Báo cáo khoa học: A novel cholesterol-based detergent potx

CHAPSTEROL A novel cholesterol-based detergent Katja Gehrig-Burger1, Ladislav Kohout2 and Gerald Gimpl1 Institute of Biochemistry, University of Mainz, Germany Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic Keywords CHAPSTEROL; detergent; photoreactive cholesterol; rafts; steroid nucleus Correspondence K Gehrig-Burger, Institute of Biochemistry, University of Mainz, Becherweg 30, 55099 Mainz, Germany Fax: +49 6131 3925348 Tel: +49 6131 3923829 E-mail: kburger@uni-mainz.de Website: http://www.bio.chemie uni-mainz.de/Gehrig/KG_de.htm (Received 19 October 2004, revised December 2004, accepted December 2004) Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol-based detergent developed for functional solubilization of cholesteroldependent membrane proteins are described To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein-coupled receptor requiring cholesterol for its high-affinity binding state Using the photoactivatable cholesterol analogue [3H]6,6-azocholestan-3b-ol[3aH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterolbinding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol-binding proteins Similar to Triton X-100 and CHAPS, CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts, i.e detergent-insoluble microdomains of the plasma membrane The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol-dependent membrane proteins and isolation of rafts doi:10.1111/j.1742-4658.2004.04517.x The plasma membrane of eukaryotic cells is a highly organized and dynamic structure Lipids and proteins are asymmetrically distributed between the leaflets of the membrane bilayer Several findings also indicate an asymmetrical distribution of lipids between lateral subdomains of the membrane One such putative subdomain represent rafts, i.e cholesterol- and sphingolipid-rich domains which can be isolated due to their resistance to solubilization with Triton X-100 at °C and their light buoyant density A special form of rafts are caveolae, flask-shaped invaginations of the plasma membrane that are enriched in the scaffolding protein caveolin Rafts and caveolae have been implicated in receptor-mediated signal transduction, receptor internalization, intracellular trafficking of cholesterol and cholesterol efflux Several G protein-coupled receptors [1–3], heterotrimeric G proteins [4] and signal transducers like the mitogen-activated protein kinase [5] and H-ras [6] are among the proteins enriched in these microdomains The study of integral membrane proteins often requires their purification The first step in purifying membrane-spanning proteins is to solubilize them from the membrane Water-soluble amphiphilic molecules (detergents) are used for this purpose At low concentrations in aqueous solution, detergents exist as monomers An important parameter of a detergent is its critical micelle concentration (CMC) Above the CMC, micelles are formed due to the amphiphilic nature of the detergent molecules Amphiphilic membrane components, such as lipids and transmembrane proteins, become incorporated into these detergent micelles, and Abbreviations CHAPSTEROL, 3-[(3-{[3b-hydroxy-19-oxocholest-5-en-19-yl]amino}propyl)(dimethyl)-ammonio]propan-sulfonate; CMC, critical micelle concentration; DPH, 1,6-diphenyl-1,3,5-hexatriene; GPI, glycosyl phosphatidyl inositol; HEK, human embryonic kidney; MbCD, methyl-bcyclodextrin; OTR, oxytocin receptor; [3H]photocholesterol, [3H]6,6-azocholestan-3b-ol[3aH] 800 FEBS Journal 272 (2005) 800–812 ª 2005 FEBS K Gehrig-Burger et al water-soluble protein–lipid–detergent complexes (mixed micelles) arise Now the protein of interest may be purified using suitable chromatography An ideal detergent solubilizes all of the protein of interest but does not interfere with the activity of the protein In reality however, during solubilization considerable amounts of cholesterol and other lipids are separated from the solubilized proteins possibly leading to inactivation or structural alterations of cholesterol-dependent membrane proteins [7] According to our knowledge, the only detergents with a steroid nucleus that might substitute in part for cholesterol are bile acid derivatives like CHAPS and CHAPSO (Fig 1A) CHAPS and CHAPSO combine the most desirable properties of different classes of detergents They are (a) nondenaturating, (b) disaggregating, i.e they break artificial aggregation of proteins, and (c) electrically neutral, i.e they not affect the charge of solubilized proteins Nevertheless, bile acids differ from cholesterol (Fig 1B) in several functional groups making them rather poor cholesterol substitutions Therefore, we decided to develop a novel detergent which is nondenaturating and electrically neutral like CHAPS and CHAPSO but bears cholesterol as the steroid core This novel detergent termed CHAPSTEROL Fig Structures of CHAPS and CHAPSO (A), cholesterol (B), and CHAPSTEROL (C) FEBS Journal 272 (2005) 800–812 ª 2005 FEBS A novel cholesterol-based detergent (3-[(3-{[3b-hydroxy-19-oxocholest-5-en-19-yl]amino}propyl)(dimethyl)ammonio]propan-sulfonate) (Fig 1C) was tested for its ability to solubilize the receptor of the labour-inducing peptide hormone oxytocin in its functional form The G protein-coupled oxytocin receptor (OTR) was chosen as it requires cholesterol for its highaffinity ligand-binding state in a highly specific manner [8] In addition, we tested whether CHAPSTEROL was able to extract cholesterol-dependent proteins from the membrane specifically For this purpose we affinitylabeled the solubilized fraction and the corresponding membrane pellets with a radioactive photoactivatable cholesterol analogue and analyzed the incorporation rates of the cholesterol probe The data suggest that the novel CHAPSTEROL is an appropriate detergent to solubilize cholesterol-dependent membrane proteins Employment of CHAPSTEROL may also be advantageous for the isolation of cholesterol-rich microdomains of membranes Results Design and synthesis of CHAPSTEROL The aim of this study was to create a nondenaturating detergent for functional solubilization of cholesterolbinding membrane proteins Our new detergent bears an unaltered cholesterol (Fig 1B) as the core structure To create an amphiphilic molecule the zwitterionic side chain of the bile salt derivative CHAPS (Fig 1A) was added at position C10 leading to 3-[(3-{[3b-hydroxy-19oxocholest-5-en-19-yl]amino}propyl)(dimethyl) ammonio]propan-sulfonate To stress both features we named the resulting detergent CHAPSTEROL (Fig 1C) The synthesis of CHAPSTEROL (Fig 2) was achieved with cholesteryl 3-acetate as educt as described in Experimental procedures Cholesteryl 3-acetate is available cost-effectively in large amounts The purified product is judged to be better than 95% pure by thinlayer chromatography on silica gel G in a methanol ⁄ ammonium hydroxide (95 : 5, v ⁄ v) solvent system The product appears as one spot (RF ¼ 0.48) that can be visualized with iodine Mass spectra analysis yielded 665.5 for the CHAPSTEROL 3-acetate (calculated: 664.9) The absorption spectrum of CHAPSTEROL in water consisted of one peak at 210 nm due to the secondary amide function (data not shown) The overall yield of the isolated product was 8% of the theoretical yield The whole procedure was developed to laboratory scale with very pure intermediates obtained with purification by several crystallizations and ⁄ or liquid chromatography For industrial production it is possible to use directly noncrystallized 801 A novel cholesterol-based detergent K Gehrig-Burger et al Fig Synthesis of CHAPSTEROL For details, see text DMF, dimethylformamide; TEA, triethylamine; THF, tetrahydrofuran reaction products (> 90% purity) that would increase the overall yield of CHAPSTEROL Determination of the CMC of CHAPSTEROL We determined the CMC of CHAPSTEROL in H2O fluorimetrically, according to Chattopadhyay et al [9] using CHAPSO as control This method utilizes the enhancement of fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) upon micellization The resulting CMC of CHAPSO was 0.47% (7.4 mm), in good agreement with the value given by the supplier (0.5%) (data not shown) The CMC of CHAPSTEROL was 1.0% (16 mm) and thus considerably higher than that of CHAPSO (Fig 3) Solubilization of the human oxytocin receptor We compared the abilities of CHAPSTEROL and CHAPSO to solubilize high-affinity oxytocin-binding sites from membranes of human embryonic kidney (HEK293) cells overexpressing the human oxytocin receptor As described earlier, CHAPSO is currently the best detergent for functional solubilization of the oxytocin receptor [10] Membranes were incubated with solubilization buffer containing 1% CHAPSTEROL or CHAPSO, respectively As control, we treated membranes with detergent-free solubilization buffer After centrifugation and before performing the ligand-bind802 Fig Determination of the critical micelle concentration (CMC) of CHAPSTEROL The determination of CMC was performed as described for CHAPS [9] using the enhancement of fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) upon micellization of the detergent Cps, counts per second ing test the samples were always diluted : 10 with detergent-free buffer (see below) The dilution step is necessary because it is known that higher concentrations of the detergent CHAPSO interfere with the ligand-binding of the oxytocin receptor [11] It is important to note that we obtained the same amount of specific binding regardless of whether the dilution below CMC was performed before or after ultracentrifugation (data not shown) This strongly suggests FEBS Journal 272 (2005) 800–812 ª 2005 FEBS K Gehrig-Burger et al that the oxytocin receptor remains in a soluble state after dilution In our first experiments, the samples were adjusted to the same detergent composition after solubilization, so that all samples contained 0.1% of both CHAPSO and CHAPSTEROL during the radioligand-binding test As shown in Fig 4A, CHAPSTEROL solubilizes slightly more functional oxytocin receptors as compared to CHAPSO [87 ± 6.5% specific oxytocin-binding compared to the CHAPSTEROL sample (100%)] In samples without detergent we found 7.7 (± 4.0%) of functional oxytocin receptors as compared with the CHAPSTEROL solubilized fraction In the next experiments, we asked if CHAPSTEROL also affects the ligand-binding of the oxytocin receptor (Fig 4B) We diluted CHAPSTEROL and CHAPSO solubilized fractions prior to the ligand-binding test, adjusting them to 0.1% of CHAPSTEROL or CHAPSO The detergent-free control was adjusted to 0.1% CHAPSO We found significantly higher oxytocinbinding in the CHAPSTEROL sample as compared to the CHAPSO sample [75.5 ± 8.8% specific oxytocinbinding compared to the CHAPSTEROL sample A novel cholesterol-based detergent (100%)] Together with the results presented in Fig 4A, these data indicate that CHAPSTEROL disturbs the ligand-binding of the oxytocin receptor to a lesser extent than CHAPSO In samples without detergent we detected 12.5 (± 3.6%) of the functional oxytocin receptors compared with the CHAPSTEROL sample A good detergent for purification of a given protein solubilizes the protein in a functional state and minimizes the solubilization of other proteins Thus, the lower the amount of total protein and the higher the amount of the desired protein in the soluble fraction, the more convenient is the detergent for purification of the protein of interest After precipitation, the protein concentration was determined in the different solubilized fractions CHAPSTEROL solubilized less protein (2.1 ± 0.67 mgỈmL)1) than CHAPSO (2.7 ± 0.60 mgỈmL)1; P < 0.05) (Fig 4C; also SDS ⁄ PAGE, Fig 4D) The relative high amount of protein in the detergent-free soluble fraction (1.6 ± 0.34 mgỈmL)1) is mainly due to the application of methyl-b-cyclodextrin (MbCD) that has been shown to substantially remove proteins from the plasma membrane [12] Cholesterol– Fig Solubilization of high-affinity oxytocin-binding sites (A, B) and protein (C, D) with CHAPSTEROL and CHAPSO Membranes of human embryonic kidney cells stably expressing the human oxytocin receptor (HEK-OTR) were incubated with solubilization buffer containing 1% CHAPSTEROL, or 1% CHAPSO or no detergent (control) at °C for 30 After centrifugation at 20 000 g for 60 the supernatant was further centrifuged at 100 000 g for 60 The supernatant (solubilized fraction) was diluted : 10 with detergent-free solubilization buffer to yield both 0.1% CHAPSO and CHAPSTEROL in each sample (A), or 0.1% CHAPSO for the CHAPSO sample and the detergent-free sample and 0.1% CHAPSTEROL for the CHAPSTEROL sample (B) Ligand-binding tests (A, B) and protein determination (C) were performed as described in Experimental procedures (D) Diluted solubilized fractions (100 lL each) were precipitated, incubated in sample buffer (100 °C, min) and separated by 10% SDS ⁄ PAGE The gel was stained with Coomassie (RotiÒ-Blue, Roth, Karlsruhe, Germany) Data represent mean values ± SD of six measurements from two independent experiments (A, B) or of 12 measurements from four experiments (C) * Indicates P < 0.05; *** indicates P < 0.001 FEBS Journal 272 (2005) 800–812 ª 2005 FEBS 803 A novel cholesterol-based detergent MbCD complexes have been applied to the solubilization buffer to increase the specific binding of the oxytocin receptor For solubilization of the functional oxytocin receptor, CHAPSO was used at a concentration of 1% which corresponds to approximately twice its CMC (0.47%) At 1%, CHAPSO solubilizes twice as many functional oxytocin receptors as compared to the solubilization at 0.47% [10] Still higher concentrations of CHAPSO lead to a higher amount of total protein in the solubilized fraction but also to a loss of binding activity of the oxytocin receptor [13] We asked if solubilization with CHAPSTEROL at twice its CMC (i.e 2%) could further increase the yield of functional oxytocin receptors However, the amount of functional oxytocin receptors was the same for both, 1% and 2% CHAPSTEROL whereas the amount of total protein in the solubilized fraction was increased (2.76 ± 0.74 mgỈmL)1 at 2% CHAPSTEROL vs 2.1 ± 0.67 mgỈmL)1 at 1% CHAPSTEROL) The purity of CHAPSTEROL exceeds 95% To exclude the possibility that contaminants from the intermediate products of the CHAPSTEROL synthesis could contribute to the solubilization of the oxytocin receptor we repeated the solubilization experiments using 5-cholesten-3b,19-diol and 5-cholesten-3b-ol-19-oic acid as detergents We tested these steroids at 0.05%, i.e the maximal amount of contaminants in the solubilization buffer when using 1% CHAPSTEROL None of these steroids led to any solubilization of functional oxytocin receptors (data not shown) underlining that CHAPSTEROL is the sole solubilizing agent Photoaffinity labeling of proteins using a tritiated photoactivatable cholesterol analogue Because CHAPSTEROL solubilized more functional cholesterol-binding oxytocin receptors and less total protein, we wondered whether CHAPSTEROL might preferentially solubilize cholesterol-binding proteins For this purpose, we employed [3H]6,6-azocholestan3b-ol[3aH] ([3H]photocholesterol), a tritiated cholesterol derivative which bears a photoreactive group at position C6 (Fig 5A) Photocholesterol reliably mimicks cholesterol [11,14–19] Upon irradiation [3H]photocholesterol becomes a reactive carbene that forms covalent bonds with neighbouring proteins We photoaffinity-labeled the soluble fraction and the membrane pellet of CHAPSTEROL, CHAPSO and detergent-free samples with [3H]photocholesterol as described in Experimental procedures The proteins were precipitated to remove unbound [3H]photocholesterol The total radioactivity covalently bound to the 804 K Gehrig-Burger et al precipitated proteins in the solubilized fraction was determined and normalized to the total amount of protein present in the solubilized fraction The amount of bound [3H]photocholesterol per mg protein in the CHAPSTEROL solubilized fraction (set to 100%) was significantly higher than in the CHAPSO solubilized fraction (46.1 ± 8.6%) or in the detergent-free sample (55.8 ± 5.9%) (Fig 5B) When the ratio of radioactivity per mg protein of the soluble fraction to the radioactivity per mg protein of the insoluble membrane pellet was determined, a value of 0.64 (± 0.107) was obtained for the CHAPSO sample This means that solubilization by CHAPSO leads to a significant (P < 0.01) depletion of cholesterol-binding proteins In contrast, the ratio for the CHAPSTEROL sample was 1.23 (± 0.182) indicating that solubilization by CHAPSTEROL even leads to a small but significant (P < 0.05) enrichment of cholesterol-binding proteins For the detergent-free control a value of 0.86 (± 0.123) was obtained, i.e the amount of cholesterolbinding proteins per mg protein did not differ significantly between the solubilized fraction and the membrane pellet (Fig 5C) It is important to note, that the amount of oxytocin receptor is negligible (0.008%, w ⁄ w) compared to the total amount of membrane proteins Thus, the labeling of the oxytocin receptor contributes only to a minor extent to the labeling of cholesterol-binding proteins During photoaffinity labeling, the detergent concentration was 0.1% in the CHAPSO as well as in the CHAPSTEROL solubilized fraction That is, the CHAPSO concentration was at one-fifth of its CMC while the concentration of CHAPSTEROL was at one-tenth of its CMC Thus, the higher photoaffinity labeling of the CHAPSTEROL solubilized fraction could be due to the fact that CHAPSTEROL was further below its CMC than CHAPSO, possibly leading to a higher degree of reassociation of lipids with proteins To test this possibility, we performed additional experiments using 2% CHAPSTEROL, 1% CHAPSO and detergent-free control After 10-fold dilution prior to photoaffinity labeling CHAPSTEROL was now at 0.2%, i.e one-fifth of its CMC The results obtained with 0.2% CHAPSTEROL extracts were very similar compared to the labeling experiments at 0.1% CHAPSTEROL The amount of bound [3H]photocholesterol in the CHAPSTEROL solubilized fraction (set to 100%) was again significantly higher than in the CHAPSO solubilized fraction (44.6 ± 2.0%) or in the detergent-free sample (64.48 ± 16.5%) Again, we observed an enrichment of cholesterol-binding proteins in the CHAPSTEROL solubilized fraction, a depletion in the CHAPSO solubilized fraction and neither FEBS Journal 272 (2005) 800–812 ª 2005 FEBS K Gehrig-Burger et al A novel cholesterol-based detergent depletion nor enrichment in the soluble fraction of the detergent-free sample (ratio of radioactivity per mg protein of the soluble fraction to the radioactivity per mg protein of the insoluble membrane pellet: 1.32 ± 0.248, CHAPSTEROL; 0.59 ± 0.092, CHAPSO; 0.82 ± 0.071, detergent-free sample) These results underline that the higher labeling of proteins with [3H]photocholesterol in the CHAPSTEROL solubilized fraction is due to an enrichment of cholesterolbinding proteins and not due to a simple reassociation of lipids with proteins after dilution of the detergent below its CMC The enrichment of cholesterol-binding proteins in the CHAPSTEROL solubilized fraction and the depletion in the CHAPSO solubilized fraction could also be due to a different competition of the detergents with protein–photocholesterol interaction In additional experiments we have incorporated [3H]photocholesterol into membranes, subjected the samples to cross-linking and solubilized them with CHAPSO or CHAPSTEROL The ratio of the radioactivity per mg protein of the soluble fraction to the radioactivity per mg protein of the insoluble membrane pellet was determined and a value of 1.4 for CHAPSTEROL and 0.58 for CHAPSO obtained This is in accordance with the results shown above and further indicates that CHAPSTEROL leads to an enrichment of cholesterol-binding proteins in the soluble fraction Isolation and characterization of detergentinsoluble membrane microdomains using CHAPSTEROL, CHAPS and Triton X-100 Fig Labeling of solubilized membranes of HEK293 cells stably expressing the human oxytocin receptor (HEK-OTR) with [3H]photocholesterol (A) Structure of [3H]photocholesterol (B) Labeling yield of the soluble fractions in relation to the protein content Membranes of HEK-OTR cells were incubated with solubilization buffer containing 1% CHAPSTEROL, 1% CHAPSO or no detergent (control) at °C for 30 After centrifugation at 20 000 g for 60 the supernatant was further centrifuged at 100 000 g for 60 The solubilized fraction was diluted (1 : 10) with detergent-free buffer and incubated with 0.6 lCi [3H]photocholesterol at 30 °C for 10 After irradiation (k > 335 nm) at 30 °C for min, proteins were precipitated and the incorporated radioactivity was measured Data are expressed as the amount of radioactivity per mg solubilized protein (determined by the Bradford assay) Radioactivity per mg protein of the CHAPSTEROL sample was set to 100% (C) The pellets of both centrifugation steps were combined and resuspended in 500 lL NaCl ⁄ Pi, incubated with 3.0 lCi [3H]photocholesterol and treated as described for (B) Data are expressed as the ratio of radioactivity per mg protein of the soluble fraction to the radioactivity per mg protein of the pellet Data represent values ± SD of two independent experiments *** Indicates P < 0.001; ** indicates P < 0.01 FEBS Journal 272 (2005) 800–812 ª 2005 FEBS Traditionally, cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) are isolated due to their resistance against solubilization with Triton X-100 at °C and their light buoyant density More recently, other detergents such as CHAPS have also been used for the isolation of rafts [20,21] For this reason, we compared the characteristics of microdomains extracted with Triton X-100, CHAPS and CHAPSTEROL with regard to caveolin, the caveolin-interacting protein H-ras and the distribution of the lipids, cholesterol and sphingomyelin Detergentinsoluble membrane microdomains were prepared by extraction in the cold (4 °C) using the various detergents followed by sucrose density gradient centrifugation We analyzed the distribution of caveolin and H-ras by Western blotting As shown in Fig 6A, the distributions of caveolin among sucrose density fractions prepared with Triton X-100 or CHAPS were similar With both detergents, enrichment of caveolin was highest in fraction although the caveolin distribution 805 A novel cholesterol-based detergent A K Gehrig-Burger et al C Fig Isolation and characterization of detergent-insoluble membrane microdomains using CHAPSTEROL, CHAPS and Triton X-100 For preparation of microdomains, membranes of HEK-OTR cells were solubilized at °C for 30 by CHAPSTEROL (‹), CHAPS (C) or Triton X-100 (T) and subjected to sucrose density gradient centrifugation After precipitation and SDS ⁄ PAGE, proteins were blotted and probed with anti-caveolin IgG (A) or stained with Ponceau S (B) Lanes 1–12: fractions of the sucrose density gradient (1 ¼ top, 12 ¼ bottom), M: prestained protein standard (SeeBlueÒ Plus2, Invitrogen, Karlsruhe, Germany) After lipid extraction the sphingomyelin and cholesterol distribution (C) were analyzed For analysis of the sphingomyelin distribution, lipids were resolved by thinlayer chromatography The relative amount of sphingomyelin was determined densitometrically The cholesterol content of the fractions was analyzed spectrophotometrically using a cholesterol oxidase kit B among the Triton X-100 fractions was broader, exhibiting substantially large amounts of caveolin in fractions 10 and 11 Using CHAPSTEROL, most of the caveolin was detected in fraction These slight differences in the distribution profile of caveolin among the different samples were consistently found in several fractionation experiments The distribution of H-ras always corresponded to the distribution of caveolin (data not shown) To examine the protein distribution in the fractions of the sucrose density gradient, the blotted proteins were stained with Ponceau S This dye stains all proteins with a sensitivity of about 50 ngỈprotein band)1, can easily be washed off with NaCl ⁄ Pi and is compatible with subsequent antibody reactions In all three samples most of the protein is detected in fraction 12 at the bottom of the gradient (shown for CHAPSTEROL in Fig 6B) The detergent-insoluble proteins are distributed almost equally between fractions 6–11 Fractions 1–5 contained very low amounts of protein Thus, the distribution pattern of the total protein among the fractions of the sucrose density gradient was very similar, regardless of the detergent used The distribution of sphingomyelin was analyzed densitometrically after separation of the lipid extracts by thin-layer chromatography as described in Experimental procedures Cholesterol was determined spectro806 photometrically by a cholesterol oxidase assay We found that the distributions of cholesterol and sphingomyelin were almost in parallel to the distribution of caveolin for all detergents tested (Fig 6C) Discussion The choice of a suitable detergent is one of the first and crucial aspects in protein purification In 1980, Hjelmeland created a novel detergent called CHAPS, a sulfobetaine-derivative of the bile salt, cholic acid [22] CHAPS is nondenaturating, disaggregating, and electrically neutral, i.e it does not interfere with conventional techniques such as ion-exchange chromatography and isoelectric focussing Due to their steroid nuclei CHAPS and the related compound CHAPSO were, up until now, the best detergents for solubilization of the G protein-coupled oxytocin receptor that binds at least five molecules of cholesterol [23] and exhibits a very stringent and unique requirement for structural features of cholesterol [8] CHAPS and CHAPSO, both bearing a steroid core, have been found to be the only detergents that solubilize a small amount of the oxytocin receptor in its high-affinity ligand-binding state [10] However, even these detergents solubilize only low amounts ( 8%) of high-affinity oxytocin-binding sites of the membrane In addition, the zwitterionic FEBS Journal 272 (2005) 800–812 ª 2005 FEBS K Gehrig-Burger et al detergent CHAPS solubilizes membrane lipids asymmetrically, leading to a depletion of cholesterol in the solubilized preparation [24] In comparison to cholesterol, CHAPS and CHAPSO have a hydroxyl group at C3 in a-configuration, a zwitterionic side chain and additional hydroxyl groups in their ring systems These features make CHAPS and CHAPSO rather poor substitutions for cholesterol during the solubilization process of cholesterol-dependent membrane proteins Therefore, we decided to create a novel detergent employing the beneficial properties of CHAPS in combination with a cholesterol molecule as a core structure Because different cholesterol-dependent proteins have different requirements for the structure of cholesterol [23], an unchanged cholesterol nucleus within a detergent should meet the requirements of many cholesterol-binding proteins The synthesis of the novel detergent CHAPSTEROL requires inexpensive starting material and can be performed by a combination of well-known and relatively simple synthetic steps [22,25–27] The absorption spectrum of CHAPSTEROL in water consists of one peak at 210 nm due to the secondary amide function This indicates no serious interferences of CHAPSTEROL with UV monitoring of proteins at this wavelength Because the CMC of CHAPSTEROL is twice as high (1.0%, w ⁄ v) as the CMC of CHAPSO, CHAPSTEROL should be easily removed from proteins by the dialysis that is necessary for reconstitution or crystallization experiments Solubilization experiments with CHAPSO and CHAPSTEROL presented herein show that CHAPSTEROL solubilizes more oxytocin receptor in its high-affinity ligand-binding state than CHAPSO When compared with CHAPSO, CHAPSTEROL interferes less with the binding of oxytocin to its receptor The exact mechanism of the interference of CHAPSO with the ligand-binding of the solubilized oxytocin receptor is not clear Reconstitution studies suggest that the interference could be due to a removal of cholesterol from the oxytocin receptor and ⁄ or denaturation of the oxytocin receptor by CHAPSO [13] At detergent concentrations leading to a maximal solubilization of functional oxytocin receptors, CHAPSTEROL solubilizes less ‘contaminating’ proteins than CHAPSO Possibly, due to its cholesterol nucleus, CHAPSTEROL might bind directly and specifically to cholesterol-binding proteins This could lead to a favoured solubilization of cholesterol-binding proteins To test whether CHAPSTEROL is able to specifically extract cholesterol-dependent proteins from the membrane, we affinity-labeled the solubilized fraction and the corresponding pellets with a tritiated photoactivatable cholesterol analogue ([3H]photocholesterol), FEBS Journal 272 (2005) 800–812 ª 2005 FEBS A novel cholesterol-based detergent and analyzed the incorporation rates of this cholesterol probe Photocholesterol has been demonstrated to reliably mimick cholesterol biophysically [16] and biochemically: it supports the high-affinity ligand-binding state of the oxytocin receptor [11] In addition, it has been used to verify the cholesterol-binding of several proteins, such as synaptophysin [15], vitellogenin [17], proteolipid protein [18], a cholesterol transporter [14], and the metabotropic glutamate receptor [19] The photoaffinity labeling approach demonstrated that CHAPSTEROL extracted 2.2-fold more cholesterolbinding proteins than CHAPSO Whilst administration of CHAPSO depletes the solubilized fraction of cholesterol [28] and cholesterol-binding proteins (this work), solubilization with CHAPSTEROL led to an enrichment of cholesterol-binding proteins Thus, CHAPSTEROL is a promising detergent for purification of cholesterol-dependent membrane proteins and in this respect clearly superior to CHAPSO Several years ago, a novel application of detergents emerged beyond protein solubilization and purification It is known that glycosyl phosphatidyl inositol (GPI)-anchored proteins are poorly solubilized with Triton X-100 (reviewed in [29]) In 1992, Brown & Rose analyzed the Triton X-100-insoluble fraction of epithelial cells and found them not only to be enriched in GPI-anchored proteins but also in sphingolipids and cholesterol [30] During the subsequent years many results were published concerning the composition of cholesterol- and sphingolipid-rich microdomains that are insoluble in Triton X-100 at °C It was concluded that these so-called ‘rafts’ are important for signal transduction and protein sorting in the cell (reviewed in [31]) Roper et al [20] demonstrated the coexistence ¨ of different cholesterol-based lipid rafts that contain various subsets of proteins by comparing sucrose density gradients following solubilization of membranes with Lubrol WX or Triton X-100 Apparently, different detergents at different temperatures lead to the preparation of different membrane microdomains Here, we compared detergent-insoluble microdomains prepared at °C by means of Triton X-100, CHAPS or CHAPSTEROL Among the fractions of the density gradients, the sharpest distribution profile for caveolin was found in the CHAPS and CHAPSTEROL samples whereas the distribution profile for caveolin in the Triton X-100 sample was considerably broader Caveolin is a cholesterol-binding membrane protein and a marker of caveolae, a subset of rafts [32] It interacts with several receptor and effector proteins such as the small GTP-binding protein H-ras [33] The distribution of H-ras as well as the distribution of the marker lipids cholesterol and sphingomyelin always followed the 807 A novel cholesterol-based detergent distribution of caveolin It is known that Triton X-100 has a limited capability to distinguish rafts from disordered membrane domains, as the ordered phase is substantially solubilized with this detergent even at °C [34] Our data show that solubilization with CHAPSTEROL maintains the localization of the marker proteins caveolin and H-ras as well as the localization of the marker lipids cholesterol and sphingomyelin, to rafts We conclude that CHAPSTEROL is an appropriate detergent for solubilization and purification of cholesterol-dependent membrane proteins and is also suitable for the study of cholesterol-rich membrane microdomains important for signal transduction and transport processes in cells Experimental procedures Materials The substances were from the following suppliers: [3H]NaBH4 and Hybond ECL nitrocellulose, Amersham Corp (Braunschweig, Germany); 1,6-diphenyl-1,3,5-hexatriene, Molecular Probes (Karlsruhe, Germany); [tyrosyl2,6–3H]-oxytocin, PerkinElmer (Jugesheim, Germany); cell ¨ culture media and supplements, PAA Laboratories (Colbe, ¨ Germany); filter safe, Zinsser Analytic (Frankfurt, Germany); cholesterol diagnostic kit, R-Biopharm (Darmstadt, Germany); silica gel G HPTLC plates and silica gel G, Merck (Darmstadt, Germany); prestained protein standard, Invitrogen (Karlsruhe, Germany); anti-caveolin IgG, Transduction Laboratories (Lexington, KY, USA); anti-H-ras IgG, Santa Cruz (Heidelberg, Germany); Bradford reagent, RotiÒ-Blue and solvents, Roth (Karlsruhe, Germany) Secondary antibodies, lipid standards, Ponceau S and all other chemicals were from Sigma (Munchen, Germany) ă Synthesis of CHAPSTEROL Starting with cholesteryl 3-acetate, 19-hydroxycholest-5-en3b-yl acetate was prepared as described [25–27] The further synthesis was similar to the synthesis of the bile acid derivative CHAPS described by Hjelmeland [22] and is shown in Fig 19-Hydroxycholest-5-en-3b-yl acetate (100 mg) was dissolved in acetone (15 mL) in an ice bath Oxidation of this steroid to 3b-acetoxy-cholest-5-en-19-oic acid was achieved by dropwise addition of Jones reagent (2.67 g of CrO3 in 2.3 mL of H2SO4 and 7.7 mL of H2O) until persistence of the orange color After leaving the sample on ice for 10 min, isopropanol (1 mL) was added, the solution was stirred at 20 °C for 15 min, H2O (20 mL) was added and the steroid was extracted with ethyl acetate The ethyl acetate was removed by distillation under reduced pressure in a rotary evaporator The dry 3b-acetoxy-cholest-5-en-19oic acid was dissolved in anhydrous tetrahydrofuran 808 K Gehrig-Burger et al (1.5 mL) in a 25 mL round-bottom flask equipped with a drying tube, and anhydrous triethylamine (32 lL, 0.227 mmol) was added The flask was gently swirled, ethyl chloroformate (22 lL, 0.227 mmol) was added, and the flask was immediately placed into an ice bath for 20 The contents of the round-bottom flask were filtered into a flask containing tetrahydrofuran (1 mL) and 3-dimethylaminopropylamine (20 lL) The filter cake obtained was washed with tetrahydrofuran (2 · mL) The tetrahydrofuran was removed under reduced pressure The residue was taken up in dichloromethane (500 lL) and the organic phase was extracted with m sodium hydroxide (200 lL) After phase separation, the dichloromethane was separated and dried over MgSO4 for 30 The dried dichloromethane solution was separated and the MgSO4 was washed with dichloromethane (2 · 500 lL) The dichloromethane solutions were combined, solvent was removed under reduced pressure in a rotary evaporator, mL of toluene and mL of ethanol were added, and the solvents were distilled off The residue was dissolved in anhydrous dimethylformamide (1.75 mL) 1,3-Propane sultone (27.7 mg) in dimethylformamide (110 lL) was added and the solution was stirred in a water bath at 60 °C for h The resulting solution was then allowed to stand at 20 °C for 15 h The solvent was removed under reduced pressure in a speed-vac concentrator The products were dissolved in methanol and fractionated on silica gel G column chromatography (column: 23 cm length, diameter: 2.1 cm, volume of gel bed: 80 mL) with methanol ⁄ ammonium hydroxide (95 : 5, v ⁄ v) The products were analyzed by thin-layer chromatography on silica gel G in a methanol ⁄ ammonium hydroxide (95 : 5, v ⁄ v) solvent system The 3-acetate of CHAPSTEROL was visualized by iodine vapor at RF ¼ 0.51 To obtain free CHAPSTEROL, alkaline hydrolysis of CHAPSTEROL 3-acetate was performed CHAPSTEROL 3-acetate (13 mg) was dissolved in water (605 lL) and NaOH (1 m, 260 lL), and the reaction mixture was heated to 60 °C for 90 After neutralization with HCl, the obtained solution was desalted on a Sephadex LH-20 column The detergent CHAPSTEROL was purified again on silica gel G column chromatography (column: 15 cm length, diameter: cm, volume of gel bed: 12 mL) with methanol ⁄ ammonium hydroxide (95 : 5, v ⁄ v) The products were analyzed by thin-layer chromatography on silica gel G in a methanol ⁄ ammonium hydroxide (95 : 5, v ⁄ v) solvent system The 3-[(3-{[3b-hydroxy-19-oxocholest-5-en-19yl]amino}propyl) (dimethyl)ammonio]propan-sulfonate (CHAPSTEROL) was visualized by iodine vapor at RF ¼ 0.48 Synthesis of [3H]photocholesterol [3H]6,6-Azocholestan-3b-ol[3aH] was synthesized as described [11,15] In brief, 6-keto-5a-cholestan-3b-ol was FEBS Journal 272 (2005) 800–812 ª 2005 FEBS K Gehrig-Burger et al converted to 6,6-azocholestan-3b-ol according to the method of Church et al [35] 6,6-Azocholestan-3b-ol was oxidized to 6,6-azocholestan-3-on using chromic acid and subsequently reduced to [3H]6,6-Azocholestan-3b-ol[3aH] ([3H]photocholesterol) (1 CiỈmmol)1) using [3H]NaBH4 Determination of critical micelle concentration The CMCs of CHAPSTEROL and CHAPSO were determined fluorimetrically according to Chattopadhyay et al [9] with slight modifications This method utilizes the enhancement of fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) upon micellization DPH stock solution (5 lL; 67 lm in 1.6% dimethylformamide) was added to various amounts of detergent dissolved in 45 lL of H2O The tubes were vortexed and incubated in the dark at 20 °C for 30 Fluorescence measurements were then performed with a spectrofluorimeter (Quantamaster, PTI Laurenceville, NJ, USA) using cm path-length quartz microcuvettes The excitation wavelength was 358 nm and the emission wavelength was 430 nm The excitation and emission slits were set at bandwidths of 1.5 and nm, respectively Fluorescence was averaged over s for each sample reading Background samples without DPH were prepared in all cases, and their fluorescence intensity was subtracted from measured values Duplicate sets of samples were prepared in each case and average fluorescence is shown in Fig To reverse any photoisomerization of DPH, samples were kept in the dark in the fluorimeter for 30 s before the excitation shutter was opened and the fluorescence was measured Preparation of steroid–methyl b-cyclodextrin inclusion complexes The cholesterol–MbCD inclusion complex was prepared as described previously [28] Briefly, 750 mg cholesterol were dissolved in mL 2-propanol at 60 °C and added dropwise to 25 g MbCD in 200 mL H2O in a water bath (80 °C) The mixture was stirred at 80 °C until the initially precipitating steroid was completely dissolved For preparation of the [3H]photocholesterol–MbCD inclusion complex the steroid (final concentration 0.3 mm) was added to an aqueous solution of MbCD (40 mgỈmL)1) The mixture was overlaid with N2, and was continuously vortexed under light protection at 30 °C for 24 h in a thermomixer Cell culture HEK293 cells stably expressing the human oxytocin receptor (HEK-OTR) [8] were cultured in monolayers in complete Dulbecco’s modified Eagle’s medium supplemented with penicillin (100 000 L)1), streptomycin 100 mgỈL)1), and 10% (v ⁄ v) fetal bovine serum at 37 °C and 5% (v ⁄ v) CO2 FEBS Journal 272 (2005) 800–812 ª 2005 FEBS A novel cholesterol-based detergent Membrane preparation Cells were centrifuged at 100 g for 10 The cell pellet was washed twice with NaCl ⁄ Pi and was resuspended in ice cold homogenization buffer containing 20 mm Hepes, pH 7.4, mm EDTA, and a protease inhibitor cocktail containing bacitracin, soybean trypsin inhibitor, leupeptin, and phenylmethylsulfonyl fluoride The suspension was homogenized with a Teflon homogenizer (1000 r.p.m., 10 strokes) Subsequently, the homogenate was centrifuged at 48 000 g for 30 min, and the pellet was washed with binding buffer (20 mm Hepes, pH 7.4, 10 mm MgCl2) Solubilization HEK-OTR membranes (700 lg) were resuspended in 70 lL of solubilization buffer (detergent, 40 mm Hepes (pH 7.4), 10% (w ⁄ v) glycerol, 300 mm NaCl, 10 mm MgCl2) with 0.1 mm cholesterol–MbCD and incubated at °C for 30 The solubilized membranes were centrifuged at 20 000 g for 60 Subsequently, the supernatant was centrifuged at 100 000 g for 60 in a Ti60 rotor at °C The supernatant (solubilized fraction) was then used for the receptor-binding assay and protein determination Protein precipitation and determination Quantitative precipitation of proteins was performed as described by Wessel & Flugge [36] Precipitated proteins ă were dissolved in 100% formic acid for protein determination or in sample buffer containing 4% (w ⁄ v) SDS, 20% (w ⁄ v) glycerol, 130 mm Tris ⁄ HCl, pH 6.7, 0.01% (w ⁄ v) bromphenol blue, and 200 mm dithiothreitol for SDS ⁄ PAGE Protein was determined by the Bradford assay [37] using bovine serum albumin as standard Receptor-binding assay Solubilized fraction (70 lL) was diluted : 10 to the following buffer composition: 20 mm Hepes (pH 7.4), 10% (w ⁄ v) glycerol, 300 mm NaCl, 10 mm MgCl2 and mm cholesterol–MbCD The respective detergent concentrations are given for each experiment in the Results section To determine the total binding, 90 lL of the diluted solubilized fraction were incubated with 10 lL [3H]oxytocin (final ligand concentration: 10 nm) at 30 °C for 30 The binding reaction was stopped by addition of mL ice cold filtration buffer (10 mm Hepes, pH 7.4, mm MgCl2) Bound ligand was separated from free ligand by rapid filtration over Whatman GF ⁄ F filters using a Brandel cell harvester Filters were washed twice with filtration buffer, placed in scintillation vials and made transparent with 10 mL of filtersafe (Zinsser Analytic, Frankfurt, Germany) 809 A novel cholesterol-based detergent Radioactivity was measured in a LKB 1215 Rackbeta liquid scintillation counter Nonspecific binding was determined in the presence of a 500-fold excess of unlabeled oxytocin Photoaffinity labeling of proteins with [3H]photocholesterol Solubilized fraction (50 lL) was diluted : 10 with detergent-free buffer [40 mm Hepes, pH 7.4, 10% (w ⁄ v) glycerol, 300 mm NaCl, 10 mm MgCl2] The membrane pellets of the two centrifugation steps of the solubilization were combined and resuspended in 500 lL NaCl ⁄ Pi [3H]Photocholesterol–MbCD was added: 0.6 lCi (for solubilized fractions) or 3.0 lCi (for membrane pellets) The mixture was continuously vortexed in a thermomixer at 30 °C for 10 Thereafter, the sample was irradiated at 30 °C for using a mercury-arc lamp (200 W, k > 335 nm) Proteins of the samples (100 lL) were precipitated to remove unbound [3H]photocholesterol The protein pellets were resuspended in 10 mL of a scintillator cocktail (filtersafe, Szinsser Analytic) Radioactivity was measured in a LKB 1215 Rackbeta liquid scintillation counter Isolation of low density detergent-insoluble membrane domains Low density detergent-insoluble membrane domains were purified from HEK-OTR membranes essentially as described [38] Membranes (500 lg) were resuspended in 100 lL ice cold solubilization buffer [1% (w ⁄ v) detergent, 40 mm Hepes, pH 7.4, 10% (w ⁄ v) glycerol, 300 mm NaCl, 10 mm MgCl2] After 30 min, the solubilized membranes were diluted : with 80% (w ⁄ v) sucrose and placed at the bottom of a 5.5 mL ultracentrifuge tube A discontinuous gradient was formed above the lysate by adding mL each of 30% and 5% (w ⁄ v) sucrose solutions in 20 mm Hepes, pH 7.4, mm MgCl2, and 150 mm NaCl The tubes were centrifuged at 190 000 g for 18 h in a SW-41 rotor at °C Fractions (350 lL) were collected beginning at the top of the gradient Western blotting Proteins in sample buffer were incubated at 100 °C for min, separated by 11% SDS ⁄ PAGE [39] and transferred to Hybond ECL nitrocellulose (Amersham Corp.) Membranes were blocked with 2.5% nonfat milk protein in 10 mm Tris ⁄ HCl, pH 7.4, 0.8% NaCl, 0.02% KCl, 0.05% (w ⁄ v) Tween-20 at °C for 16 h Membranes were probed with anti-caveolin or anti-H-ras IgG, respectively, at 20 °C for h Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents Total protein was visualized by incubating the membrane with 810 K Gehrig-Burger et al Ponceau S red at 20 °C for and subsequent washing with distilled water to remove background Lipid analysis The samples were extracted with chloroform ⁄ methanol ⁄ water according to the method of Bligh & Dyer [40], with slight modifications Briefly, fractions of the caveolae preparation (350 lL each) and 1310 lL chloroform ⁄ methanol (1 : 2, v ⁄ v) were vigorously mixed in a thermomixer at 30 °C for 10 and centrifuged at 21 000 g for 10 The supernatant was mixed with 440 lL of chloroform and 440 lL of water and was centrifuged at 21 000 g for 30 The bottom lipid phase was evaporated under N2 and used for sphingomyelin and cholesterol determinations For determination of sphingomyelin the lipids were resolved by thin-layer chromatography on Silica Gel G plates in chloroform ⁄ methanol ⁄ water (65 : 25 : 4, v ⁄ v) Cholesteryl palmitate, cholesterol, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin were used as lipid standards The plates were developed by spraying with sulfuric acid ⁄ methanol (50 : 50, v ⁄ v) and charring at 120 °C for 30 To determine relative amounts of sphingomyelin densitometric analysis was performed using aida (advanced image data analyzer, version 3.50, Raytest, Straubenhardt, Germany) Cholesterol was assayed spectrophotometrically using the cholesterol oxidase-based cholesterol diagnostic kit from R-Biopharm (Darmstadt, Germany) Statistics Tukey’s post-test was performed using graphpad prism version 4.01 for Windows (GraphPad Software, San Diego, CA, USA) Acknowledgements This work 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Nature 227, 680–685 40 Bligh ED & Dyer WJ (1959) A rapid method of total lipid extraction and purification Can J Biochem Physiol 37, 911–917 FEBS Journal 272 (2005) 800–812 ª 2005 FEBS ... pellets with a radioactive photoactivatable cholesterol analogue and analyzed the incorporation rates of the cholesterol probe The data suggest that the novel CHAPSTEROL is an appropriate detergent. .. vials and made transparent with 10 mL of filtersafe (Zinsser Analytic, Frankfurt, Germany) 809 A novel cholesterol-based detergent Radioactivity was measured in a LKB 1215 Rackbeta liquid scintillation... using aida (advanced image data analyzer, version 3.50, Raytest, Straubenhardt, Germany) Cholesterol was assayed spectrophotometrically using the cholesterol oxidase-based cholesterol diagnostic