Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

BB Biochif ic~a

et Biophysica A~ta

ELSEVIER

Biochimica et Biophysica Acta 1259 (1995) 245-253

Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein Burkhard Rolf

a,

Elke Oudenampsen-Kriiger a, Torsten BSrchers a,b, Nils Joakim F~ergeman Jens Knudsen c, Axel Lezius aT Friedrich Spener a,b,*

C

a Department ofBiochemistr3', Unit,ersi~' ofMiinster, Wilhelm-Klemm-Str. 2, 48149 Miinster, Germany Institute of Chemical and Biochemical Sensor Research, Mi~nster, Germany c Institute ofBiochemisto', Odense Unicersio', Odense, Denmark

Received 3 April 1995; accepted 3 August 1995

Abstract

The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coil (E. coil) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Ko of 0.26 tzM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay and a fluorescence displacement assay. In none of the assays binding of cholesterol to L-FABP was observed. Keywords: Liver-type fatty acid binding protein; Titration calorimetry; Ligand binding

1. Introduction

Fatty acid binding proteins (FABP) belong to the family of 14-15 kDa cytosolic proteins that bind hydrophobic ligands. This family consists of 8 different FABPs with a sequence homology of between 20 and 70%, of four proteins that bind retinal, retinol and retinoic acid and of one protein that preferably binds bile acids. Members of this protein family are expressed in a variety of mammalian tissues, some tissues contain more than one FABP

Abbreviations: ELISA, enzyme-linked immunosorbent assay; RFI, relative fluorescence intensity; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RT-PCR, reverse transcription-polymerase chain reaction; LPA, lysophosphatidic acid (l-oleoyl-sn-glycerol3-phosphate); L-FABP, liver-type fatty acid binding protein; ACBP, acyl-CoA binding protein; SCP 2, sterol carrier protein 2 * Corresponding author (at address a). Fax: + 4 9 251 838358; e-mail: spener @uni-muenster.de. t The nucleotide sequence data reported in this paper appear in the EMPL, GenBank and DDBJ nucleotide sequence databases under the accesion number: X86904 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 1 7 0 - 0

[1,2]. The epidermal [3] and brain type [4] FABPs and a testis lipid binding protein [5] have only recently been described, the latter thus far only by sequence homology. The liver-type FABP (L-FABP) is abundantly expressed in the liver up to 5% of the total cytosolic proteins and to slightly lower amounts in the intestine [6]. L-FABP binds fatty acids like the other types of the FABP subgroup, however in addition it also binds lysophosphatidic acid [7], heme [8] and some prostaglandins [9,10]. The isoelectric heterogeneity for L-FABP isolated from bovine liver [11] has been shown to depend on the state of lipidation and on an amino acid exchange at position 105 (Asn vs. Asp), as well as on a covalent modification of the protein's sole cysteine [12]. The proposed function of these proteins is to mediate the intracellular transport of fatty acids, the formation of an intracellular pool of fatty acids, the protection of enzymes and membranes from the detergent effect of fatty acids and the modulation of enzymatic activities by the control of the concentration of free fatty acids. Binding of acyl-CoA's to L-FABP has been controversially discussed in the literature since an acyl-CoA binding protein (ACBP),

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a 10 kDa protein not related to the FABPs was discovered [13] and shown to efficiently compete with L-FABP for acyl-CoA binding [14]. The recent report of Hubbel et al. [15] on the binding of acyl-CoA's to L-FABP demonstrated that the existence of a specific cytosolic transporter for acyl-CoA's is still a matter of debate. Better preparations of the respective proteins and a new method for measuring binding constants should improve our understanding of the physiological function of L-FABP and ACBP. Besides acyl-CoA, cholesterol has also been discussed as a possible ligand for L-FABP [16]. In several cholesterol and fatty acid binding assays, Scallen et al. [17] compared L-FABP to SCP2, the other proposed cholesterol binding protein present in liver cytosol [18], finding no affinity of L-FABP for cholesterol. In contrast to these studies Nemecz and Schroeder [19] found an affinity of L-FABP for cholesterol and the fluorescent cholesterol analogue dehydroergosterol, which is comparable to that for fatty acids. Another class of interesting ligands for L-FABP are peroxisomal proliferators such as fibrates. Although these xenobiotics are used as drugs to lower lipid levels, little is known about the mechanism by which they activate genes of the fatty acid metabolism. Several data recently indicated that the peroxisomal proliferator activated receptor (PPAR), a member of the nuclear hormone receptor superfamily [20], is activated not only by peroxisomal proliferators, but also by fatty acids [21,22]. To define the role of a FABP in the cytosol parallel to the putative fatty acid activated receptor in the nucleus on the regulation of gene expression, information about the affinity of L-FABP for fatty acids and for peroxisomal proliferators are necessary. Titration calorimetry is a method for the determination of thermodynamic parameters and was recently used for the determination of binding constants of long chain acyl-CoA's to ACBP [23] and for the investigation on the binding of oleic acid to rat intestinal and liver FABP [24]. Here we describe the application of this method for the comparison of a variety of ligands in their affinity for recombinant bovine L-FABP.

2. Materials and methods

2.1. Materials Radiolabelled [l-14C]oleic acid (53,8 Ci/mol) and [1 a2a(n)-3H]cholesterol (46 Ci/mmol) were from Amersham, Braunschweig, Germany. Restriction enzymes, calf intestinal phosphatase and T4 DNA ligase were from Boehringer Mannheim, Mannheim, Germany. Taq DNA polymerase and Chromatography media were from Pharmacia, Freiburg, Germany and Lipidex-1000 was from Canberra Packard, Groningen, The Netherlands. All other chemicals were of analytical grade or better.

2.2. Cloning of the cDNA First strand cDNA was synthesised using 0.5 /zg bovine liver poly-A mRNA (Renner, Dannstadt, Germany), MMuLV reverse transcriptase (New England Biolabs, Schwalbach/Taunus, Germany) and oligo dTl5 as primer in a final volume of 20 /xl. 2 /zl of this reaction was used as template for a PCR carried out in a final volume of 50 /xl containing 200 /xM of each dNTP, 5 /zl 10 X PCRbuffer and 2.5 u Taq DNA polymerase (all Pharmacia, Freiburg, Germany), 10 pmol upstream primer (5'CCTCATTGCCCATATGAACTTCTCCGG-3') and 10 pmol downstream primer (5'-AAATACAGCGGATCCTAAATTCTCTFGCTGACTC-3'). Primers were designed according to the known rat L-FABP sequence [25] because the N-terminal and C-terminal amino acids of both proteins are identical. The recognition sequences for the restriction endonucleases NdeI (upstream primer) and BamHI (downstream primer) that were incorporated for our cloning strategy are in italics. They were included for the expression of the protein using the pET-system [26]. The reaction mix was overlaid with mineral oil and PCR was carried out with the following parameters: 1 min 94°C, 1 min 58°C, 1 rain 72°C, 30 cycles and a final extension step at 72°C for 10 rain. The resulting 400 bp fragment was isolated from a preparative agarose gel, blunt ended and cloned into the Sinai site of pT7/T13a-18 according to standard procedures [27]. After sequencing with Sequenase Version 2.0 DNA Sequencing Kit (USB, Cleveland, OH, USA), the BamHI/NdeI fragment was subcloned in the pET-3c expression vector (Novagen, Madison, WI, USA) and used for the transformation of E. coli BL21(DE3)pLysS.

2.3. Expression and purification of recombinant bovine L-FABP Growth of E. coli cells and the expression of the protein was carried out as described previously for recombinant bovine heart-type FABP [28]. Cells from a 2 1 culture were harvested by centrifugation (15 min, 4°C, 6000 x g) and stored overnight at -20°C. The frozen pellets were resuspended in the 3-fold ( w / v ) volume of buffer A (10 mM Tris/HCl (pH 7.4), 30 mM NaCI) containing 500 u Benzonase (Merck, Darmstadt, Germany), incubated for 10 min at room temperature and then disrupted by sonication (3 × 15 s, 0°C, 25 W). After centrifugation (1 h, 4°C, 100000 × g) for the removal of the cell debris, soluble proteins were desalted by gel filtration on a Sephadex G-25 (5 X 28 cm, 5 ml/min) column equilibrated in buffer A. Protein containing fractions were pooled (G-25 protein) and chromatographed on a Q-Sepharose Big Beads column (5 X 30 cm, 5 ml/min) in buffer A. Under these conditions, L-FABP did not bind to the column material and could be separated from most of the E. coli proteins. Fractions containing L-FABP were pooled

B. Roll et al. / Biochimica et Biophysiea Acta 1259 (1995) 245-253

(Q-BB protein) and concentrated by ultrafiltration in a stirred Amicon cell (Amicon, Witten, Germany). The concentrated solution was subjected to a final gel filtration on Sephadex S-100 HR (2.6 X 80 cm, 2 ml/min) in phosphate-buffered saline (pH 7.4). The single peak was pooled (S-100 protein) and its purity checked by means of SDSPAGE in a 13.5% gel [29]. For binding assays protein was delipidated in a Lipidex column [30]. Briefly, protein in a concentration of 1 m g / m l in 20 mM potassium phosphate, 50 mM KCI (pH 7.2) (buffer B) was run twice through the Lipidex column (1 ml of gel/mg of protein) at 37°C with a flow rate allowing a contact time of the protein to the column of at least 3 h each time. All chromatography steps were carried out with the FPLC-system (Pharmacia, Freiburg, Germany) and at room temperature, except for the Lipidex column. 2.4. Protein determination

The concentration of pure L-FABP was determined using the extinction coefficient of 0.31 ml • mg- 1 . c m at 278 nm determined gravimetrically for the p l 7.0 L-FABP isolated from bovine liver. Total protein content was measured using the bicinchonic assay and ovalbumin as standard [31]. The generation of antibodies and ELISA were performed as described by Nielsen et al. [32] for the heart-type FABP. Recombinant L-FABP was used as standard. 2.5. Titration calorimetry

Experiments were performed with a Microcal Omega titration microcalorimeter (Microcal, Northhampton, MA, USA) [33]. The solution of the delipidated protein in buffer B in the sample cell was 0.13 mM in all experiments. Titration was performed at 37°C. The ligand solution was about 6 mM in buffer B and injected in 28 aliquots (4 ~1, each) in 3 min intervals. The ligand solution was prepared as followed: 0.5 to 1 mmol of ligand was weighed in a 50 ml tube and dissolved in 25 ml of water by adding 1.1 tool per mol KOH. Then 25 ml of 2 X buffer B was added and the final concentration was adjusted by dilution with buffer B. In a typical experiment, the final ligand solution was 6 mM. All ligand solutions were prepared daily and stored at room temperature during the day. Oleoyl-CoA solution was prepared by dissolving oleoyl-CoA directly in buffer B and stored at 4°C during the day. The synthesis of oleoyl-CoA is described elsewhere [34]. The concentration of oleoyl-CoA was determined using the extinction coefficient of 14700 M -~- cm-J at 260 nm. The sample cell was stirred with 400 rpm. The reference cell contained a solution of 0.02% sodium azide in water. Calibration was done with standard electrical pulses. The raw data were processed using the Origin software supplied by the manufacturer. After the heat of dilution was subtracted from the

247

heat of binding, the data were fitted to a model assuming two independent binding sites according to [35]. During the fit, all variable parameters (binding stoichiometry, binding enthalpy and affinity constants of both binding sites) were floated. 2.6. Lipidex assay

The binding of cholesterol and oleic acid to the protein was also determined by the Lipidex method [36]. The protocol was identical to the protocol of Specht et al. [28] used for recombinant bovine heart-type FABP. Briefly, in an assay volume of 250 /zl, 0.4 to 0.6 /zM L-FABP was incubated in a buffer containing 10 mM Tris/HCl, 100 # M Triton X-100 (pH 8.0) together with radiolabelled ligand in a concentration range from 0.24 to 2.4/zM in ten steps for 1 h at 37°C. From these samples 50/~1 were used for the determination of the actual ligand concentration by means of scintillation counting. Each value was determined 5 times. Then 50 /zl of a 50% lipidex suspension (for the oleic acid binding assay) or 200 /xl of lipidex suspension (for the cholesterol binding assay) were added to the prechilled tubes and incubated at 0°C for 30 min. After centrifugation (5 min, 14000 X g) radioactivity, in 100 /zl of these solutions, was determined by scintillation counting. Each value represents a mean of 5, corrected against a blank determined 3-fold under identical conditions without protein. The blank was always below 5% of the total amount of iigand in both cases. [la-2c~(n)3H]cholesterol was added from an ethanolic stock solution to the assay tubes. The determination of the actual ligand concentration in the tubes prior to the addition of the lipidex suspension revealed no significant loss of cholesterol via precipitation. The amount of lipidex necessary to bind 95% of the cholesterol added was derived from titration experiments without protein. 2.7. Liposome binding assay

The preparation of the liposomes was done by the method of Brecher et al. [37] as described by Rasmussen et al. [14]. Briefly, 77 mg of phosphatidylcholine dissolved in chloroform and 500 nmol of radiolabelled ligand dissolved in ethanol were added into a 2 ml tube and the solvents were evaporated under a nitrogen stream. Residual solvent was removed by lyophilisation in a vacuum centrifuge for 2 h at 37°C. After resuspension in 1 ml buffer C (10 mM Tris/HCl (pH 7.4), 0.1 M NaC1) and 12 h shaking, the suspension was centrifuged for 2 min at 9000 X g. The supernatant was discarded and the liposomes were resuspended in 2 ml of the buffer C. The exact amount of ligand present in the final liposome solution was determined by liquid scintillation counting. For the binding assay, this stock solution was used to prepare 8 working solutions with ligand concentrations between 0.5 and 8 /xM by

B. Rolf et al. / Biochimica et Biophysica Acta 1259 (1995) 245-253

248

dilution with buffer C. 400 /xl of the working solutions were added to 100 /xl of a protein solution (2.5 /zM) and incubated for 30 min with shaking at room temperature. After centrifugation for 5 min at 11 000 × g, radioactivity in 200 /xl of the supernatant was measured by liquid scintillation counting. Each value was determined 4 times, the amount of liposomes that could not be spun down under these conditions was determined 3-fold in blanks without protein. 2.8. Fluorescence binding assay L-FABP (0.5 /xM) in phosphate-buffered saline was incubated with increasing amounts (0-2.6 /xM) of transparinaric acid (Molecular Probes, Eugene, OR, USA). The excitation wavelength was 324 nm (slit 2.5 nm) and the relative fluorescence intensity (RFI) was measured at 430 nm (slit 10 nm). trans-Parinaric acid was added from an ethanolic stock solution, the final ethanol concentration did not exceed 2%. All fluorescence experiments were carried out using a Perkin Elmer LF50B. 2.9. Fluorescence displacement assay L-FABP (0.5 /zM) and trans-parinaric acid (0.5 /xM) were incubated together and the relative fluorescence intensity at 430 nm was determined. The excitation wavelength was 324 nm. This solution was then titrated with the respective tigand from an ethanolic stock solution up to a 6-fold molar excess in six steps. After each addition of the competing ligand RFI was determined again. The final ethanol concentration was below 2%.

3. Results 3.1. Cloning, expression and isolation The cloning strategy for bovine L-FABP by PCR with primers deduced from the homologous rat cDNA sequence [25] and from the primary structure of the bovine protein [12] allowed us to insert restriction sites for NdeI and BamHI for the subsequent expression in the pET system [26]. Fig. 1 shows the sequence of the coding part of the cDNA and of the deduced amino acid sequence. Seven of the resulting clones were sequenced and revealed identity except for position 273, where G or C was found. This exchange has no effect on the protein sequence deduced, which correlates with the protein sequence of the p l 7.0 isoform we reported earlier [12]. The exchange NI05D yielding the p l 6.0 isoform was not detected in any of the seven clones. The covalent modification of the cysteine reported for part of the authentic bovine liver protein [12] was not observed in the recombinant protein, as all of the cysteine was accessible for the Ellman's reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (data not shown). For overexpression of recombinant bovine L-FABP we used the pET-system which we [28] and others [38,39] have successfully applied earlier for different FABPs. After subcloning of the cDNA in the pET3c-vector, we were able to express bovine L-FABP abundantly in E. coli. By sandwich-ELISA the amount was estimated at 25% of the soluble E. coli proteins. Fig. 2 shows a SDS-PAGE with proteins from different steps of the isolation process. Due to the high rate of overexpression L-FABP is almost pure after the anion exchange chromatography employing QSepharose. Routinely, a gel filtration step was employed

1 1

ATG AAC

T T C TCC GGC AAG TAC CAA GTC CAG ACC CAG GAG AAC TAT Met Asn Phe Ser Gly Lys Tyr Gln Val Gln Thr Gln Glu Asn Tyr

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GAG GCC TTC ATG AAG GCA GTC GGG ATG CCC GAT GAC ATC ATC CAG GIu Ala Phe Met Lys Ala Val Gly Met Pro Asp Asp Ile Ile Gln

91 31

AAG GGG AAG GAT ATC AAG GGG GTG TCG GAA ATC GTG CAG AAT GGG Lys Gly Lys Asp Ile Lys Gly Val Ser Glu Ile Val Gln Asn Gly

136 AAG CAC TTC AAG TTC ATC ATC ACC GCT GGC TCC A A A GTG ATC CAG 46 Lys His Phe Lys Phe Ile Ile Thr Ala Gly Ser Lys Val Ile Gln 181 AAT GAG TTC ACC TTG GGG GAG GAG TGT GAG A T G GAG TTC ATG ACT 61 Asn Glu Phe Thr Leu Gly Glu Glu Cys Glu Met Glu Phe Met Thr 226 GGG GAG AAG ATC AAG GCA GTG GTT CAG CAG GAA GGT GAT AAT A A A 76 Gly Glu Lys Ile Lys Ala Val Val Gln Gln Glu Gly A s p Asn Lys 271 CTG GTG A C A ACT TTC AAG GGC ATC AAG TCT GTG ACT GAA TTC AAT 91 Leu Val Thr Thr Phe Lys Gly Ile Lys Ser Val Thr Glu Phe Asn 316 GGT GAC ACT GTT ACC AGT ACC ATG ACG AAG GGC GAC GTT GTC TTC 106 Gly Asp Thr Val Thr Ser Thr Met Thr Lys Gly Asp Val Val Phe 361 A A G A G A G T C A G C A A G A G A A T T T A G 121 Lys Arg Val Ser Lys Arg Ile

Fig. I. Sequence of the coding part of bovine L-FABP cDNA and of the deduced amino acid sequence. Bovine liver mRNA was reverse transcribed and amplified with primers deduced from the protein sequence corresponding to the 5'- and the Y-end of the coding part of the cDNA (see Section 2). The sequence introduced by the primer is typed in bold.

B. Rolf et al. / Biochimica et Biophysica Acta 1259 (1995) 245-253

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time [min] -10

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Fig. 2. Purification progress of the recombinant bovine L-FABP. SDSPAGE, 13.5% Coomassie stain. Lane 1: 1 0 0 0 0 0 × g supernatant, lane 2: G-25 protein, lane 3: Q-BB protein, lane 4 : S - 1 0 0 protein. "~ -2 ,

for polishing and buffer adjustment. The high yield of the purified recombinant protein (about 70 mg per 2 1 of culture) allowed us to perform several binding assays, titration calorimetry as reported here and, in addition, crystallisation studies (unpublished results).

3.2. Titration calorimetry. The binding of oleic acid, oleoyl-CoA, arachidonic acid, lysophosphatidic acid and bezafibrate to L-FABP was investigated using isothermal titration calorimetry. A constant amount of protein was titrated with the ligand in all cases. Fig. 3 (upper panel) shows the raw data obtained from the titration with oleic acid. After integration of the peaks, data were fitted to the model assuming two independent binding sites. The fitted curve is shown in the lower panel of Fig. 3. The two binding sites of the protein

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oleic acid/L-FABP [mol/mol] Fig. 3. Titration calorimetry data for the binding of oleic acid to recombinant bovine L-FABP. The upper panel shows the raw data, in the lower panel the data were corrected for the heat of dilution and integrated ( • ). The curve is the result of the fit using a model assuming two independent binding sites. The concentration of the protein in the sample cell was 0.13 mM and the concentration of the ligand in the syringe was 6 mM.

can be easily distinguished in the curve due to the opposite signs of the binding enthalpy. Until the 1:1 stoichiometry is reached, the first, high affinity binding site (K d 0.26 /xM, Table 1) is loaded with oleic acid and due to the negative binding enthalpy heat is generated. Between the 1:I and 2:i stoichiometry, the reaction heat is positive

Table 1 Binding constants and enthalpies for the binding of different ligands to L-FABP determined by titration calorimetry

nl: Kdt [/.tM]: AG I [kJ/mol]: /IH~[kJ/mol]: TAS~ [kJ/mol]:

n2: Kj2 [/xM]: AG 2 [ k J / m o l ] : AH 2 [kJ/mol]: TAS z [ k J / m o l ] :

Oleic acid

Oleoyl-CoA

LPA

Arachidonic acid

Bezafibrat

1 + 0.05 0.26 + 0.01 - 39.1 ___0.2 -16+1 23 _ 1 0.9 + 0.03 4.9±0.3 - 31.5 ± 0.1 22 ± 4 53.4 ± 4

0.96 + 0.04 1 + 0.1 - 35.6 + 0.2 -18+1 18 ± 1 0.7 -t- 0.1 7.4 ± 0.4 - 29.5 ± 0.2 17 ± 4 47 ± 4

1 _ 0.03 1.3 ± 0.3 - 35 _ 0.6 -12+ 1 23 _+ 1 0.8 ± 0.2 18_+5 - 28 ± 1 8+ I 36 ± I

0.91 + 0.02 0.54 + 0.07 - 37.2 + 0.4 -16+3 23 + 3 1.7 ± 0.1 3_+ 1.6 - 33 4- 2 -3 ± 3 30 ± 4

0.79 + 0.06 0.8 + 0.1 - 36 + 1 -15.9+0.4 20+ 1 0.8 ± 0.4 12+ 1 - 29.2 ± 0.2 -5 ± 1 24 ± 1

The concentration of the delipidated L-FABP in the sample cell was 0.13 mM in all experiments, the ligand concentration at the end of the experiment in the sample cell was 4 - 5 - f o l d over the protein concentration in all cases, nil 2 is the stoichiometry for the first and the second binding site, respectively. Data are means of two experiments _ difference of the mean from the measured values.

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leading to the positive binding enthalpy for the second, low affinity binding site of bovine L-FABP ( K d 5 /zM, Table 1). In Table 1 the binding constants and binding enthalpies, free enthalpies and entropies derived from the titration of the L-FABP with the different ligands are compared. The phenomena described above for the oleic acid binding could also be observed upon the binding of oleoyl-CoA and lysophosphatidic acid. In contrast, arachidonic acid and the peroxisomal proliferator bezafibrate are also bound in a 2:1 stoichiometry, but both binding sites exhibit a negative binding enthalpy. In all cases the dissociation constants for the first ligand were all in the micro molar range, oleic acid having the highest affinity. As cholesterol cannot be used for titration calorimetry because of its low solubility, we investigated the binding of cholesterol in several other assays.

150 =o 100 .~.

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1000

2000

3000

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Fig. 5. Binding isotherms determined by the liposome assay. L-FABP (0.5 /xM) was incubated with oleic acid ( I ) or cholesterol (O) in multilamellar liposomes (0.5-8 /zM of the ligand) in a final volume of 0.5 ml for 1 h at 25°C. Liposomes were precipitated at 11000×g. All values are corrected against a blank without protein.

3.3. Lipidex assay The Lipidex assay is widely used for the measurement of affinity constants for the binding of fatty acids to FABPs. In this assay, unbound ligand is removed from the binding solution after cooling to 0°C by the Lipidex-1000 material, which is a hydrophobic derivative of Sephadex. Because of the relative low affinity of the Lipidex material for cholesterol, the amount of Lipidex in the assay had to be increased 4-fold in order to guarantee complete removal of unbound radioactive ligand applied. Analysis of the binding of oleic acid to L-FABP revealed a K d of 0.7/zM and a stoichiometry of 1.2 mol fatty a c i d / m o l protein (Fig. 4). Apparently only the high affinity binding site of L-FABP has been detected due to the only moderate excess of fatty acid used in this experiment. In contrast to others [19], who for cholesterol binding to L-FABP have determined binding parameters comparable to those for fatty acids using the Lipidex assay, we could not show any affinity of cholesterol for L-FABP.

tions. To circumvent this problem, we employed liposomes as a donor for cholesterol. With the use of multilamellar liposomes, separation of bound and unbound ligand can be achieved by centrifugation. L-FABP exhibited affinity for the membrane bound oleic acid, but no release of cholesterol from the membranes (Fig. 5) was observed. Whether this variance reflects a different affinity of the protein for cholesterol and oleic acid or a difference in the affinity of the liposomes for both molecules is not clear. Nevertheless, the results of Nemecz and Schroeder, who carried out these experiments under similar conditions and observed binding of cholesterol to the rat L-FABP [19], could not be reproduced. 3.5. Binding assay with fluorescentfatty acid trans-Parinaric acid is a fluorescent fatty acid analogue which is known to bind to bovine [40] and rat L-FABP [41]. The advantage of this binding assay is that no separa-

3.4. Extraction o f ligands from liposomes One major problem in binding studies with cholesterol is the very low solubility of cholesterol in aqueous solu-

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Fig. 4. Binding isotherms determined by the Lipidex assay. L-FABP (0.5 p,M) and oleic acid (11) or cholesterol (Q) were incubated for 1 h at 37°C, unbound ligand was removed at 0°C.

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Fig. 6. Displacement of L-FABP bound trans-parinaric acid by different ligands. Fluorescence assay (Aex 324 nm, Ae,. 430 nm); L-FABP (0.5 /zM) was charged with trans-parinaric acid (0.5 /xM) and incubated with increasing amounts (0-3 /~M) of cholesterol (O), lysophosphatidic acid (A) and oleic acid (11) at 25°C. The inset shows the binding of trans-parinaric acid to L-FABP (0.5 /zM) 25°C.

B. Rolf et al. /Biochimica et Biophysica Acta 1259 (1995) 245-253

tion between bound and unbound ligand is necessary. Bound and unbound ligand can be distinguished because free trans-parinaric acid has a negligible fluorescence intensity in aqueous solutions (Fig. 6, inset). Binding constants determined with trans-parinaric acid are consistent with those of the other assays. In competition experiments the L-FABP bound trans-parinaric acid was displaced by the addition of oleic acid and of lysophosphatidic acid as shown by the decrease of fluorescence intensity (Fig. 6). However, under identical conditions cholesterol could not displace bound fatty acid, strongly indicating that L-FABP is unable to bind cholesterol.

4. Discussion In the E. coli expression system used for bovine LFABP, the amount of recombinant protein was approximately 25% of the soluble E. coli proteins. Due to the high rate of overexpression, about 35 mg of pure recombinant protein could be obtained from 1 1 of culture (A600 = 3). This is about 20 times higher than the yield of protein from the expression systems described for human L-FABP [39] and for rat L-FABP [42]. A synthetic cDNA for rat L-FABP expressed in E. coli yielded 10 mg of pure protein per liter of culture [43]. The differences in the expression rate of bovine L-FABP and rat L-FABP might be due to the use of a different expression systems. The recently described expression of the human L-FABP [39] was carried out in the same expression system we used, in this case other reasons like RNA secondary structure or codon usage may be responsible for the low yield. The enthalpy for the binding of the second oleic acid molecule to bovine L-FABP determined by titration calorimetry is positive (Table 1) in contrast to earlier titration calorimetric studies on the binding of oleic acid to rat L-FABP [24]. But for the latter protein too the enthalpic contribution of the second ligand was lower than that of the first one. Thus the binding of the second oleic acid molecule to bovine L-FABP is, to a great extent, driven by an increase in the entropy. This may be explained by the displacement of ordered water molecules from the binding pocket of the protein and the release of these water molecules into the bulk of solvent as the main driving force. Nevertheless, not all of the ligands for bovine L-FABP investigated show this effect (Table !). Enthalpy and entropy appear to contribute both to the binding of the second molecule of arachidonic acid or bezafibrate to the second binding site. The thermodynamics of the binding of lysophosphatidic acid and oleoyl-CoA are more comparable to the binding of oleic acid, although both ligands are bound with a lower affinity than oleic acid. The binding constants for oleic acid determined by titration calorimetry are consistent to those determined earlier by the Lipidex procedure [44].

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The role of L-FABP in the intracellular acyl-CoA metabolism has been a matter of debate since the discovery of ACBP [13]. The present data unambiguously show that L-FABP is able to bind oleoyl-CoA. However, the binding affinity ( K d 1.1 /xM) is four times lower than the binding affinity for oleic acid (Ka 0.26 /zM) and five to six orders of magnitude lower than the binding of oleoylCoA to ACBP (K d 10-~3). Although the concentration of L-FABP in the liver cytosol is about ten times higher than the concentration of ACBP, L-FABP is only able to play a role in acyl-CoA metabolism under conditions when the concentrations of cytosolic long chain acyl-CoA exceeds that of ACBP. The concentration of total acyl-CoA and ACBP in rat liver have been reported to be 52 and 53 nmol/g tissue, respectively [45]. However, in the fasted rat the total acyl-CoA concentration in liver exceeds 2.5 times the ACBP concentration [46]. Therefore under these conditions L-FABP could play a role in acyI-CoA metabolism. The difference between the binding constants of acyl-CoA's to L-FABP and ACBP [23] also explains the data of Rasmussen et al. [14] obtained with the Lipidex procedure and the liposome binding assay. Both assays apparently underestimate the binding constant of acylCoA's to ACBP, thus the weaker binding of this ligand to L-FABP could not be observed. Nevertheless, binding of the bulky acyl-CoA's to the 14 kDa L-FABP may allow to develop a preliminary model of the way L-FABP binds its ligands. We have recently been able to identify Arg 122 as the cationic counterpart of the headgroup of the ligand in bovine L-FABP by modification of the arginine with phenylglyoxal [47]. In rat L-FABP, different mutants of Arg 122 were found to have a 2-4-fold reduced affinity for the fluorescent ligand dansylamino undecanoic acid (DAUDA) [48]. Knowing that this interaction of the negatively charged head group of the ligand is important for the binding process, as for example fatty acid methyl esters or monoolein are not bound (Meyjohann and Spener, unpublished results), one could imagine that this important ionic interaction takes place at the surface of the protein, an argument also favoured by Thumser et al. [49]. Binding of acyl-CoA to L-FABP would then occur via hydrophobic interaction of the hydrophobic tail of the ligand inside the protein and via ionic interaction of the pyrophosphate or the ribose 3' phosphate at the border between the interior cleft and the surface of the protein, without any contribution of the sugar and the adenine residue to the binding. This model is supported by further evidences. Cistola et al. [50] compared rat I-FABP and L-FABP fatty acid complexes at different pH values with ~3C-NMR and found that the carboxylic headgroup of the fatty acid bound to L-FABP is more solvent accessible than the headgroup of the I-FABP bound fatty acid, where it is known from the crystal structure that the headgroup of the ligand is completely buried inside the protein [51]. The above model could explain the different behaviour of the apo and holo forms of heart-, intestinal- and liver-type FABP in isoelec-

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tic focusing. Both the heart-type [52] and the intestinal-type (Meyjohann and Spener, unpublished results) exhibit the same p l whether they are lipidated or not and in both proteins the headgroup of the ligand interacts with an arginine inside the protein [51,53]. In contrast, when LFABP binds its ligands, the p I shifts to a more acidic pH [1 1,t2], possibly because a surface charge contributed by Arg 122 is complexed by the fatty acids carboxylic headgroup. The dissociation constants determined are all in t h e / x M range and the differences in the affinity of the protein for the first ligands are small (Table 1). Compared to the other members of the FABP family, which only bind fatty acids, L-FABP seems to be a more unspecific hydrophobic ligand binding protein, responsible for the transport of a variety of negatively charged, hydrophobic ligands in liver cytosol and intestinal mucosa cells. In the past and also recently a new role for L-FABP as a cholesterol binding protein was proposed [16,19]. We therefore performed several independent binding assays to test this possibility. However, in none of the experiments presented here L-FABP exhibited any affinity for cholesterol, thus an involvement of L-FABP in the transport and the metabolism of cholesterol seems unlikely. These findings differ to those of Nemecz and Schroeder [19], who determined a K d of 1.6 p~M and a binding stoichiometry of 0.83 m o l / m o l for the binding of cholesterol to recombinant rat L-FABP in a liposome assay and a Ko of 0.78 /zM and a binding stoichiometry of 0.47 in a Lipidex assay. The reason for these contradictory results is not clear, especially as both assays were performed under conditions similar to those used by Nemecz and Schroeder [19]. Rat and bovine L-FABP have a sequence identity of 80%. Although we have not studied rat L-FABP, we do not expect species specific binding properties since fatty acid binding experiments with rat and bovine L-FABP, using the same techniques, revealed similar results [24] (and this work). However, we cannot rule out that different evolutionary needs could explain a different binding behaviour for cholesterol. SCP 2, a 12 kDa protein also present in the liver and not related to the family of FABPs is another possible candidate for this function. Although direct binding of cholesterol to SCP 2 has been reported only once [17], a stimulation of enzymes of the cholesterol metabolism as the microsomal conversion of dehydrocholesterol to cholesterol by SCP 2 was reported [54]. However, although we have been unable to observe binding of cholesterol to L-FABP, one has to consider that all cholesterol binding assays are hampered by the very low solubility of cholesterol. Unfortunately, no positive control for a cholesterol binding assay is available. The binding of the peroxisomal proliferator bezafibrate to L-FABP with an affinity comparable to fatty acids, is interesting in as much as the peroxisomal proliferators are believed to bind to the peroxisomal proliferator activated receptor (PPAR), a member of the steroid and thyroid

hormone receptor superfamily [55]. As the natural ligand of the PPAR is still unknown, one may speculate whether the activation of this receptor occurs via the binding of the drug bezafibrate itself or by the binding of fatty acids which are displaced from the L-FABP by bezafibrate. The latter model has been proposed by Isseman et al. [21]. As the affinity of L-FABP for bezafibrate is approximately 3-fold less than for oleic acid, this model would require a high excess of the drug over the fatty acid. It has been shown that peroxisomal proliferators also induce the transcription of L-FABP in addition to certain enzymes of the peroxisomal lipid metabolism [56]. Kahn and Sorof [57] demonstrated that various peroxisomal proliferators induce mitogenesis only in rat hepatoma HTC-R~T3-cells that have been transfected with L-FABP cDNA in sense orientation. However, the elucidation of the exact mechanism by which L-FABP is involved in the stimulation of transacting factors and perhaps in its own expression requires further investigation.

Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 3 1 0 / A 4 ) and by the Fonds der Chemischen Industrie. B.R. is a recipient of the Graduiertenf'6rderung from the State of Northrhine-Westfalia and for his stay in Denmark he was supported by an EMBO short term fellowship. This work is part of the PhD-thesis of B.R.

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