219
Biochimica et Biophysica Acta, 1087 (1990) 219-225 Elsevier
BBAEXP 92165
Partial apolipoprotein E-fl-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor Agn~s Ribeiro 1, Philippe Cardot 1,., Richard Benarous 2, Patrick Pernas 1, Dominique Pepin a, Tania Rybkine 1, Jean Chambaz 1 and Gilbert Bereziat 1 I UPA CNRS 1283, CHUSaint-Antoine, Paris and 21NSERM U15, CHU Cochin, Paris (France)
(Received 23 February 1990) (Revised manuscript received 20 June 1990)
Key words: Fusion protein; XGT11 expression vector, Apolipoprotein E; LDL(B/E) receptor; Fibroblast
A partial rat apo E-fl-galaetosidase fusion protein was produced in Escheriehia coli Y1089 infected with recombinant ?~GTll obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for aim E binding domain towards the LDLOB/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a K d of 5 • 10 -s M and 4.1 • l0 s sites per cell. Fusion protein binding to fibroblasts was mediated by their aim E moiety and not by fl-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) fl-galactosidase did not compete with fusion protein binding;, and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in L D L ( B / E ) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native aim E. However, compared to full-length native or engineered aim E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the L D L ( B / E ) receptor and for mapping residues and domains involved in the binding process.
Introduction
Plasma low-density lipoproteins (LDL) are the major lipoprotein class involved in cholesterol supply to various non hepatic tissues and cells in the body. The delivery of cholesterol to the cells through the LDL(B/E) receptor pathway accounts for the regulation of hydroxymethylglutaryl CoA reductase and acyl CoA:cholesterol acyltransferase in cultured human fibroblasts [1,2]. LDL internalization and cholesterol processing are initiated after LDL bind to a specific high affinity receptor on the cell surface (for review, see Ref. 3). It has been shown that lipoproteins containing apolipoprotein B (apo B) [4] as well as the arginine-rich apolipoprotein E (apo E) [5] bind to the same cell surface receptor named LDL(B/E) receptor. The receptor
* Present address: INRA, 63122 Ceyrat, France. Correspondence: A. Ribeiro, URA CNRS 1283, CHU Saint-Antoine, 27 rue de Chaligny, 75012 Paris, France.
purified from bovine adrenal cortex membranes binds HDL¢, an HDL class containing apo E, with a 20-fold higher affinity than LDL but with a total binding 4-fold lower [6]. Large triacylglycerol-rich lipoproteins from hyperlipidemic patients bind to the LDL receptor but normal very low density hpoproteins (VLDL) do not [7]. Apo E has been shown to mediate this binding [8], whereas apo B is unnecessary [9]. Though apo E is responsible for the binding, native delipidated apo E does not bind to the L D L ( B / E ) receptor and must be reeombined with phospholipids to recover its binding activity [10]. The main binding domain of apo E has been identified by various studies including arginine [11] or lysine [12] chemical modifications, sequencing of human apo E2, E3 and E4 isoforms [13,14], binding experiments of apo E fragments to the L D L ( B / E ) receptor [15] and inhibition of the binding by monoclonal antibodies [16]. It is located between residues 140 to 150 of human apo E and residues Arg-145, Lys-146 and Arg-158 play a crucial role in the binding. By comparing human [14], mouse [17] and rat [18] sequences, the murine apo E binding domain has been located between residues 132
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
220 to 142 with Arg-137, Lys-138 and Arg-150 as important residues. In the present paper we report the binding properties of a partial apo E-B-galactosidase fusion protein produced in E. coli infected with recombinant )~GTll clone selected by screening of a rat liver c D N A library [19]. Though displaying a partial sequence of apo E overlapping the apo E sequence coding for the binding domain to the L D L ( B / E ) receptor, the fusion protein retained functional properties of apo E and was used for assaying binding of apo E to the L D L ( B / E ) receptor. Materials and Methods
Chemicals. Restriction endonuclease EcoRI, the nick translation labelling D N A kit, Na125I (13-17 mCi/mg), [a-35S]dATP aS ( > 6 0 0 Ci/mmol) and [c~-32P]dCTP ( > 3000 Ci/mmol), were from Amersham (U.K.). All the enzymatic reactions were performed according to the supplier's instructions. Nitrocellulose membranes for R N A and protein blotting (BA 85, 0.45 mm) were purchased from Schleicher and Schuell (Dassel, F.R.G.). Protosorb lacZ immuno-adsorbent was purchased from Promega (Madison WI), E. coli /3-galactosidase, from Sigma (St Louis, MO) and Human fetal lung fibroblasts (MRCS), from Biomerieux (France). Dulbecco's modified Minimum Essential Medium (DMEM) with Earle's salts and fetal calf serum (FCS) were from Gibco (Grand Island, NY), Ultroser G, from IBF (France) and bovine serum albumin (BSA), from Boehringer (Mannheim, F.R.G.). Apo E cDNA cloning Preparation of anti-rat LDL antiserum. LDL were isolated from rat serum by sequential ultracentrifugations performed successively at densities 1.006 and 1.063 g / m l as described by Lindgred [20]. The L D L fraction was washed under the isolation conditions and dialyzed against 0.15 M NaC1 buffer (pH 7.4) containing 0.01% EDTA and 0.02% N a N 3. L D L were emulsified in Freund's adjuvant then administered to rabbit by successive Booster injections. Antiserum samples, stored at - 2 0 ° C , were titrated by ELISA procedure and their specificity was checked by polyacrylamide gel electrophoresis (PAGE) and Western blotting with rat serum lipoproteins. The anti-rat LDL antiserum reacted mainly to apo B, but also very weakly to apo E (Fig 1, lane C). Screening of the rat liver eDNA library. The screening was performed according to Young and Davis [19] using a rat liver XGTll c D N A expression library (kindly given by J. Tamkun, MIT) expressed in E. eoli Y1090 [21]. 250 000 recombinants were screened with the antirat LDL antiserum diluted to 1:500. To eliminate the antibodies directed against E. coli or coliphage X proteins, the antiserum was previously treated by affinity
adsorption according to De Wet et al. [22]. Clones were purified by successive screening. Selected positives clones were checked as follows: fusion proteins were secreted onto nitrocellulose filters by dot cultures containing 103 recombinants. These filters were probed with the anti-LDL antiserum either previously incubated with or without an excess of rat serum LDL (results not shown). Epitope selection from anti-LDL serum using fusion proteins [23]. Fusion proteins secreted by E. coli Y1090 infected with recombinants clones were blotted onto nitrocellulose filters, then incubated with the anti-LDL antiserum diluted at 1:200 in Tris-saline buffer (TBS) (pH 8), for I h. After three washings with 0.5% Triton X-100 in TBS to remove non-specifically retained antibodies, the specific anti-fusion protein antibodies were eluted with a 5 mM glycine-HC1 buffer (pH 2.3) containing 150 mM NaC1, 0.5% Triton X-100 and 0.1 g/1 albumin. After neutralization with 1 M Tris-base (1 : 20, v/v) and concentration in dialysis tubing embedded in polyethylene glycol 20 M, anti-fusion protein antibodies were titrated by ELISA procedure. Their specificity was characterized by SDS-PAGE and Western blotting. cDNA characterization, cDNAs were obtained from the selected recombinants by EcoRI digestion. An aliquot was labelled with [32 P]dCTP by nick translation [24] and used for Northern Blotting [25] of total polyA R N A prepared from rat liver according to Chirwing et al. [26]. Hybridization and washings were carried out at 65°C. Another aliquot of c D N A was incorporated into the M13 mpl8 vector according to Messing and Vieira [27] and D N A sequencing was performed using deoxyadenosine 5' [a-35S]dATP 0~S as described by Sanger et al. [28].
Preparation of fusion protein Lysogens. Lysogenic E. coli Y 1089 were prepared from recombinant clones according to Young and Davis [19]. To produce fusion protein, lysogenic cells were first cultured in L Brooth medium at 30°C until absorbance at 600 nm reached 0.5. The lytic cycle was then triggered by raising quickly temperature up to 4 2 ° C for 15 min and fusion protein synthesis was induced with 10 mM isopropyl /3-D-thiogalactopyranoside (IPTG). Isolation of fusion protein. After a 1 h incubation at 37°C, cells were centrifuged for 15 rain at 10000 × g at 3 7 ° C and were resuspended in TEP buffer: 100 mM Tris-HC1 (pH 7.4), 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell disruption was completed by freezing in liquid nitrogen and sonication. Sonicated extracts were centrifuged for 10 min at 10000 × g at 4°C. Surpenatants were treated with three volumes of saturated ammonium sulfate and centrifuged for 20 min at 10000 × g at 4°C. Pellets were dissolved in cold TEP buffer at a concentration of 20
221 mg protein/ml. Samples were finally diluted 5-fold with 50 mM Tris-HC1 (pH 7.4), 150 mM NaC1, 0.2% NP40. Fusion protein was purified by immunoaffinity adsorption on Protosorb lacZ and eluted by raising the pH to 10.8 using 0.1 M bicarbonate buffer according to the manufacturer. After concentration, the purity of fusion protein preparation was checked by SDS-PAGE. Polyacrylamide gel electrophoresis (PAGE) and Western blotting. Electrophoresis was performed in the presence of 1% sodium dodecyl sulfate (SDS) according to Laemmli [29]. Samples of rat lipoproteins were preincubated for 1 h at 37 ° C in 0.5 M Tris buffer (pH 6.8) containing 20% sucrose, 0.05% bromophenol blue and 1.8% SDS. Samples of fusion protein and standard proteins were preincubated for 2 min at 95°C in the same buffer containing 5% fl-mercaptoethanol. Western blotting was done according to Burnette [30]. Electrophoretic transfer onto nitrocellulose membranes was carried out for 16 h at a constant voltage of 20 V in 25 mM Tris-base buffer (pH 8.3) containing 0.192 M glycine. The antigen-antibody complex was revealed either with peroxidase-labelled anti-rabbit IgG antiserum or with 125I-labelled protein A (700 000 c p m / m l ) for 1 h. Proteins were assayed according to Lowry et al. [311.
Binding experiments with fusion proteins Protein labelling. Human LDL were prepared as described above. Fusion protein, fl-galactosidase and human LDL were iodinated using the technique of Bilheimer et al. [32]. 125I-labelled proteins were extensively dialyzed against saline-buffer to remove free iodine. Specific activity was 600-900 dpm/ng. Binding assays. Experiments were carried out with human fetal lung fibroblasts (MRC5) or with skin fibroblasts from a patient with homozygous familial hypercholesterolemia (kindly given by J. Chapman, Paris). Cells were maintained in D M E M medium supplemented with 10% FCS at 37°C under 5% CO 2 humid atmosphere. Cells were grown for 24 h in DMEM medium supplemented with 2% Ultroser (lipoproteinfree serum substitute) in 30 mm Petri dishes, in order to overexpress LDL(B/E) receptor. This preincubation was followed by three washings with PBS (pH 7.4). Binding studies were then carried out with the indicated amounts of 125I-labelled fusion protein, a25I-fl-galactosidase or human t25I-labelled LDL in 0.5 mL DMEM, 10 m M Hepes medium, (pH 7.4) for 1 h at 4°C, according to Goldstein and Brown [33]. After four washings with PBS (pH 7.4), cells were harvested using a rubber policeman, centrifuged for 5 min at 400 × g and radioactivity associated with the pellet was measured using a gamma counter. Competition experiments were carried out by adding indicated amounts of unlabelled fusion protein, human
LDL or fl-galactosidase to cells at 4 o C, 30 rain prior to the addition of 12 or 16 pmol iodinated ligands. Results
Isolation and characterization of apo E cDNA clones h G T l l is a general recombinant DNA expression vector capable of producing polypeptides specified by inserted DNA. Recombinants comprise insert D N A at the EcoRI site of lacZ gene, and produce fl-galactosidase fused to the polypeptide of interest, depending on foreign D N A orientation and reading frame with respect to those of lacZ. Initial plaque-screening of rat liver cDNA library was performed with 250 000 recombinants using anti-rat LDL antiserum. Clones were isolated after four rounds of purification and their identity was confirmed by immunological and RNA hybridization studies. Antibodies which specifically bound fusion proteins expressed by recombinant clones were isolated from total anti-LDL antiserum by epitope selection [23]. The MW kDa
C
R
;',,
apoB 200 116 92 67
45
apoE 30
A
B
A
B
A
B
Fig. 1. Characterization of recombinant clone by epitope-selection. Fusion protein secreted by recombinant clones was characterized by a two-step procedure: 1 - they were used to select specific anti-fusion protein antibodies from the anti-LDL antiserum. 2 - the specifity of these anti-FP antibodies was identified by Western blotting with rat lipoproteins. 1 mg of either rat L D L + VLDL (B) or rat H D L (A) were submitted to electrophoresis with 12% polyaerylamide in presence of 1% SDS, then to Western blotting. Immunodetection was performed with a 1:10000 dilution of either crude anti-LDL antiserum (lane C) or antibodies obtained by epitope selection from recombinant clone (lane R) or from a non-recombinant X G T l l clone (lane ~). Antigen-antibody complexes were revealed with 12~I-protein A.
222
A Ioe Z
Biochimica et Biophysica Acta, 1087 (1990) 219-225 Elsevier
BBAEXP 92165
Partial apolipoprotein E-fl-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor Agn~s Ribeiro 1, Philippe Cardot 1,., Richard Benarous 2, Patrick Pernas 1, Dominique Pepin a, Tania Rybkine 1, Jean Chambaz 1 and Gilbert Bereziat 1 I UPA CNRS 1283, CHUSaint-Antoine, Paris and 21NSERM U15, CHU Cochin, Paris (France)
(Received 23 February 1990) (Revised manuscript received 20 June 1990)
Key words: Fusion protein; XGT11 expression vector, Apolipoprotein E; LDL(B/E) receptor; Fibroblast
A partial rat apo E-fl-galaetosidase fusion protein was produced in Escheriehia coli Y1089 infected with recombinant ?~GTll obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for aim E binding domain towards the LDLOB/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a K d of 5 • 10 -s M and 4.1 • l0 s sites per cell. Fusion protein binding to fibroblasts was mediated by their aim E moiety and not by fl-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) fl-galactosidase did not compete with fusion protein binding;, and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in L D L ( B / E ) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native aim E. However, compared to full-length native or engineered aim E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the L D L ( B / E ) receptor and for mapping residues and domains involved in the binding process.
Introduction
Plasma low-density lipoproteins (LDL) are the major lipoprotein class involved in cholesterol supply to various non hepatic tissues and cells in the body. The delivery of cholesterol to the cells through the LDL(B/E) receptor pathway accounts for the regulation of hydroxymethylglutaryl CoA reductase and acyl CoA:cholesterol acyltransferase in cultured human fibroblasts [1,2]. LDL internalization and cholesterol processing are initiated after LDL bind to a specific high affinity receptor on the cell surface (for review, see Ref. 3). It has been shown that lipoproteins containing apolipoprotein B (apo B) [4] as well as the arginine-rich apolipoprotein E (apo E) [5] bind to the same cell surface receptor named LDL(B/E) receptor. The receptor
* Present address: INRA, 63122 Ceyrat, France. Correspondence: A. Ribeiro, URA CNRS 1283, CHU Saint-Antoine, 27 rue de Chaligny, 75012 Paris, France.
purified from bovine adrenal cortex membranes binds HDL¢, an HDL class containing apo E, with a 20-fold higher affinity than LDL but with a total binding 4-fold lower [6]. Large triacylglycerol-rich lipoproteins from hyperlipidemic patients bind to the LDL receptor but normal very low density hpoproteins (VLDL) do not [7]. Apo E has been shown to mediate this binding [8], whereas apo B is unnecessary [9]. Though apo E is responsible for the binding, native delipidated apo E does not bind to the L D L ( B / E ) receptor and must be reeombined with phospholipids to recover its binding activity [10]. The main binding domain of apo E has been identified by various studies including arginine [11] or lysine [12] chemical modifications, sequencing of human apo E2, E3 and E4 isoforms [13,14], binding experiments of apo E fragments to the L D L ( B / E ) receptor [15] and inhibition of the binding by monoclonal antibodies [16]. It is located between residues 140 to 150 of human apo E and residues Arg-145, Lys-146 and Arg-158 play a crucial role in the binding. By comparing human [14], mouse [17] and rat [18] sequences, the murine apo E binding domain has been located between residues 132
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
220 to 142 with Arg-137, Lys-138 and Arg-150 as important residues. In the present paper we report the binding properties of a partial apo E-B-galactosidase fusion protein produced in E. coli infected with recombinant )~GTll clone selected by screening of a rat liver c D N A library [19]. Though displaying a partial sequence of apo E overlapping the apo E sequence coding for the binding domain to the L D L ( B / E ) receptor, the fusion protein retained functional properties of apo E and was used for assaying binding of apo E to the L D L ( B / E ) receptor. Materials and Methods
Chemicals. Restriction endonuclease EcoRI, the nick translation labelling D N A kit, Na125I (13-17 mCi/mg), [a-35S]dATP aS ( > 6 0 0 Ci/mmol) and [c~-32P]dCTP ( > 3000 Ci/mmol), were from Amersham (U.K.). All the enzymatic reactions were performed according to the supplier's instructions. Nitrocellulose membranes for R N A and protein blotting (BA 85, 0.45 mm) were purchased from Schleicher and Schuell (Dassel, F.R.G.). Protosorb lacZ immuno-adsorbent was purchased from Promega (Madison WI), E. coli /3-galactosidase, from Sigma (St Louis, MO) and Human fetal lung fibroblasts (MRCS), from Biomerieux (France). Dulbecco's modified Minimum Essential Medium (DMEM) with Earle's salts and fetal calf serum (FCS) were from Gibco (Grand Island, NY), Ultroser G, from IBF (France) and bovine serum albumin (BSA), from Boehringer (Mannheim, F.R.G.). Apo E cDNA cloning Preparation of anti-rat LDL antiserum. LDL were isolated from rat serum by sequential ultracentrifugations performed successively at densities 1.006 and 1.063 g / m l as described by Lindgred [20]. The L D L fraction was washed under the isolation conditions and dialyzed against 0.15 M NaC1 buffer (pH 7.4) containing 0.01% EDTA and 0.02% N a N 3. L D L were emulsified in Freund's adjuvant then administered to rabbit by successive Booster injections. Antiserum samples, stored at - 2 0 ° C , were titrated by ELISA procedure and their specificity was checked by polyacrylamide gel electrophoresis (PAGE) and Western blotting with rat serum lipoproteins. The anti-rat LDL antiserum reacted mainly to apo B, but also very weakly to apo E (Fig 1, lane C). Screening of the rat liver eDNA library. The screening was performed according to Young and Davis [19] using a rat liver XGTll c D N A expression library (kindly given by J. Tamkun, MIT) expressed in E. eoli Y1090 [21]. 250 000 recombinants were screened with the antirat LDL antiserum diluted to 1:500. To eliminate the antibodies directed against E. coli or coliphage X proteins, the antiserum was previously treated by affinity
adsorption according to De Wet et al. [22]. Clones were purified by successive screening. Selected positives clones were checked as follows: fusion proteins were secreted onto nitrocellulose filters by dot cultures containing 103 recombinants. These filters were probed with the anti-LDL antiserum either previously incubated with or without an excess of rat serum LDL (results not shown). Epitope selection from anti-LDL serum using fusion proteins [23]. Fusion proteins secreted by E. coli Y1090 infected with recombinants clones were blotted onto nitrocellulose filters, then incubated with the anti-LDL antiserum diluted at 1:200 in Tris-saline buffer (TBS) (pH 8), for I h. After three washings with 0.5% Triton X-100 in TBS to remove non-specifically retained antibodies, the specific anti-fusion protein antibodies were eluted with a 5 mM glycine-HC1 buffer (pH 2.3) containing 150 mM NaC1, 0.5% Triton X-100 and 0.1 g/1 albumin. After neutralization with 1 M Tris-base (1 : 20, v/v) and concentration in dialysis tubing embedded in polyethylene glycol 20 M, anti-fusion protein antibodies were titrated by ELISA procedure. Their specificity was characterized by SDS-PAGE and Western blotting. cDNA characterization, cDNAs were obtained from the selected recombinants by EcoRI digestion. An aliquot was labelled with [32 P]dCTP by nick translation [24] and used for Northern Blotting [25] of total polyA R N A prepared from rat liver according to Chirwing et al. [26]. Hybridization and washings were carried out at 65°C. Another aliquot of c D N A was incorporated into the M13 mpl8 vector according to Messing and Vieira [27] and D N A sequencing was performed using deoxyadenosine 5' [a-35S]dATP 0~S as described by Sanger et al. [28].
Preparation of fusion protein Lysogens. Lysogenic E. coli Y 1089 were prepared from recombinant clones according to Young and Davis [19]. To produce fusion protein, lysogenic cells were first cultured in L Brooth medium at 30°C until absorbance at 600 nm reached 0.5. The lytic cycle was then triggered by raising quickly temperature up to 4 2 ° C for 15 min and fusion protein synthesis was induced with 10 mM isopropyl /3-D-thiogalactopyranoside (IPTG). Isolation of fusion protein. After a 1 h incubation at 37°C, cells were centrifuged for 15 rain at 10000 × g at 3 7 ° C and were resuspended in TEP buffer: 100 mM Tris-HC1 (pH 7.4), 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell disruption was completed by freezing in liquid nitrogen and sonication. Sonicated extracts were centrifuged for 10 min at 10000 × g at 4°C. Surpenatants were treated with three volumes of saturated ammonium sulfate and centrifuged for 20 min at 10000 × g at 4°C. Pellets were dissolved in cold TEP buffer at a concentration of 20
221 mg protein/ml. Samples were finally diluted 5-fold with 50 mM Tris-HC1 (pH 7.4), 150 mM NaC1, 0.2% NP40. Fusion protein was purified by immunoaffinity adsorption on Protosorb lacZ and eluted by raising the pH to 10.8 using 0.1 M bicarbonate buffer according to the manufacturer. After concentration, the purity of fusion protein preparation was checked by SDS-PAGE. Polyacrylamide gel electrophoresis (PAGE) and Western blotting. Electrophoresis was performed in the presence of 1% sodium dodecyl sulfate (SDS) according to Laemmli [29]. Samples of rat lipoproteins were preincubated for 1 h at 37 ° C in 0.5 M Tris buffer (pH 6.8) containing 20% sucrose, 0.05% bromophenol blue and 1.8% SDS. Samples of fusion protein and standard proteins were preincubated for 2 min at 95°C in the same buffer containing 5% fl-mercaptoethanol. Western blotting was done according to Burnette [30]. Electrophoretic transfer onto nitrocellulose membranes was carried out for 16 h at a constant voltage of 20 V in 25 mM Tris-base buffer (pH 8.3) containing 0.192 M glycine. The antigen-antibody complex was revealed either with peroxidase-labelled anti-rabbit IgG antiserum or with 125I-labelled protein A (700 000 c p m / m l ) for 1 h. Proteins were assayed according to Lowry et al. [311.
Binding experiments with fusion proteins Protein labelling. Human LDL were prepared as described above. Fusion protein, fl-galactosidase and human LDL were iodinated using the technique of Bilheimer et al. [32]. 125I-labelled proteins were extensively dialyzed against saline-buffer to remove free iodine. Specific activity was 600-900 dpm/ng. Binding assays. Experiments were carried out with human fetal lung fibroblasts (MRC5) or with skin fibroblasts from a patient with homozygous familial hypercholesterolemia (kindly given by J. Chapman, Paris). Cells were maintained in D M E M medium supplemented with 10% FCS at 37°C under 5% CO 2 humid atmosphere. Cells were grown for 24 h in DMEM medium supplemented with 2% Ultroser (lipoproteinfree serum substitute) in 30 mm Petri dishes, in order to overexpress LDL(B/E) receptor. This preincubation was followed by three washings with PBS (pH 7.4). Binding studies were then carried out with the indicated amounts of 125I-labelled fusion protein, a25I-fl-galactosidase or human t25I-labelled LDL in 0.5 mL DMEM, 10 m M Hepes medium, (pH 7.4) for 1 h at 4°C, according to Goldstein and Brown [33]. After four washings with PBS (pH 7.4), cells were harvested using a rubber policeman, centrifuged for 5 min at 400 × g and radioactivity associated with the pellet was measured using a gamma counter. Competition experiments were carried out by adding indicated amounts of unlabelled fusion protein, human
LDL or fl-galactosidase to cells at 4 o C, 30 rain prior to the addition of 12 or 16 pmol iodinated ligands. Results
Isolation and characterization of apo E cDNA clones h G T l l is a general recombinant DNA expression vector capable of producing polypeptides specified by inserted DNA. Recombinants comprise insert D N A at the EcoRI site of lacZ gene, and produce fl-galactosidase fused to the polypeptide of interest, depending on foreign D N A orientation and reading frame with respect to those of lacZ. Initial plaque-screening of rat liver cDNA library was performed with 250 000 recombinants using anti-rat LDL antiserum. Clones were isolated after four rounds of purification and their identity was confirmed by immunological and RNA hybridization studies. Antibodies which specifically bound fusion proteins expressed by recombinant clones were isolated from total anti-LDL antiserum by epitope selection [23]. The MW kDa
C
R
;',,
apoB 200 116 92 67
45
apoE 30
A
B
A
B
A
B
Fig. 1. Characterization of recombinant clone by epitope-selection. Fusion protein secreted by recombinant clones was characterized by a two-step procedure: 1 - they were used to select specific anti-fusion protein antibodies from the anti-LDL antiserum. 2 - the specifity of these anti-FP antibodies was identified by Western blotting with rat lipoproteins. 1 mg of either rat L D L + VLDL (B) or rat H D L (A) were submitted to electrophoresis with 12% polyaerylamide in presence of 1% SDS, then to Western blotting. Immunodetection was performed with a 1:10000 dilution of either crude anti-LDL antiserum (lane C) or antibodies obtained by epitope selection from recombinant clone (lane R) or from a non-recombinant X G T l l clone (lane ~). Antigen-antibody complexes were revealed with 12~I-protein A.
222
A Ioe Z
