Purification and Characterization of Recombinant Human Apolipoprotein A-II Expressed in Escherichia...

Eur. J. Biochem. 225, 1141-1150 (1994) 0 FEBS 1994

Purification and characterization of recombinant human apolipoprotein A-I1 expressed in Escherichia coli JosC LOPEZ’, Martine LATTA’, Xavier COLLET3, Berlinda VANL004, GCrard JUNG’, Patrice DENEFLE’, Maryvonne ROSSENEU4 and Jean CHAMBAZ’ ’ URA CNRS 1283, Institut des Cordeliers, Paris, France

’ U 326 INSERM, HGpital Purpan, Toulouse, France Laboratoire de Biotechnologie, Rh6ne-Poulenc Rorer, Vitry, France

Laboratory of Biochemistry, A. Z. Sint-Jan, Brugge, Belgium

(Received May 27/August 22, 1994) - EJB 94 0762/1

We have expressed recombinant human apolipoprotein A-I1 (apoA-11) in Escherichia coli, as a fusion protein with Schistosorna juponicum glutathione-S-transferase (GST). The GST-A11 fusion protein was recovered by affinity chromatography using glutathione as a ligand. After thrombin cleavage and removal of the GST carrier, recombinant apoA-I1 was obtained in a highly purified form and was exclusively composed of dimeric apoA-11. Kinetics of association to dimyristoylglycerophosphocholine (Myr,GroPCho) vesicles showed that recombinant apoA-I1 exhibited the same pattern of association as human plasma apoA-11. Electron microscopic analysis of the complexes showed a typical pattern of rouleaux, characteristic of stacked discs, with a diameter similar to that determined by gradient-gel electrophoresis. Circular dichroism measurements showed that the a- helical content of both plasma and recombinant apoA-I1 increased similarly when the proteins associated with Myr,GroPCho vesicles, at the expense of a random-coil stmcture. Lipid-bound apoA-I1 consisted of 70-72% a helices, suggesting the presence of three 18-residue a helices/apoA-I1 monomer. Cross-linking experiments indicated that Myr,GroPCho complexes contained two molecules dimeric apoA-IVvesicle. Recombinant apoA-I1 was as efficient as plasma apoA-I1 in associating with HDL subclasses, and in displacing apoA-I from dipalmitoylglycerophosphocholine/cholesterol/ apoA-I complexes, most likely due to its highly ordered secondary structure when associated with Myr,GroPCho vesicles. These findings demonstrate that recombinant apoA-I1 exhibits the same structural and functional properties as human plasma apoA-11. Thus, the expression system utilized is appropriate to produce mutagenized forms to further structure/function analysis.

Human apolipoprotein A-I1 (apoA-11) is a major component of high-density lipoproteins (HDL). It is synthesized by the liver and, to a much lesser extent, by the intestine (Schon- feld et al., 1982; Hussain and Zannis, 1990). Following secretion, apoA-I1 is mainly incorporated into lipoprotein particles (Lp) containing both apolipoprotein A-I (apoA-I) and apoA-I1 (LpA1:AII; Cheung and Albers, 1984). In human plasma, apoA-I1 occurs as a dimer consisting of two identical 77-residue subunits linked by an intra-disulfide bond at residue 6 (Brewer et al., 1972). Recent advances in apoA-I1 genetics (Lusis, 1988; Doolittle et al., 1990) have suggested that apoA-I1 plays a significant role in HDL metabolism. Overexpression of mouse apoA-I1 in transgenic

Correspondence to J. Chambaz, Centre National de la Recherche Scientifique Unit6 1283, Institut des Cordeliers, 15 rue de 1’Ecole de MCdecine, F-75006 Paris, France

Abbreviations. ApoA-I, apolipoprotein A-I ; ApoA-11, apolipoprotein A-11; CETP, cholesterol ester transfer protein; Gdn/HCl; guanidine hydrochloride ; GST, glutathione-S-transferase ; GST- AII, glutathione-S-transferase-apolipoprotein-A-I1 fusion protein; HDL, high-density lipoprotein; IPTG, isopropylthiogalactoside; LCAT, lecithine cholesterol acyl transferase ; Lp, lipoparticule ; Myr,GroPCho, dimyristoylglycerophosphocholine; Pam,GroPCho, dipalmitoylglycerophosphocholine; PhMeSO’F, phenylmethylsulfonyl fluoride.

mice results in increased HDL cholesterol and in more extensive fatty streak lesions, compared to those in normal mice (Warden et al., 1993). Moreover, transgenic mice overexpressing both human apoA-I and apoA-I1 seem less protected against atherosclerosis in response to an atherogenic diet than transgenic mice overexpressing only apoA-I (Schultz et al., 1993). In vitro experiments have recently shown that apoA-11-containing lipoproteins do not activate lecithin cholesterol acyltransferase (LCAT) and are poor sub- strates for cholesterol ester transfer protein (CETP), as compared to apoA-I-containing lipoproteins (Vanloo et al., 1992 ; Lagrost et al., 1994). ApoA-I1 is actually able to displace apoA-I from the HDL surface (Lagocki and Scanu, 1980), which might account for its ability to impair reverse cholesterol transport induced by apoA-I, and, therefore, to enhance the risk of developing coronary heart disease. The elucidation of the structure/function relationship of apoA-I1 will require further studies with apoA-I1 mutants produced by site-directed mutagenesis.

The present study describes the expression of human apoA-I1 cDNA in Escherichia coli, as a fusion protein with Schistosorna jupunicum gluthatione-S-transferase (GST). Af- ter thrombin cleavage and removal of the GST carrier, the recombinant human apoA-I1 has been obtained in a highly

1142

purified form. The physico-chemical properties of recombinant apoA-11, its ability to displace apoA-I from lipid-lipoprotein complexes, and to associate with HDL subclasses are compared to those of human plasma apoA-11.

MATERIALS AND METHODS Synthetic apoA-I1 gene and plasmid construction

A synthetic gene encoding mature human apo-A11 was prepared by ligation of three double-stranded synthetic oligo- nucleotide cassettes and subcloning of ligated cassettes into a M13mp19 plasmid. A Met-1 residue was designed in front of the Gln+l residue to direct correct translation of apoA-I1 mRNA in different procaryotic expression systems. Since these attempts failed to produce apoA-11 in a free soluble form, mature human apoA-I1 was expressed as a fusion protein with GST, using the pGEX-2T vector developed by Smith and Johnson (1988). This vector comprises the GST gene under the control of the Tnc promoter inducible by isopropylthiogalactoside (IPTG), and the termination site of the gene is substituted by a polylinker including the sequence encoding the proteolytic recognition site of thrombin. The synthetic human apoA-I1 gene was excised from the M13mp19 plasmid and subcloned in the pGEX-2T vector. The insertion and the orientation of the synthetic fragment into the recipient vector was controlled by DNA sequencing. The recombinant plasmid, designated pGEX2T-AI1, was used to transform E. coli strain TGl. Transformants were selected by their resistance to ampicillin, and designated pXL2132.

Production and purification of fusion protein GST-A-I1 pXL2132-transformed E. coli cells were cultured in a

100-L fermenter. First, 0.3 mL pXL2132 glycerol stock was cultured in 600 mL inoculum medium, containing 6 g/L Na,HPO,, 3 g/L KH2P04, 0.5 g/L NaC1, 1 g/L NH,Cl, 10 g/ L yeast extract, 0.015 g/L CaCl,, 5 g/L glucose, 0.24 g/L MgSO, and 0.1 g/L ampicillin. After a 16-h incubation at 37 "C under agitation, the culture reached an A,,, of approxi- matively 5 and was used to inoculate the 100-L fermenter. The culture medium contained 330 g Na,HPO,, 165 g KH,P04, 27.5 g NaCl, 55 g NH,Cl, 220 g casamino acids and 5 mL antifoam. After sterilisation and addition of 0.91 g CaCl,, 302 g glucose, 14.5 g MgSO, and 0.6 g thiamine/HCl, the culture volume was 60 L. Addition of 600 mL inoculum culture lead to pH 6.8 and an A,,, of 0.12. Culture was grown for 3 h at 37°C to reach an A,,, of 0.7-0.9, then the production of the fusion protein was induced by adding 1 mM IPTG for 2 h. Culture medium was cooled to 12-14"C, and concentrated to about 2 L on a Microgon cartridge. All the following steps were performed at 4°C. Cells were pelleted by centrifugation at 8000 rpm for 15 min (JA14 Beckman rotor), resuspended in 1 L lysis buffer A [50 mM Tris/HCl, pH 7.5, 10 mM EDTA, phenylmethylsulfonyl fluoride (PhMeSO,F)] and completely disrupted using a French pres- sure cell (4.136 MPa). After addition of PhMeSO,F, the cell lysate was centrifuged at 8000 rpm, and the clear supernatant was applied at 60 mL/h overnight to a 50-mL column of glutathione-agarose beads which had previously been swollen and equilibrated in buffer A. After the A,,, had returned to baseline values, the column was depacked and beads were washed several times in 50mM TrisMCl, pH7.5, until all the non-specifically absorbed material was removed. The amount of fusion protein coupled to the beads was assayed

from aliquots eluted with 1OmM reduced glutathione in 50 mM Tris/HCl, pH 7.5, using the Biorad kit assay and its purity was estimated by SDSPAGE.

Cleavage of fusion protein and purification of apoA-I1 Cleavage was operated on the beads in an equal volume

of 50 mM Tris/HCI, pH 8.0, using human plasma thrombin at an enzymehubstrate ratio of 1 : 100 by mass. After 3 h at room temperature, cleavage was stopped by adding 1 mM PhMeSO,F, and the beads were centrifuged for 5 min at 5000 rpm and washed three times in the same buffer. Recom- binant human apoA-I1 was then purified by FPLC (Phar- macia) on a monoQ column (16 cmXlO cm) equilibrated with buffer B (10 mM Tris/HCl, pH 8.2 and 7 M urea) at a flow rate of 2 mL/min. The column was eluted with elution buffer C (0.15 M NaCI, 10 mM Tris/HCI, pH 8.2 and 7 M urea) with a linear gradient (0-20%) for 75 min, followed by 40 min at a 20% plateau. Elution was carried on up to 100% buffer C to eluate GST. Collected fractions were analyzed by SDS/PAGE. Fractions corresponding to apo A-I1 were pooled and dialyzed/concentrated using a micro-prodi- con system (Spectrum).

Isolation of plasma apoA-I1 and apoA-I Plasma apoA-I1 and apoA-I were obtained by ultracen-

trifugal isolation of HDL from fresh plasma obtained from normolipemic donors. The HDL fraction was subsequently delipidated and apoA-I and apoA-I1 were isolated by ion- exchange chromatography according to standard techniques (Blaton et al., 1979). The purity of the proteins was checked by SDSPAGE and by amino acid analysis.

SDSPAGE and Western blotting Polyacrylamide gel electrophoresis (PAGE) was per-

formed in the presence of 1% SDS according to Laemmli (1970). Cleavage and purity of the isolated proteins were assessed using 14% and 18% cast Tricine polyacrylamide gels (Novex Experimental Technology). Western blotting was carried out according to Burnette (1981), using a rabbit polyclonal anti-(human apoA-11) serum. The antigen-anti- body complex was revealed with horseradish-peroxidase-labeled anti-rabbit IgG (Nordic Immunological Laboratories), using enhanced chemiluminescence detection reagents and ECL-Hyperfilms (Amersham).

Preparation and characterization of phospholipidprotein complexes

For association experiments, recombinant or plasma apoA-I1 was complexed with dimyristoylglycerophosphocholine (Myr,GroPCho) at a Myr,GroPCho/protein ratio of 2 : 1, by mass, as previously described (Vanloo et al., 1992). For displacement experiments, complexes were prepared by incubation of plasma apoA-I with dipalmitoylglycerophosphocholine (Pam,GroPCho) and free cholesterol, at a Pam,GroPCho/protein/cholesterol ratio of 3 : 1 :O. 15, by mass, using the cholate dialysis procedure (Matz and Jonas, 1982). Complexes were isolated by gel filtration on a Su- perose 6 PG column in 5 mM Tris/HCl, pH 8.1,0.15 M NaCl and 0.2 g/L NaN,, in a FPLC system (Waters). ApoA-II-containing complexes were detected by measuring the A,,, and the Tyr emission at 305 nm on a Jasco SP500 spectrofluorim-

1143

eter. The composition and size of the complexes were determined on the fraction with maximal UV absorption in the elution profile. Phospholipids were measured using an enzymic assay kit (Biomerieux) and the proteins were assayed by phenylalanine quantification by reverse-phase HPLC (Brasseur et al., 1990). The sizes of the complexes were estimated by non-denaturating gel electrophoresis in a 8 - 25 % polyacrylamide gradient (Pharmacia). Gels were scanned using a laser densitometer (Ultroscan XL), and the Stockes radii were estimated in comparison with high-molecular- mass protein standards (Pharmacia). Electron microscopic analysis of the complexes was performed at a protein concentration of 150 pg/mL as previously described (Vanloo et al., 1992). Particle size was determined by measuring 120 discrete particles for each sample. The mean diameter and the size distribution of the complexes were calculated.

Cross-linking studies To calculate the number of moles proteidcomplex, cross-

linking was performed with dithiobisuccinimidyl propionate (Pierce Chem.) followed by 8 -25 % gradient SDSPAGE according to Swaney (1986). Cross-linked apo-A-11-comprising monomer, dimer, trimer and oligomeric forms characteristic of this protein, was used to calibrate the gels for molecular- mass determination.

Circular dichroism measurements CD spectra of the proteins and the isolated complexes

with Myr,GroPCho were measured at room temperature at a protein concentration of 0.2 mg/mL in 10 mM sodium phos- phate, pH 7.4, at room temperature in a Jasco 600 spectropo- larimeter (Vanloo et al., 1992). Nine spectra were collected and averaged for each sample.

Displacement of apoA-I from Pam,GroPCho/apoA-I complexes

Complexes equivalent to 50 pg apoA-I were incubated with various amounts of apoA-I1 for 2 h at 25"C, then separated by gel filtration on a Superose 12HR column as previously described (Vanloo, 1992). Complexes were detected by measuring the A,,, and the Trp emission at 330 nm. Phos- pholipids were quantified as described above. ApoA-I and apoA-I1 were assayed by immunonephelometry using mo- nospecific antisera raised in the rabbit and concentration standard curves were established using purified plasma apoA-I and apoA-I1 or recombinant apoA-I1 for calibration.

Association of recombinant apoA-I1 to human plasma lipoproteins

High-density lipoprotein fractions HDL, and HDL, were isolated from the plasma of normolipemic healthy human donors by sequential flotation ultracentrifugation as previously described (Weisgraber and Malhey, 1980). Labeling of recombinant apoA-I1 with Iz5I was performed by the N-bromosuccinimide method according to Sinn et al. (1988). Specific radioactivity was 8000 c p d n g protein. The preparation of labeled recombinant apoA-I1 (5 pL) was added to either human plasma (2 mL) or to HDL, or HDL, (300 pg protein). The mixture was ajusted to a density of 1.21 g/mL with solid KBr, and subjected to discontinuous density gradient ultracentrifugation according to Terpstra et al. (1981). Gradient

A

kDa 1 2 3 4 5 6

96

66

45

31

21

14.4

31 - <-

21 - 14.4 -

t

Fig. 1. Time-course of expression of GST-A11 fusion-protein in E. coli. GST-AII-expressing E. coli cells were cultured in a fermenter (A) or in a I-L flask (B). Samples were collected before (lane 2) and after induction with 1 M IPTG at the following times: 30 (lane 3), 60 (lane 4), 90 (lane 5) , and 105 min (lane 6), and were analyzed by 12.5 % PAGE followed by staining with Coomassie blue (A) or transferred onto nitrocellulose and immunodetected with an anti-(human apoA-11) serum (B). The position and size of protein markers are indicated lane 1. The arrow indicates the position of the fusion protein.

fractions (500 pl) were collected for measurement of density and radioactivity.

RESULTS

Expression of GST-A11 fusion protein

Human apoA-I1 was expressed in E. coli TG1 cells as a fusion protein with GST. Fig. 1 shows the accumulation of a protein migrating at the expected molecular mass of GST- AII, i.e. 34.5 kDa, resulting from the fusion of GST (26 kDa) with monomeric zuman apoA-I1 (8.5 m a ) , when cells were induced with IPTG at a low culture density (A,,, = 0.8 - 1 .O). In addition, several bands ranging over 26 - 33 kDa were present (Fig. 1 A), and reacted with an anti-(human apoA-11) serum (Fig. lB), suggesting that these bands corresponded to degradation products of the fusion protein. Extending the induction period over 2 h, or culturing cells at high cell density (up to A,, = 27 in a fermenter; results not shown) lead to a major degradation of GST-AII. Therefore, cultures were induced at low density with 1 mM IPTG for 105 min. Under these conditions, GST-A11 accounted for about 5-10% of the total bacterial proteins. GST-A11 fusion protein remained stable during the different steps of preparation from fermenter culture and was recovered as a soluble protein from bacterials extracts (results not shown).

Purification and proteolytic cleavage of GST-A11 fusion protein

About 80 % of the proteins purified from the supernatant of disrupted cells by affinity chromatography on glutathione- agarose beads corresponded to full-length GST-A11 (Fig. 2A,

1144

A

1 2 3 4 kDa

96 66 45

31

21

14.4

B

1 2 3 kDa

106 - 80 -

49.5 - 32.5 - 27.5 - 18.5 -

Fig. 2. Purification and protease cleavage of GST-A11 fusion protein. GST-A11 expressing E. coEi cells were grown in a fermenter and disrupted as described in Fig. 1. Supernatants collected after centrifugation of the disrupted cells were purified by affinity chromatography and purified fusion proteins were cleaved with thrombin as described in Materials and Methods. Samples of proteins retained on glutathione-agarose beads before (lane 2) and after (lane 3) cleavage and recovered in the supernatant after cleavage (lane 4) were analyzed by 18 % PAGE followed by staining with Coomassie blue (A) or transferred onto nitrocellulose and probed with an anti-(human apoA-11) serum (B).

lane 2 and B, lane 1). The cleavage of purified fusion proteins with thrombin was completed at an enzyme/substrate ration of 1 : 100 in 3 h at room temperature under mild agitation (Fig. 2A, lane 3 and B, lane 3). Recombinant apoA-I1 was recovered in the supernatant collected after centrifugation of thrombin-treated beads. It was partially contaminated with GST (Fig. 2A, lane 4), most likely partially detached from agarose-SH beads when the pH of the solution was raised from 7.5 (the optimal pH for stability of GST-glutathione beads complexes) to 8.0, which is the optimal pH for thrombin activity.

Purification of recombinant apoA-I1 Recombinant apoA-I1 eluted in two peaks when superna-

tants were applied to a preparative monoQ column in 7 M urea (Fig. 3A). The first peak, eluting at 18-20% 0.15 M NaCl, consisted mostly of monomeric apoA-11, whereas the second peak, eluting at a 20% plateau, was exclusively composed of dimeric apoA-11 (Fig. 3 B). ApoA-I1 was recovered in a dimeric form after dialysiskoncentration (Fig. 3 C, D). The N-terminal amino acid sequence of purified recombinant apoA-TI was GSMQAKEPCVESLV, in line with the known sequence of human plasma apoA-I1 (Lackner et al., 1984). The recombinant apoA-II contained three additional N-terminal residues, Gly-Ser-Met. Gly-Ser were on the C-terminal side of the scissile bond of thrombin and Met-' was designed in the synthetic apoA-I1 gene, as explained in Materials and Methods. The experimental molecular mass determined by mass spectrometry (17965 Da) was identical to the calculated data, and differed from human plasma apoA-I1 by approxi- mately 600 Da, corresponding to the three extra residues on each monomer. Both recombinant and human plasma apoA- 11 displayed a retention time of 50 min on HPLC chromatography (Brownlee RPC4). Table 1 summarizes the yield of production of recombinant apoA-TI. After affinity chromatography of the supernatant from E. coli cell lysate, 108 mg GST-A11 fusion protein were obtained/g lysate supernatant proteins. After thrombin cleavage, preparative monoQ purifi-

cation, dialysis and concentration, 12.0 mg recombinant apoA-I1 were recovered/g lysate supernatant proteins. These results were confirmed by calculation of the phenylalanine content of recombinant apoA-I1 at A,,, with a molar absorption coefficient of 0.74. The overall yield of purification of recombinant apoA-I1 procedure was estimated as 45 %, tak- ing into account the 4 : 1 mass ratio between GST-A-I1 fusion protein and recombinant apoA-I1 (Table 1).

Characterization of the Myr,GroPCho/apoA-I1 complexes

The ability of recombinant apoA-I1 to associate with Myr,GroPCho vesicles to form small discoidal complexes was estimated by measuring the turbidity decrease at 325 nm as a function of temperature, as previously described (Vanloo et al., 1992). Transition curves of identical shape and mid- point temperature (23.1 "C) were obtained for plasma and recombinant apoA-11, suggesting that recombinant apo A-I1 can, similar to plasma apoA-11, associate with Myr,GroPCho to form discoidal complexes (results not shown). The complexes generated between Myr,GroPCho and plasma or recombinant apoA-I1 were fractionated on a Superose 6 PG column (Fig. 4). Both complexes eluted at the same volume. The yield of complex formation was 100% for the two forms of apoA-11, since we did not observe any peak containing either free protein or free lipid. The phospholipid/protein mass ratio was found to be similar in the complexes formed with plasma and recombinant apoA-I1 (Table 2). Gradient gel electrophoresis of the elution peak (inserts of Fig. 4) showed the presence of two populations of complexes with a diameter of 9.4 and 10.5 nm, for recombinant as well as for plasma apoA-IINyr,GroPCho complexes (Table 2). Plasma apoA-I1 was mainly incorporated in the larger complexes, whereas recombinant apoA-I1 was mainly recovered in smaller complexes (inserts of Fig. 4).

The electron microscopic analysis of the chromato- graphic fractions corresponding to the complexes showed a typical pattern of rouleaux characteristic of stacked discs (inserts of Fig. 5) . Particles formed with plasma (Fig. 5A) and recombinant apoA-I1 (Fig. 5B) exhibited similar size distribution and mean diameters. The mean diameters calculated from the electron micrographs were larger than those calculated from gradient gel electrophoresis (Table 2), maybe due to electron irradiation and flattening of the complexes during sample preparation.

Stability of the phospholipid-protein complexes

Circular dichroism measurements showed that the a-helical content of both plasma and recombinant apoA-I1 increased when the proteins associate with Myr,GroPCho to form complexes (Table 3). This suggests that the structure of the two apoproteins in these complexes is more ordered than that in the native protein. This was confirmed by denaturation experiments carried out by titration with increasing guanidine hydrochloride (Gdn/HCl) concentrations (Vanloo et al., 1992). The mid-point of GddHC1 denaturation, corresponding to the helix-coil transition, increased in the complexes generated with Myr,GroPCho and recombinant or plasma apoA-I1 (results not shown), suggesting that both forms of apoA-I1 were protected from denaturation by association with phospholipids.

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A 1 2 3 4 5

Dimeric Apolipoprotein A-ll

Monomeric Apolipoprotein A-11

90 96 102 110 118 123 137

i . . . . * . . . I , 50 65 80 95 110 125 140 155 170

Fraction number

Dimeric Apolipoprotein A-ll

Monomeric Apolipoprotein A-I1

D

1 2 3 4 5

Dimeric Apolipoprotein A-I1

Monomeric Apolipoprotein A-ll

Fraction number

Fig. 3. Purification of recombinant apoA-11. Recombinant apoA-I1 was purified by mono Q preparative FPLC from supernatants recovered after cleavage of fusion proteins as described in Fig. 4. (A) displays the elution profile of recombinant apoA-11. (B) Fractions corresponding to the peaks were analyzed by 18% PAGE followed by staining with Coomassie blue. Fractions containing pure recombinant apoA-I1 were pooled, submitted to dialysiskoncentration and analyzed by 18% PAGE followed by staining with Coomassie blue (C) or transferred onto nitrocellulose and probed with an anti-(human apoA-11) serum (D). Lanel, proteins recovered in the supernatant after cleavage of fusion proteins; lanes 2 and 3, purified recombinant apoA-I1 run without or with 2-mercaptoethanol, respectively; lanes 4 and 5 , human apoA-I1 (Sigma) run in the absence or presence of 2-mercaptoethanol, respectively. The position of monomeric and dimeric apoA-I1 is indicated in the margin.

Table 1. Purification yield of recombinant apoA-11. Proteins were assayed using Biorad kit with BSA as standard.

Step Component Protein Yield

g1100 g moVlOO mol Affinity purification GST-A11 108.0 3.13 100 100

Dialysis rAII 12.0 1.40 11.1 44.7

Thrombin cleavage GST-A11 75.5 2.19 70 70 Preparative monoQ rAII 13.0 1.52 12 48.6

Displacement of plasma apoA-I by recombinant apoA-I1 nant apoA-11, a second peak appeared with an elution volume of 14 mL containing apoA-I (Fig. 6B, C and D), while re-

The ability of recombinant apoA-11 to displace apoA-1 combinant apoA-I1 coeluted with the complexes at 12 mL. from phospholipid complexes was monitored by gel-filtration At an apoA-II/apoA-I molar ratio of 3, apoA-I completely chromatography on a Superose 12 HR column (Fig. 6). Na- dissociated from the complexes, whereas apoA-I1 eluted in tive human apoA-I/Pam,GroPCho/cholesterol complexes two peaks (Fig. 6D). The first peak, eluting at 12 mL, corre- eluted as a single peak at an elution volume of 12 mL sponded to the maximal amount of recombinant apoA-I1 in (Fig. 6A). In the presence of increasing amounts of recombi- the complexes, and the second peak, eluting at 16 mL, cor-

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800 E : 600

' 400

= . c s P

1000 r I10

.

-

-

I *

200 } 0 L

1000

800 E : 600

4 400

200

P . c P

0

60 70 80 90 100 110

Elution volume (ml)

60 70 80 90 100 110

Elution volume (ml)

Fig. 4. Elution profile of plasma (A) and recombinant (B) apoA- IUMyr,GroPCho complexes, separated on a Superose 6PG column. The presence of apoA-I1 (+), and of phospholipids (U) in the elution fractions was monitored by measurement of the Tyr fluores- cence intensity for apoA-11, and by an enzymic colorimetric assay for phospholipids. Inserts represent the separation of the complexes corresponding to the top fraction of the chromatography peak by a 4- 30% polyacrylamide gradient gel electrophoresis. Diameters were determined by comparison with known standards after scan- ning of the gels. Standards of interest are 669, 440, 232 and 140 kDa.

responded to the lipid-free recombinant apoA-11. Our results demonstrate that the capacity of recombinant apoA-I1 to displace apoA-I from the complexes is comparable to that of plasma apoA-I1 (Fig. 6E). Plasma apoA-I was completely displaced by either plasma or recombinant apoA-11, at a dimeric apoA-II/apoA-I molar ratio of 2 : 1 (Fig. 6F), indicat- ing that two molecules of apoA-I1 can substitute for one mol- ecule of apoA-I, in agreement with previously published data (Lagocki and Scanu, 1980).

A

x 30

s I 20 6

- U

10

8 9 10 11 12 13 14 15

Size (nm)

8 9 10 11 12 13 14 15

Size (nm)

Fig. 5. Electron micrographic analysis of negatively stained plasma (A) and recombinant (B) apoA-IUMyr,GroPCho complexes. The mean diameter and distribution, for n = 120 particles are given for each sample. Inserts, electron micrographs of the negatively stained complexes.

Table 3. Amount of the secondary structure of the complexes determined by circular dichroism.

Complex Helix D sheet ,8 turn Coil

%

Plasma apoA-I1 44 12 18 26

plasma apoA-I1 78 1 13 5 Myr,GroPCho/

Recombinant apoA-I1 42 10 20 28

Myr,GroPCho/ recombinant apoA-I1 72 3 13 13

Table 2. Composition and size of the complexes generated between Myr,GroPCho and recombinant or plasma apoA-11.

M yr,GroPCho/ Myr,GroPC ho/ Diameter apo A-I1 apoA-I1

by GGE by elecron microscopy

by mass nm

Plasma apoA-TI 2: 1 68:l 9.4, 10.5 (?3) 11.1 Recombinant apoA-TI 2: 1 72:1 9.4, 10.5 ( 2 3 ) 11.4

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6 8 10 12 14 16 18 20 6 8 10 12 14 16 18 20

Elution v o l u m e (ml) Elution v o l u m e (ml)

6 8 10 12 14 16 18 20

0.50

0.40

i! 0.50

!! 0.20 0.10 z

58 0.00

40 D1

80

6 8 10 12 14 16 18 20

Elution v o l u m e (ml) Elution v o l u m e (mi)

. - a 8

0 1 2 3

apo A-ll/apo A- l (rnollmol)

0 0.5 1 2 3 4

apo A-lllapo A.1 (mollmol)

Fig. 6. Displacement of plasma apoA-I by recombinant apoA-11. Plasma apoA-I was associated with Pam,GroPCho and cholesterol at a 3: 1. by mass, ratio and complexes were separated by gel filtration on a Superose 12HR column before (A) and after incubation with recombinant apoA-11. ApoA-IUapoA-I molar ratios are 1 : 1 (B), 2 : 1 (C) and 3 : 1 (D). Phospholipids (0) were assayed in the eluted fractions by an enzymic colorimetric assay, and apoA-I (+) and apoA-I1 (0) by immunonephelometry. The displacement of apoA-I (E) and the corresponding incorporation of recombinant (+) apoA-I1 (F) in the complexes was calculated from A-D. Similar data were obtained for plasma apoA-I1 (0).

Association of recombinant apoA-I1 with human plasma HDL

In order to assess the ability of recombinant apoA-I1 to associate with human lipoproteins, the protein was labeled and subjected to discontinuous-density-gradient ultracentrifugation in the presence of human plasma, or human HDL, or HDL, (Fig. 7). When incubated with HDL, or HDLs, labeled recombinant apoA-I1 was recovered as a single peak at den- sities of 1.11 g/mL or 1.145 g/mL, corresponding to those of HDL, and HDL,, respectively (Patsh et al., 1980). When incubated with total plasma, radioactivity was found in a

broader peak at the density range of HDL,, which is consis- tent with the fact that apoA-I1 mainly associated to the HGL, fraction in human plasma (Cheung and Albers, 1982). In any case, radioactivity was detected at a density < 1.080 g/mL. These data demonstrate that recombinant apoA-I1 associates completely and specifically to HDL (Lagocki and Scanu, 1980).

DISCUSSION Soluble apolipoproteins share common lipid-binding

properties and structural organization due to the occurence

1148

X

0 In

7

1.00 1.05 1 .10 1.15 1.20 1 .25 1.30

Average density (glrnl)

Fig. 7. Association of recornhinant apoA-I1 with human plasma HDL. '251-labeled recombinant apoA-I1 was incubated with HDLL (a), or HDL, (+), or total human plasma (0), then submitted to discontinuous-gradient ultracentrifugation. Radioactivity was measured in 500 pL fractions of increasing density.

of 22-residue repeats within their sequence (Segrest et al., 1992). Besides, the apolipoproteins exert specific functions as lipid carriers, enzyme activators and receptor ligands. The common and the specific functions of the different apolipoproteins could be traced back to specific features of their primary and secondary structure. Recent advances in apoA- I1 genetics, studies in transgenic animals and in vitro experiments strongly suggest that apoA-I1 plays a significant role i n HDL metabolism, possibly by impairing the reverse cholesterol transport induced by apoA-I. ApoA-I1 is actually able to displace apoA-I from the HDL surface (Lagocki and Scanu, 1980). Although the helical segments of apoA-I and apoA-I1 share common shapes and dimensions, differences in the molecular hydrophobicity potential around the helical segments could account for a greater stability of the association of apoA-IUphospholipid complexes (Brasseur et al., 1992).

This hypothesis can be tested by generating mutant forms of apoA-I1 by site-directed mutagenesis, provided that suffi- cient amounts are produced in recombinant expression systems. Since human apoA-I1 is 0-glycosylated and undergoes a complex array of intracellular and extracellular post-translational modifications (Hussain and Zannis, 1990 ; Remaley et al., 1993; Lopez et al., 1994), it would have been prefera- ble to use a mammalian cell expression system. Unfortu- nately, the permanent C127 cell line expressing human apoA- I1 that we previously generated (Lopez et al., 1994) produced apoA-IT in insufficient amounts, apoA-I1 being more rapidly degraded in C127 as well as in HepG2 cells compared to apoA-I and apoE (Lopez et al., 1994). Moreover, our attempts to express human apoA-I1 in a free soluble form in E. coli yielded extremely low levels of recombinant apoA- 11.

We, therefore, turned to the pGEX-2T system developed by Smith and Johnson (1988) which allows the expression of heterologous proteins in E. coli as C-terminal fusion proteins with S. juponicum GST. Optimization of GST-A-IT fusion- protein production was obtained at a low culture density and for induction periods exceeding 2 h. Even under these conditions, we found significant amounts of degradation products of the fusion protein ranging over 26-33 kDa, as was pre-

viously reported for GST fused with apoA-I or viral proteins (Brissette et al., 1991; Weiss et al., 1992). The purified recombinant apoA-I1 was completely recovered as a dimer, demonstrating that it was correctly folded using the pGEX- 2T expression system in E. coli, and that dimerization oc- curred spontaneously, as was previously shown for the mouse mammary tumor virus protease (Menendez-Arias et al., 1992). The yield of recombinant apoA-I1 production was in the same range as that of the proteins previously expressed as GST fusion proteins (Brissette et al., 1991 ; Menendez- Arias et al., 1992; Weiss et al., 1992).

Our data clearly establish that recombinant apoA-I1 be- haves similarly to human plasma apoA-I1 with regard to its association with phospholipids, with human plasma HDL and to the displacement of apoA-I from lipid-apolipoprotein complexes. The recombinant apoA-I1 contains three additional residues at the N-terminus, and thus differs slightly from human plasma apoA-11. Our results indicate that the addition of this three-residue-long N-terminus had negligible influence on the function of apoA-11. As a consequence, one can hypothesize that the pyroglutamate N-terminus of human plasma apoA-11, which results from the cyclization of the N- terminal Gln, may not be involved in the function(s) of human apoA-11. The recombinant apoA-I1 produced in E. coli was not subjected to the sialylation-desialylation process leading to the mature form of apoA-I1 present in human plasma (Hussain and Zannis, 1989; Remaley et al., 1993). Remaley et al. (1993) have shown that the non-sialylated 8.5- kDa isoform of apoA-I1 was produced by CHOldlD cells, defective in 0-glycosylation, was able to associate with HDL throughout the HDL density range, whereas sialylated apoA- TI selectively associated with HDL; (Remaley et al., 1993). These data are further supported by our results demonstrating that the 8.5-kDa recombinant apoA-I1 produced in E. coli readily associates with phospholipid vesicles and with HDL fractions similarly to native apoA-11, which is mostly present in the desialylated isoform in human plasma (Brewer et al.. 1972).

The structure of recombinant apoA-I1 in solution was similar to that of human plasma apoA-11, i.e. 44% and 42% a helix, respectively. In both cases, the association with MyrzGroPCho resulted in increased a-helical content, i.e. 72% and 78% respectively, in accordance with previous studies (Massey et al., 1981). Several helical segments have been predicted in the apoA-I1 sequence at residues 9- 29(11-28), 37--50 and 52-70(52-69) (Massey et al., 1989; Brasseur et al., 1992). The experimental values of helical content estimated from circular dichroism data in the present study and by Massey et al. (1 989) support the hypothesis that apoA-I1 contains three a helices, when associated with Myr,GroPCho.

The complexes generated through the association of recombinant apoA-I1 with Myr,GroPCho vesicles have similar characteristics as those formed with human plasma apoA- 11. Cross-linking experiments indicated that Myr,GroPCho complexes contained two molecules of dimeric apoA-II/particle, i.e. 12 a helices, assuming 3 helices/monomeric apoA- TI. These results agree with previous experimental and theo- retical data reporting 12 helices/MyrZGroPCho/apoA-II complex (Edelstein et al., 1982; Brasseur et al., 1992). In the present study, the Myr,GroPCho/apoA-I1 complexes consisted of 70 mol phospholipid/mol dimeric apoA-11, as compared to 55-69 mol (Brasseur et al., 1992) and 45-72 mol (Massey et al., 1980) in previous reports.

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Recombinant apoA-I1 was able to displace apoA-I from Pam,GroPCho/cholesterol/apoA-I complexes in the same way as plasma apoA-11. ApoA-I was displaced by 2 mol dimeric apoA-11, as previously reported for Myr,GroPCho/cho- 1esteroVapoA-I vesicles (Van Tornout e t al., 1979) and native HDL (Edelstein e t al., 1982).

In this paper, w e demonstrated that, despite its high hy- drophobic content and its susceptibility to intracellular degradation in eukaryotic and prokaryotic systems, recombinant apoA-I1 could be produced in significant amounts as a fusion protein with GST in E. coli. Recombinant apoA-I1 exhibited the same structural and functional properties as human plasma apoA-11. Thus, the expression system utilized is appropriate and can be employed to produce mutagenized forms of human apoA-I1 for further structure-function analysis.

This work was supported in part by grants from the Bioavenir program (RhBne Poulenc Rorer), from INSERM (910105), and from ARCOL. We are grateful to D. Fauchet for mass spectroscopy measurements and N-terminus sequencing, Nadine Fromage and Vassilis Zannis for helpful discussions.

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