11,12 As sst(s) are overexpressed in neuroendocrine tumors, this inhibitory effect on cell-growth upon SST treatment is utilized for treatment of endocrine tumors and acromegaly13 and also for imaging and diagnosis of neuroendocrine cancers using radiolabeled SST analogues.14,15 In most studies addressing tissue localization and trafficking of the somatostatin receptors, radioactively labeled SST peptide complexes were employed.1618 The intrinsic resolution limit of the radiolabeling approaches preclude capturing these processes with sufficient level of morphological and biomolecular detail. Moreover, radioactive techniques are potentially hazardous and demand setting up cumbersome laboratory safety procedures. In comparison, fluorescence labeling and optical imaging offers submicrometer resolution and noninvasiveness under nonhazardous conditions and provides high optical contrast in near-native Received: February 27, 2011 Revised: August 7, 2011 Published: August 08, 2011 1768
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Bioconjugate Chemistry cellular physiological environments. In addition, a specific spectroscopic signature of the fluorescent label allows efficient suppression of the cells morphological and autofluorescence background, pushing the sensitivity toward visualizing single ligand receptor events. Multiplexed labeling of several proteins involved in a concerted molecular event is another attractive capability of fluorescent labels. This approach has thus far been restricted to labeling the sst (receptors) rather than SST (ligands), predominantly by immunofluorescence techniques.16,19,20 As a result, important receptor functionality questions, for example, related to the postendocytosis fate of SST in certain cell types, whether the ligand is dissociated from the receptor, remains unresolved. A fluorescent label that renders SST observable will provide a comprehensive insight into the interaction of the ligand and the receptor, in combination with the existing methods of visualizing sst. Development and applications of a few types of fluorescent somatostatin analogues have been reported in the literature. Nouel et al., Sarret et al., and Stroh et al. have reported employment of a Bodipy-conjugated [D-Trp8]SST analogue to investigate the ligand internalization in COS-7,21 AtT-20 cells,22 and neurons,23 respectively. The former investigated the differential internalization of the ligand by heterologously expressed sst1 and sst2A subtypes, and the latter two proposed sst2A to be the subtype that contributed most to the ligand internalization in AtT-20 cells and neurons. Licha et al. and Becker et al. developed a new, cyanine dye-conjugated, octreotate analogue (a stabilized analogue of SST) and demonstrated labeling of the transfected somatostatin receptors in vitro24 and in tumors expressing somatostatin receptors in vivo.25 Kostenich et al. have also reported on labeling tumors using several types of fluorescent analogues of somatostatin pharmacore (a small peptide chain that facilitates the receptor binding).26 Fluorescein-conjugated SST was employed to investigate the expression of somatostatin receptors in the immune system,27 whereas fluorescein isothiocyanate (FITC)-conjugated octreotide was used for observation of its reuptake by the proximal tubular cells to be mediated by the scavenger receptors rather than sst.28 Another fluorescent version of the octreotide aided elucidation of the mechanisms that dictated low permeation of the somatostatin-analogues and other drugs through the blood brain barrier.29 However, the chemical attachment of a fluorescent organic dye to an SST molecule is prone to disruption of the optical or the biological activity, demanding a careful design of the conjugation procedure, with reproducibility of the chemical reaction yield being an inherent problem.21,24,26 In this paper, we report on the design and production of a robust, reproducible, genetically engineered fluorescent somatostatin analogue, somatostatin-monomeric red fluorescent protein (SST-mRFP), and evaluation of its fluorescent and pharmacological properties. The functionality of SST-mRFP in both endogenous and transfected cells expressing sst2A is characterized. We also report, for the first time to the best of our knowledge, on an undissociated postendocytotic fate of the SST-sst2A (ligand receptor) complex in transfected Chinese Hamster Ovary (CHO-K1) cells.
’ EXPERIMENTAL PROCEDURES Data analysis and curve fittings were performed either in Matlab R2009B or Microcal Origin academic version 8.1. Images were processed using Image J.
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Genetic Engineering of Fluorescent Somatostatin (SSTmRFP). The genetic engineering procedure, microbial culturing,
and cell lysis were carried out as per the standard protocols. In order to produce the chimeric protein, His-tagged SST-mRFP, the DNA fragment encoding SST (AGCKNFFWKTFTSC) was reconstituted using a pair of primers 50 -TCATGGGTACCGGAGGTGGAGGTTCCGGAGGTGGAGGATCTGCTGGATGCAAGAACTTC-30 and 50 -ATGACAAGCTTATCAGCAGGATGTAAAGGTCTTCCAGAAGAAGTTCTTGCATCCAGCAG-30 followed by fusing to mRFP30 using a flexible peptide (Gly4Ser)2 linker. This construct was subsequently cloned into pQE-30 vector (QIAGEN) using BamHI and Hind III sites. This pQE-His-mRFP-SST plasmid was transfected to E. coli BL21 strain and incubated in LB (lysogeny broth) at 30 °C to express this recombinant protein. On reaching an optical density of 0.8 at 550 nm, the bacterial suspension was induced with 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside), and incubated for another 12 h. Purification of the recombinant protein was carried out, as described earlier.31 SDS/PAGE gel electrophoresis was performed in 12% polyacrylamide gel according to the standard protocol, and the resultant solution stored at 4 °C. The concentration of the recombinant protein in terms of mg/mL was estimated using absorption measurements at a wavelength of 280 nm. Prior to the cell experiments, the SST-mRFP solution was desalted with PD SpinTrap G-25 columns (GE Healthcare), pre-equilibrated with PBS (10 stock purchased from Invitrogen), and used within two months. The molar concentrations of SST and SST-mRFP were calculated based on the individual molecular masses of SST (1.63 kDa) and SST-mRFP (29.73 kDa). Mass Spectroscopy. Mass spectroscopy analysis of the recombinant protein was carried out using Australian Proteome Analysis Facility (APAF), Sydney, Australia. In brief, two batches of the SST-mRFP solution were prepared in ammonium bicarbonate. One of the batches was reduced using dithiothreitol followed by alkylation using iodoacetamide. Later, both the batches were digested using trypsin, concentrated and desalted using zip-tip extraction, and analyzed by means of matrix assisted laser desorption/ionization (4800 Plus MALDI TOF/TOF). As a result, a number of peptides of known amino acid sequences (inferred from the DNA sequence template used for the SSTmRFP production) were produced. An Nd:YAG laser (355 nm) was used to irradiate the sample. Mass spectra were acquired for the mass range of 5003500 Da. The near point calibration provided a mass accuracy greater than 500 ppm. The data was exported to a database search program, Mascot (Matrix Science Ltd., London, UK). The peaklists of the two analyzed batches were benchmarked against the fragments and their corresponding molecular masses determined a priori. Spectral Measurements. The optical absorption spectrum of 1 μM SST-mRFP solution prepared in PBS was acquired using a spectrophotometer (Cary 5000 UVvis-NIR, Varian Inc.). The spectrum was baseline corrected and presented here. The fluorescence spectra of SST-mRFP and mRFP prepared in PBS were acquired using a fluorimeter (Fluorolog Tau3 system, JY Horiba). The mRFP fluorescence immunity to the cell preparation chemical procedures was evaluated by adding paraformaldehyde (final concentration of 3.7%) to the SSTmRFP solution and spectral acquisition after 20 min, followed by the spectral data analysis. The change in SST-mRFP concentration during this procedure was accounted for using a negative control experiment. 1769
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Bioconjugate Chemistry Cell Culture. The mouse AtT-20 neuroblastoma cell line was obtained from ATCC (VA, USA) and were grown in culture media composed of 90% DMEM (Dulbecco’s modified Eagles Medium), 10% FBS (fetal bovine serum), and penicillin/streptomycin, 50/5 μg/mL, respectively (all reagents purchased from Invitrogen), in tissue culture treated flasks, inside an incubator maintained at 37 °C and 5% CO2. AtT-20 cells are known to endogenously express sst2A at a density of 1.2 pmol/mg.32 For the membrane potential assay, they were grown on a 96-well glass bottomed, black-sided culture plate (Costar) until reaching confluency. For imaging, cells were seeded into an 4-well slide (BD Falcon) one day before the experiment. The CHO-K1 cell line stably expressing the human somatostatin receptor type 2A (sst2A) at a density of 10 pmol/mg was obtained from PerkinElmer Life and Analytical Sciences. The cells were grown in culture media composed of 90% F-12 Ham’s, 10% FBS, and 400 μg/mL G-418 (all reagents purchased from Invitrogen), in tissue culture treated flasks (BD Falcon) inside an incubator maintained at 37 °C and 5% CO2. For imaging, cells were seeded into an 8-well slide (BD Falcon) one day before the experiment. Cells used for the intracellular Ca2+ measurements were grown on 96-well plates until reaching confluency. Membrane Potential Assay. A membrane potential change in the AtT-20 cells upon SST-induced activation was monitored using a fluorescence-based membrane potential assay (FLIPR bulk assay kit, Molecular Devices). AtT-20 cells grown in a 96well plate were incubated overnight in Lebovitz L-15 media with 1% FBS (100 μL/well) inside a 37 °C incubator. The proprietary blue dye supplied with the FLIPR membrane potential assay kit was dissolved in a potassium-free modification of the recommended HEPES (N-[2-hydroxyethyl]piperazine-N0 -[2-ethanesulphonic acid]) buffered Hank’s balanced salt solution (in terms of mM: HEPES 18.5; Na2HPO4 0.34; NaHCO3 4.17; KH2PO4 0.44; MgSO4 0.83; MgCl2 1.05; NaCl 143.7; CaCl2 1.26; Glucose 5.5). 100 μL dye solution was added to each well in the 96-well plate and placed at 37 °C, 30 min prior to the experiment. The sst agonists or drugs (SST and SST-mRFP) of 5 final concentration were prepared in the above-described buffer and distributed in a 96-well plate (clear, v-bottom). The 96-well cell and drug plates were placed inside the flexstation (FlexStation 3, Molecular Devices). The membrane potential changes corresponding to the various drug concentrations were obtained by analyzing the recorded fluorescence signals (excitation/ emission wavelengths, 530 nm/565 nm, respectively). Data fitting was performed by the following inbuilt logistic equation: ymin ymax þ ymax y¼ 1 þ ðx=EC50Þn
where ymin and ymax are the minimum and saturation response, x is the concentration of the drug, EC50 is the drug concentration required to stimulate half-maximal response, and n is the fitting parameter that determines a slope for the fit. Ca2+ Mobilization Assay. The CHO-K1 cells heterologously expressing sst2A were used to characterize SST-mRFP activity in comparison with that of SST, using the Ca2+ mobilization assay. Fluorescence dye (Fura2-AM, Invitrogen) loaded into the cells reported the concentration of cytoplasmic Ca2+ ([Ca2+]). The buffer used in [Ca2+] measurements contained, in terms of mM, the following: NaCl 140, KCl 2, CaCl2 2.5, MgCl2 1, HEPES 10, D-Glucose 10, and 0.05% BSA (pH and osomolality adjusted to 7.4 ( 0.1 and 330 ( 40 mosmol/kg, respectively), hereafter referred to as HBS. HBS-Ca was same as the above except that
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Figure 1. Mass spectrum of the trypsin-digested SST-mRFP (a) before and (b) after further reduction and alkylation. (a) A peak at 2180.93 Da and the peak absence at 1625.73 Da confirm the presence of disulfide bond in the SST module of the SST-mRFP complex. (b) Reduction of the disulfide bond diminished the peak at 2180.93 Da; instead, a new peak at 1682.73 Da appears.
2.5 mM CaCl2 was replaced with 10 mM EGTA (ethylene glycolbis[b-aminoethyl ether]-N,N,N0 ,N0 -tetraacetic acid). HBS+Ca was similar to HBS, except for 20 mM of CaCl2. Cells were washed once with HBS prior to loading with 2 μM Fura2-AM prepared in HBS. After 1 h of Fura2-AM incubation at 37 °C, the cells were washed 4 times with HBS and incubated again for 30 min at 37 °C. The cells were washed once immediately before the flexstation measurements. Drugs (SST and SST-mRFP), at 5 the final concentration, were prepared in HBS and loaded into the built-in drug chamber in a 96-well plate (clear, v-bottom). The instrument was programmed to record fluorescence emission at 510 nm under two consecutive excitations at wavelength bands centered at 340 and 380 nm, repeatedly every 4 s. The fluorescence was recorded before and after the addition of the drug. The autofluorescence background, acquired from cells lacking dye, was subtracted from the fluorescence assay signals. Minimum and maximum fluorescence measurements, acquired from the cells treated with HBS-Ca and HBS+Ca, respectively, in the presence of 50-μM digitonin, were used to calculate the exact intracellular [Ca2+] using the equation derived by Grynkiewicz et al.33 The data were exported and fit to the logistic equation. Cell Imaging. The CHO-K1 cells were serum-starved overnight, with 1% FBS in the growth media. For the experiment, the cells were incubated with 100 nM SST, 100 nM mRFP, or 100 nM SST-mRFP prepared in PBS, supplemented with 0.1% bovine serum albumin (BSA), 20-mM D-glucose, 0.9-mM CaCl2, and 0.5-mM MgCl2 (hereafter referred to as PBS+CM), for 1770
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Figure 2. (a) Optical absorption spectrum of 1 μM SST-mRFP with a cuvette path length of 4.5 mm (baseline subtracted). (b) Normalized fluorescence spectra of mRFP and SST-mRFP. Excitation spectra of mRFP (open circles, purple) and SST-mRFP (thick dashed line, red). Emission spectra of mRFP (solid circles, purple) and SST-mRFP (thick solid line, red). Emission spectra of SST-mRFP in paraformaldehyde, thin solid line, black.
30 min at 37 °C to stimulate the cells. This step was avoided to evaluate the sst2A distribution in the unstimulated cells. For the antagonist competition experiment, 10 μM BIM23627 was coincubated together with 100 nM SST-mRFP under the same conditions. Immunocytochemistry (ICC) analysis of the cells under investigation was carried out as follows. The cells were fixed with 3.7% paraformaldehyde solution, prepared in PBSCM (same as PBS+CM, without glucose and BSA), for 20 min at room temperature. After washing once with PBSCM, the cells were permeabilized for 15 min with 0.2% Triton X-100, prepared in PBSCM, at room temperature. Subsequently, the cells were washed with PBSCM and blocked with 1% BSA solution prepared in PBSCM for 1 h at room temperature in order to reduce nonspecific binding of the antibodies to the substrates. As the next step, the rabbit-serum-derived primary antibody, against the carboxyl-terminal of sst2A (diluted 1:125 times from the stock solution) (Biotrend Chemikalien GmBH), prepared 2 h prior to this step in PBSCM containing 1% BSA, was added to the cells and incubated for 1 h at room temperature while shaking. The cells were then thoroughly washed with PBS containing 1% BSA and incubated with antirabbit antibody conjugated with fluorescence dye FITC (Jackson Immunoresearch), prepared in PBS containing 1% BSA, for 1 h at room temperature while shaking. Finally, the cells were washed once with PBS containing 1% BSA, and sealed with a coverslip for imaging under either Leica TCS SP2 or Leica SP5 fluorescence confocal microscopes. The AtT-20 cells were incubated with either 1 μM SST-mRFP or 1 μM mRFP (control) prepared in PBS+CM for 1 h at 37 °C. The cells were fixed with 3.7% paraformaldehyde solution, prepared in PBS, for 20 min at room temperature. Subsequently, 10 μM Hoechst solution, prepared in PBS, was added to the cells and incubated for 12 min, at room temperature, for nuclear labeling. The cells were washed two times with PBS and were prepared for imaging under the fluorescence confocal microscope.
Figure 3. (a) Membrane potential response of the wild-type AtT-20 cells, containing endogenous sst and (b) intracellular [Ca2+] response of the sst2A-expressing CHO-K1 cell under stimulation by SST [solid black circles (mean ( SEM), solid black-sigmoid, logistic equation fit], and SST-mRFP [open red triangles (mean ( SEM), solid red-sigmoid, logistic equation fit]. The data shown here are representative of the four independent experiments, each performed in quadruplicate. The error bars, if not displayed, are smaller than the symbol size.
’ RESULTS AND DISCUSSION The recombinant protein, SST-mRFP, molecular composition was characterized using the mass spectroscopy based analytical instrumentation. The activity of the mRFP module of the SST-mRFP was characterized by means of optical spectroscopy, while the biological activity of the SST group was characterized using several cell model experiments. In particular, the membrane hyperpolarization and fluorescence imaging reported on the endogenous receptor activity in the AtT-20 cells activated by SST-mRFP, whereas the intracellular Ca2+ mobilization and fluorescence imaging with auxiliary ICC reported on the transfected receptor activity of the SST-mRFP activated CHO-K1 cells. Biochemical Characterization. The hairpin bend in a native SST molecule is stabilized by the disulfide bond between the amino acids Cys-3 and Cys-14 (amino acid numbers as per the native SST numbering). This disulfide bond has been shown to facilitate high biological activity; consequently, its absence results in reduced potency.24 The correct disulfide bond was formed in the genetically engineered chimeric protein, SST-mRFP, as confirmed by the mass spectroscopy analysis. Prior to the analysis, a trypsin digestion procedure resulted in a controlled disintegration of the SST-mRFP recombinant protein. Among a number of resultant peptide fragments with amino acid sequence known a 1771
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Figure 4. Fluorescence confocal images of CHO-K1 cells transfected with sst2A receptors. mRFP and immunolabeled sst2A fluorescence signals are color-coded red and green, respectively. ICC images of cells in (a) basal state or (b) treated with SST. (c) Cells treated with mRFP under 584 nm excitation that highlights mRFP. (df) Cells treated with SST-mRFP under (d) 488 nm excitation that highlights FITC, (f) 584 nm excitation that highlights mRFP, and (e) overlaid. (gi) Cells treated with SST-mRFP along with BIM23627 under (g) 488 nm excitation, (i) 584 nm excitation, and (h) overlaid. Yellow arrows point to the sst2A localized on the membrane; purple arrows point to the internalized sst2A or sst2A-ligand complex, localized in the perinuclear region. Scale bar, 50 μm.
priori, the following two peptides would report on the presence [HSTGAPSTPPGGGGSAGTFTSC=CK (FW = 2180.95 Da)] or absence [HSTGAPSTPPGGGGSAGCK (FW = 1625.73 Da)] of the disulfide bond. As shown in Figure 1a, a peak at 2180.93 Da on the mass spectrum revealed the presence of the characteristic disulfide-bound peptides proving the hairpin bend in the recombinant SST-mRFP. The identity of this fragment was further confirmed by the disulfide reduction and alkylation of the trypsin-digested SST-mRFP. The resultant mass spectrum is shown in Figure 1b. The peak at 2180.93 Da is diminished and a new peak at 1682.73 Da, corresponding to the alkylated form of HSTGAPSTPPGGGGSAGCK, is clearly observable. Optical Spectral Characterization. The absorption and excitation/emission spectra of SST-mRFP and pristine mRFP in PBS are shown in Figure 2a,b, respectively. The similarities in both absorption coefficient and the fluorescence spectra overlap suggest that the fluorescence property of the mRFP moiety in SST-mRFP was unaffected by the fused oligopeptide. The effect of the fixative, i.e., paraformaldehyde, was also evaluated to assess its potential influence on the mRFP spectral properties in the cell internalization experiments. The fluorescence emission of SSTmRFP was found to be reduced only by 40% under the presence of 3.7% paraformaldehyde in comparison with that in buffer (c.f., Figure 2), without any noticeable changes in the spectral profile, which was deemed acceptable for this investigation. Membrane Potential Assay. The activity of SST-mRFP toward the endogenous sst in the wild-type AtT-20 was compared to that of the native SST by monitoring the Gi/o-protein induced membrane hyperpolarization, presumably, by means of
activating the GIRK channels (G-protein coupled inwardly rectifying K+ channels).34 Figure 3a shows a representative SST and SST-mRFP doseresponse curve activating the membrane potential response. The data from the 4 independent experiments, each performed in quadruplicate, for each ligand were analyzed. pEC50 values, defined as the negative log of drug concentration expressed in M that is required to reach half the maximum of the SST-induced saturation response, were found to be 8.4 ( 0.3 and 6.1 ( 0.2 for SST and SST-mRFP, respectively. It is likely that the lower pEC50 value of SST-mRFP compared to SST was due to the natural propensity for oligomerization of the coelenterate fluorescent proteins, despite the recent progress toward their monomerization.25 Size-exclusion gel chromatography analysis of the SST-mRFP solution revealed the presence of dimers in the solution (detailed in the Supporting Information). Optimization of the purification and storage conditions of this recombinant protein is likely to diminish the SST-mRFP dimerization. Ca2+ Mobilization Assay. Quantitative characterization of the activity of SST-mRFP toward activating the heterologously expressed sst2A in CHO-K1 cells was carried out using the Ca2+ mobilization assay. Representative [Ca2+] doseresponse curves for SST and SST-mRFP are shown in Figure 3b. The calculated pEC50 values (from the data obtained from the four independent experiments, each in quadruplicate) for SST and SST-mRFP were found to be 7.8 ( 0.5 and 6.3 ( 0.3, respectively. The data point at the highest dose of SST-mRFP closely approaches the saturated SST doseresponse. Sampling of the saturation region of the SSTmRFP doseresponse curve was precluded by the SST-mRFP availability. 1772
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nuclear penetration, as shown in the Figure 5a. The specificity of this labeling can be seen from Figure 5b, where mRFP showed no internalization.
Figure 5. Fluorescence confocal images of wild-type AtT-20 cells expressing sst (receptors) treated with (a) SST-mRFP or (b) mRFP. The cell nuclei were stained with Hoechst fluorescent dye, for visual aid. mRFP and Hoechst fluorescence signals are color-coded red and cyan, respectively. Scale bar, 15 μm.
Further, the signaling pathway of SST-mRFP leading to the Ca2+ mobilization was found to be PTX sensitive (data not shown), in agreement with the literature. Cell Imaging. It was previously demonstrated that heterologous sst2A expressed in CHO-K1 cells were endocytosed upon the SST treatment within 15 min after stimulation.9 This was confirmed by our immunocytochemical experiments, where the transfected CHO-K1 cells were subjected to SST treatment (results shown in Figure 4). The sst2A receptors (color-coded green) were localized in the perinuclear region (Figure 4b, violet arrows) following the SST treatment, whereas they were found to be membrane-bound without the SST stimulation (Figure 4a, yellow arrows). Despite the usefulness of ICC in receptor localization assessment, it provides little insight into the ligand pathway and fate. In order to demonstrate the potential of our recombinant fluorescent SST analogue for molecular trafficking studies, we performed the following experiment. The CHO-K1 cells were incubated with the recombinant SST-mRFP for 30 min under the conditions described above, which induced SST-mRFP internalization, with subsequent translocation to the perinuclear regions, as observed under SST activation. Due to the fluorescent nature of the SST analogue, it was clearly visible in the cells (Figure 4f, bright red color), relaxing the need to perform a cumbersome, time-consuming ICC procedure. At the same time, mRFP showed little internalization demonstrating the specificity of SST-mRFP toward sst2A (Figure 4c). The possibility of dissociation of SST-mRFP from sst2A postinternalization was investigated by ICC. The images displayed clear spatial overlap of the fluorescence from SST-mRFP (red) and sst2A (green, immunolabeled) confirming the common intracellular fate of sst2A and SST-mRFP, in the range of time scales investigated (Figure 4e, violet arrows). Importantly, this proves the specificity of the interaction between our novel, recombinant SST-mRFP complex and the sst2A receptors. It is worthwhile to note that SST-mRFP at the concentration of 100 nM resulted in maximum internalization, with relatively low background overhead, and is useful for designing SST-mRFP imaging experiments. Co-incubation of the antagonist BIM23627 along with SST-mRFP inhibited the agonist internalization (as seen in Figure 4gi), in accordance with previous studies,35,36 again confirming the interaction specificity between SST-mRFP and sst2A. The sensitivity/specificity of SST-mRFP for labeling somatostatin receptors in wild type cells is demonstrated in Figure 5. The AtT-20 cells, known to express several receptor subtypes, sst1, sst2, sst4, and sst5,22 when treated with SST-mRFP showed bright spots, mostly discretized in the cytoplasm, with no signs of
’ CONCLUSION We report on the design, production, and biochemical, optical, and pharmacological characterization of a genetically engineered somatostatin molecule fused with a monomeric red fluorescent protein (SST-mRFP). SST-mRFP was found to maintain its mRFP-derived fluorescent properties in the presence of chemical fixatives. The SST moiety in the SST-mRFP complex was demonstrated to be active and potent for triggering signaling in both wild-type and transfected cells expressing somatostatin receptors, although its potency was lower than that of SST, as confirmed by several bioassaying methods. Pinpointing possible reasons for the lower potency, we confirmed the complex integrity associated with SST’s disulfide bond, but found the profound oligomerization of SST-mRFP, which lends itself straightforward improvement of the analogue performance. This analogue was demonstrated to be a useful fluorescent biomarker with potential to elucidate the intracellular fate of the somatostatin ligand receptor complexes in transfected and wild-type cells. In perspective, fluorescent SST/analogues can serve as a model of a drug/gene delivery vehicle, since this small molecular weight peptide is highly mobile, with good prospects of surviving the blood enzymatic degradation.37 ’ ASSOCIATED CONTENT
bS
Supporting Information. Experimental methods and results from the characterization of SST-mRFP oligomerization. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Department of Physics and Astronomy, Macquarie University, Herring Road, Sydney, NSW, Australia 2109. E-mail: andrei.
[email protected]. Phone: 0061 2 9850 7760. Fax: 0061 2 9850 8115.
’ ACKNOWLEDGMENT This research was primarily supported by a grant from the Macquarie University Research Innovation Fund. S.D. and O.S acknowledge the Russian Foundation of Basic Research and RAS Program Molecular & Cellular Biology for support of the recombinant protein synthesis. The authors express gratitude to Ms. Alisa Knapman for assistance with Flexstation 3 measurements. The mass spectroscopy analysis was undertaken at APAF, the infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS). ’ ABBREVIATIONS BSA, bovine serum albumin; CHO-K1, Chinese hamster ovary; FITC, fluorescein isothiocyanate; GIRK, G-protein coupled inwardly rectifying K+; ICC, immunocytochemistry; LB, lysogeny broth; PBS, phosphate buffered saline; PBS+CM, PBS containing CaCl2, MgCl2, BSA, and D-Glucose; PBSCM, PBS containing 1773
dx.doi.org/10.1021/bc200104u |Bioconjugate Chem. 2011, 22, 1768–1775
Bioconjugate Chemistry CaCl2 and MgCl2; SST, Somatostatin-14; SST-mRFP, somatostatinmonomeric-red fluorescent protein; sstx, somatostatin receptor of subtype x; PTX, pertussis toxin
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