Identification of Specific Amphipathic a-Helical Sequence of Human

THE JOWALOF B I O ~ I CCHEMISTRY AL 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 47, Issue of November 25, pp. 29883-29890, 1994 Printed in U.S.A.

Identification of Specific Amphipathic a-Helical Sequence of Human Apolipoprotein A-IV Involved in Lecithin:Cholesterol Acyltransferase Activation* (Received for publication, June 7, 1994, and in revised form, August 30, 1994)

Florence EmmanuelS, Armin SteinmetzOfl, Maryvonne Rosseneull, Robert Brasseur**, Nicole GosseletS, Florence AttenotS, St6phan Cuin&, Sandrine SBguretS, Martine LattaS, Jean-Charles FruchartSS, and Patrice DenBfleSOO From the $Biotechnology Department, RhBne-Poulenc Rorer S.A., France, the QUniuersitat Marburg, Germany, the iklgemeen Ziekenhuis St. Jan,Department of Clinical Chemistry, Brugge, Belgium, the **Free University of Brussels, Laboratory of macromolecules at interfaces, Belgium, and the SvnstitutPasteur Lille, France

To investigate the structure-function relationshipof neal macrophages, mouse adipose cells, and human skinfibroenzyme 1ecithin:cholesterol human apolipoprotein A-IV (apoA-IV), several deletion blasts (7-9), andactivatesthe acyltransferase (LCAT) (10-12). apoA-IV is found in the premutants of this protein were constructed by sequentially removing pairs of 22-residue repeats, potentially p-migrating HDL fraction of normal individuals and in the very having an amphipathica-helicalconformation. The mu- high density lipoprotein (VHDL) fraction of apoA-I knock-out tants, produced as recombinant poly-histidine-tagged mice, suggesting that it plays a role in the initial step of the apolipoproteins (t-apo) in Escherichia coli, assembled reverse cholesterol transport process in vivo (13, 14). apoA-IV with phosphatidylcholine(i.e.dimyristoylphosphatidyl- might alsobe involved in regulatingtriglyceride metabolism by choline, palmitoyloleoylphosphatidylcholine, or egg modulating lipoprotein lipase activation (15, 16). lecithin)as didnativeapoA-n7.Lecithin:cholesterol Multiple 22-residue repeats have been identified in apoA-IV, acyltransferase (LCAT) cofactor function, measured as which can form amphipathic a-heliceshomologous to those of cholesterolesterificationoccurringwhent-apo-phosapoA-I and apo E (17). Amphipathic a-helical repeated strucphatidylcholine-cholesterol complexes were incubated tures are one prerequisite for the cofactor function of a n apowithpurifiedenzyme,decreasedsignificantlywhen pairs of repeats between residues117 and 248 were de- lipoprotein for LCAT (18); experimental results suggest that specific regions, at least of apoA-I, are also involved in LCAT leted and most markedly when residues 117-160 were cofactor activity (19-21). byand 76%, deleted. LCAT cofactor activity decreased 90 respectively, when egg lecithin or palmitoyloleoylphos- The genecoding for apoA-IV is closely linked to thoseencodphatidylcholine was used to form the particles with theing apoA-I and apo C-I11 on chromosome 11 (22-24), but its Aaa 117-160 mutant. Thus,on the basisof deletion scan- regulation is not yet understood. Even thoughapoA-IV seems to be highly polymorphic (see Lohse and Brewer (25) for rening of t-apo, residues 117-160 seem to be involved in the view), its primary function has not been established by studyLCAT cofactor function of apoA-IV. ing natural mutants of this protein. There are still conflicting data on the effects of naturally occurring apoA-IV mutations on lipoprotein and apolipoprotein metabolism in humans (seeTenApolipoprotein (apo)’ A-IV), a M, 46,000 protein, is mainly for review). For example, the insynthesized in enterocytesin humans and is secreted into the kanen and Ehnholm (26) creased hydrophobicity of the apoA-IV (G1uSo + His) mutant, lymph inassociation with chylomicron particles (1,2). The first such protein characterized was isolated from rat plasma (3).In resulting in a higher affinity for phospholipid surfaces and an plasma, apoA-IV is associated, at least partly, with high density increased efficiency of LCAT activation in vitro (27), may be responsible for the slower i n vivo metabolism of this mutant, lipoprotein (HDL) particles (4-6). Many observations suggest that apoA-IV is likely to be in- due to increased association with HDL (28). In the presentwork we propose a new strategy for studying volved in the reverse cholesterol transport process. Analogously of apoA-IV. As only a few of to apolipoprotein A-I (apoA-I), apoA-IV forms HDL-like par- the structure-function relationship the natural variantsof apoA-IV having mutations thatsignifiticles in vitro, promotes cholesterol efflux from mouse peritoas the plasmalevel cantly affect its functions are available and is low, we used a recently described, highly of this protein * This work was supported by the Bio Avenir program, co-financedby Rhbne-Poulenc S. A. and the French ministry of Research and Indus- productive recombinant expression system (8)to construct and try. The costsof publication of this article were defrayed in part by the express several variantsof apoA-IV. These variantswere made payment of page charges. Thisarticle must therefore be hereby marked by sequentially deleting specific secondary structures, includ“aduertisement”in accordancewith 18 U.S.C.Section 1734 solelyto ing the 22-residue repeats, along the protein. These variants indicate this fact. ll Supported by grants from the Deutsche Forschungsgemeinschaft. were poly-histidine-tagged. The i n vitro investigation of the biophysical and physiological properties of thesevariants § Q To whomcorrespondenceshould be addressed:Atherosclerosis Dept., Centre derecherche de Vitry-Alforville, 13 quai Jules Guesde-BP showed that a specific domain of the protein sequence is re14, 94403, Vitry sur Seine Cedex, France. Tel.: 33-1-45-73-77-11; Fax: quired for the optimal LCAT cofactor activity of apoA-IV. 33-1-45-73-77-96. The abbreviations used are: apo, apolipoprotein; t-apo, poly-histiEXPERIMENTALPROCEDURES dine-tagged apolipoprotein;HDL, high density lipoprotein;LCAT, leciMaterials thin:cholesterolacyltransferase; DMPC, dimyristoylphosphatidylcholine; POPC, palmitoyloleoylphosphatidylcholine; aa, amino acid; Egg yolk phosphatidylcholine (egg lecithin), dimyristoylphosphatiPAGE, polyacrylamide gel electrophoresis; ATP& adenosine-5’-0- dylcholine(DMPC), palmitoyloleoylphosphatidylcholine (POPC), iso(1-thiophosphate). propyl P-D-thiogalactoside,rifampicin,and all other chemicalswere

29883

29884

ApoA-N Helical Domain (aa 117-160) and LCAT Activation

salted on a Tris-acryl GF-05 column equilibrated with 100 nm phosphate buffer, pH 8. The collected protein fractions were pooled, diluted with phosphate buffer, pH8, to obtain a final concentration of 4 mg/ml, and supplemented with Hecameg (powder)to a final concentration of 25 m ~The . mixture was then deposited on the Ni-nitrilotriacetic acid column, whichhad been equilibrated with 100 mM phosphate buffer, pH 8, containing Hecameg 25 mM. Unbound proteins were eliminated by washing the column with the same buffer, weakly boundproteins were Methods eluted by a phosphate/citrate buffer, pH 6, and the recombinant t-apos MolecularBiology-Standardmolecularbiology techniques were were eluted by a phosphate/citrate buffer, pH 5. All buffers were suppleused (29). DNA was sequenced by using the M13 universal primer, mented with Hecameg 25mM. apoA-IV specific primers, and either [35S]ATPaSand a kit (ref. 70750) Protein fractions obtained at pH 5 were neutralized by the addition from U. S. Biochemical Corp. or fluorescently labeled primers and an of 1 M NaOH (30 pl/ml) supplemented with two protease inhibitors: 0.1 Applied Biosystems model 370ADNA sequencer. A kit (ref. RPN1523) M EDTA and 0.2 M phenylmethylsulfonyl fluoride (20 and 5 $ml, refrom Amersham Corp. (Les Ulis, France) was used for site-directed spectively). Fractions were then analyzed by polyacrylamide gel elecmutagenesis. The NH,-terminal amino acid sequence was determined trophoresis in the presence of SDS (SDS-PAGE)containing 15%acrylby using an Applied Biosystems protein sequencer 477A (30). amide, bis-acrylamide according to the procedure of Laemmli (37), and Mutagenesis of t-ApoA-N-Three plasmid clones(pXL1694, pXL the more pure and concentrated fractions were pooled.The pooled frac1696, and pXL 1866)(8) containing the coding sequenceof Met-apoA-1V tions were incubated in the presence of 50 m~ (final concentration) in whole or part were used for site-directed mutagenesis. Plasmid pXL L(-)-histidine (powder) for 1 h at 4 "C to disrupt the Ni:His-protein 1694 contains a KpnI-Hind111fragment corresponding to the 3' end of binding. Nickel and histidine were removedby desalting on a Tris-acryl the human apoA-IV coding sequence (24). Plasmid pXL 1696 contains GF-05 columnequilibrated with phosphate-bufferedsaline, pH 7.4 conthe 5' half of this sequence (555 base pairs) inserted between its NdeI taining 2 m~ EDTA. and EcoRI sites. Plasmid pXL 1866 contains the integral Met-apoA-IV Purified recombinant proteins were analyzed by PAGE under noncoding sequenceinserted between its NdeI and BamHI sites. pXL 1696 denaturing conditions, SDS-PAGE, and immunoblotting. A prepacked was used to create deletion derivatives baa 1-12 (pXL 2168), Aaa 13-61 polyacrylamide gradient gel (4-20%) (Novex, Prolabo, France) was used (pXL 21831, Aaa 62-116 (pXL 2346), and Aaa 117-160 (pXL 2185). pXL for electrophoresis under non-denaturing conditions. Monoclonal anti1694 was used to create deletion derivative Aaa 333-376 (pXL 2182). bodies raised against human plasma apoA-IV and a peroxidase-conjuThe entire sequence wasreconstituted in each case by inserting into the gated goat anti-mouse antibody were used for immunoblotting. The modified expression vector the mutated half and the other, intact half of activity of bound peroxidasewas revealed by incubation with a solution the sequence. pXL 1866 was used to create deletion derivatives Aaa of 4-chloro-1-naphtholas enzyme substrate. 161-204 (pXL 2184), Aaa 205-248 (pXL 2215), baa 249-288 (pXL 2186) The chromatograms were monitored by UV detection at 280 nm. Protein concentrations were determined by the method of Bradford (38) and Aaa 289-332 (pXL 2217). The altered sequenceswere then inserted between the NdeI and BamHI sites of the expression vector pET-Ba, with a Bio-Rad reagent. Isolation of LCAT from Human Plasm-LCAT was isolated from which had been modified to contain a Met-Arg-Gly-Ser-(His)6 coding fresh human plasma by a combination of ultracentrifugation, affinity sequence (poly-histidinetag) at the NdeI site. pXL 2102consisted of the non-deleted Met-apoA-IV sequenceinserted into the modified vector.In chromatography on blue Sepharose, DEAE-Sephacel chromatography, and chromatography on hydroxyapatite, as described previously each case, the deletion was checked by sequencing. (39, 40). Production of t-Apos-Expression plasmids (pXL2102,pXL2168, Lecithin:Cholesterol Acyltransferase Activity Assay-LCAT activity pXL 2183, pXL 2346, pXL 2185, pXL 2184, pXL 2215, pXL 2186, pXL 2217, and pXL 2182) were introduced into Escherichia coli was measured by the conversion of ['4Clcholesterol to ['4Clcholesteryl BLal(DEB)(pLys)(32) by a simple transformation procedure (33). Bac- ester over time. Substrate complexes containing t-apoA-IV/ lecithin) and ['4Clcholesterol wereprepared teria weregrown by one of the followingmethods: 1) Shaker flask phospholipid (POPC or egg cultures. An overnight culture (37 "C) in M9 medium (salt mix 10 x (29) by the detergent (cholate) dialysis procedure described for apolipoproteins (41,421.The ratio of t-apoA-IV to phospholipid was kept at 1:150, 100 mMiter; thiamine 1%, 1 ml/liter; glucose 20%, 20 mMiter; MgSO, 100 mM, 10 mMiter; CaCl, 10 nm, 10 ml/liter; casaminoacids 20%, 20 mol/mol. Substrate complexes were incubated at 37"C with purified mMiter) supplemented with ampicillin (100 pg/ml) and chlorampheni- enzyme in the presence of 2-p-mercaptoethanol and fatty acid-free bocol (50 pg/ml) was used to inoculate production medium (34) in 2-liter vine serum albumin. The conditions of the assay were chosen so as to fermentation flasks. Cultures were grown at 37 "C until their absorb- warrant linearity over time and proportionality to the enzyme concenance was between 0.5 and 1 OD unit at 610 nm. Isopropyl P-D-thioga- tration. Reaction kinetics were measured at constant enzyme/substrate lactoside was then added, at a final concentration of 1mM, followed by ratios over time, as well as a t varying apolipoprotein concentrations in a 15-min incubation at 37"C, to induce T7 polymerase expression. a constant enzyme reaction mixture. The reactions were run for 10 to 45 Rifampicin (100 pg/ml, final concentration) was added and the fermen- min and were stopped with chlorofodmethanol(2:1, v:v). After extractation was carried out for 60 min. 2) High cell density cultures. These tion, ['4C]cholesterol wasseparated from [14Ckholesterylesters by thin were set up as described elsewhere (34) and grown at 37"C to an layer chromatography. Kineticdata were subjected to linear regression absorbance of about 50 OD units at 610 nm; 0.1 M isopropyl p-D-thio- analysis by the Lineweaver-Burk method. Characterization of LCAT-SubstrateComplexes-Substrate comgalactoside was then added to induce T7 polymeraseexpression.After a 20-min induction, rifampicin (200 pg/ml) was added and the fermenta- plexes prepared by the cholate dialysis procedure were characterized by density gradient ultracentrifugation, as described by Redgrave et al. tion was left to proceed for a further 60 min. The cells were then harvested by centrifugation and frozen. (43). This procedure yielded information about the density of the comRecombinant protein expression was controlled bySDS-PAGE and plexes, their resistance to ultracentrifugal force and the degree of incorporation of the apolipoprotein.Aliquots of the fractions of a gradient immunoblotting. Purification of t-Apos-Recombinant proteins were extracted from a were counted for radioactivity (14C)and also pipetted onto nitrocellufrozen bacterial pellet, as follows. Cells were suspended in lysis buffer lose. The spots wereallowed to dry and were then developed with (100 mM phosphate, 2 m~ EDTA, 1 mM phenylmethylsulfonylfluoride, antibodies to apoA-IVto localize boundand unbound protein. Biophysical Characterization of DMPC.t-ApoA-N Complexes10 mM 2-P-mercaptoethanol, pH 7.4; 50 mug cells) and disrupted by three cycles (5 min each) of sonication with a Branson sonifier (250-watt Complexes were prepared differently from those prepared for LCAT mode-pulsed, 50%duty cycle, 60 output control).Whole cells and cellu- activity assays. They wereobtained by incubating t-apoA-IV ordeletion lar debris were removed by centrifugation at 4 "C for 1h at 11,000 x g variants with sonicated DMPC vesicles a t 23 "C for 16 h. The DMPC/ t-apoA-IV molar ratio in the incubation mixture was 20011. Complexes in a Beckman 52-21 MIE centrifuge. Nucleic acids in the supernatant fraction were precipitated by the addition of 10% (w/v) streptomycin were isolated by gel chromatography on a Superose 6HR column in 0.01 sulfate (10 mug of protein) followedby incubation for 30 min at M Tris-HC1 buffer, 0.15 M NaCl, pH 8.1, in an fast protein liquid chro4 "C; they were then removed by centrifugation for 1 h at 4 "C and matography system (Waters). To detect the complexes, the absorbance a t 280 nm was monitored continuously and the Trp emission of the 11,000 x g. Recombinant proteins in the supernatant were purified by metal fractions was measured at 335 nm on an Aminco SPF500 spectrofluochelate affinity chromatography (35, 36) on a Ni-nitrilotriacetic acid- rimeter. To further measure the composition and size of the complexes, agarose resin, according to the manufacturer's (QIAGEN) procedure the two fractions with maximal UV absorption in theelution peak of the with the following minor modifications. The protein solution was de- complexes in the chromatographic runs were collected. from Sigma Chimie (La Verpillihre, France). LCAT was isolated from fresh plasma of healthy blood donors. ['4ClCholesterolwas purchased from New England Nuclear (Dreieich,West Germany).Adenosine 5"athiotriphosphate ([35SlATPaS) was obtained from Amersham Corp. (Les Ulis, France). DNA restriction enzymes, T4 DNA kinase and T4 DNA ligase were all from New England Biolabs (Beverly, MA). Acrylamide was purchased from Bio-Rad.

ApoA-IV Helical Domain (a11 7-1 60) and LCAT Activation

E'IO.1. Apolipoprotein A-IV alignm a t eequmcei To the alignment 86quences (blue letters) of apoA-W,excerpted from Ponnuswamy and Selvarqj (17),we have added the sequences of the non-helicoidaldomains (greenl e t t e r s ) and the non-degenerated helices (red letters), determined by the Chou and Fasman method (31).

29885

1-39 4-61 62-83 95-116 117-138 139-160

161-lS2 183-204 205-226 227-248

249-270 289-310 311 -332 333-376 The compositionof the complexes was determined by quantitation of the phospholipids with an enzymatic assay (BiomBrieUx, France). The size of the complexes was estimated by gradient gel electrophoresis in a 440% polyacrylamide gradient in a Phast system. The gels were scanned by using a laser densitometer (Pharmacia, Uppsala, Sweden), and the Stokes radii were estimated from protein standards

(Pharmacia). For electron mimscopy, DMPC.t-apoA-W complexes, at a protein concentration of 150 &ml, were negatively stained with a solution of potassium phosphotungstate (20 g/liter, pH 7.4). Seven pl of the samples were applied to Fomvar carbon-wated grids and examined with a zeies EM 1OC transmission electron microscope operating at 60 kV. One hundred twenty discrete particles were measured for each sample, and the mean diameter and the size distribution of the complexes were calculated. For the fluoreseenea polarization measurements, the Aminco SPF500 spectrofluorimeter was fitted with a special adapter (AmincoJp 9501).The fluorescencepolarization of the DMPC-t-apoA-IV complexes, labeled with diphenyl hexatriene (molar ratio of lipid-diphenyl hmtriene 50011,was measured as a function of temperature in order to detect changes in the fluidity of the phospholipid acylchains due to lipi&apoA-Wassociation. Temperature was increased from 15 to 40 "C at a rate of 0.6 "amin by using a circulatingwater bath (Julabo). The excitationwavelengthwas set at 365 nm and emission was detected at 427 nm.

TABLEI Description of t - a p d - N mutants and their purity Plaemid no.

Deleted amino acids

protein

pXL 2102 a2168 pXL 2183 pXL 2346 pXL 2185 pXL 2184 pXL 2215 pXL 2186 pXL 2217 pxL 2182

None Aaa 1-12 Baa 13-61 baa 62-116 Aaa 117-160 Aaa 161-204 A a a 205-248 Baa 249-288 Aaa 289-332 Aaa 333-376

98 98 90 63 87 87 83 75 94 95

Purity %

M r 1 2 3 4 5 6 7 8 9 1 0 M r

RESULTS

Before performingthe following experiments withthe tagged apoA-IV mutants, we verified that undeleted t-apoA-IV had properties similar to those of apoA-IV extracted from plasma (44)(see Tables11-V). Production of t-Apo-Wlth the T7 expression system described above, recombinant t-apolipoproteins accumulated in E. coli at levels of 6 1 0 % of the total bacterial protein in high cell density cultures and at levels of up to 30% in shaker-flask

cultures. FIG. 2. SDS-PAGE of purified undeletad t-apoA-IV and muPurifiation of t-Apos-t-ApoA-IV and mutants, recovered as soluble proteins from bacterial extracts, were purifiedby metal tants. Samples containing 10 pg of p d e d protein were m on a 0.1% SDS, 15% polyacrylamide gel. Proteins were revealed with Coomaseie t ychromatography on a Ni-nitdotiacetic acid Blue. chelate & Lane 1, undeleted t-apoA-m lane 2,bapoA-W Baa 1-12;lane 3, agarose resin, as described above. A a a 13-61; lane 4, A a a 62-116; lane 5, Aaa 117-160; lane 6,Aaa 161The mutants were constructedby deleting non-helicoidaldo- 204,lane 7, Baa 205-248, lane 8,baa 249-288;lane 9, A a a 289-332;and mains (aa1-12 and aa 333-376) or helicoidal domains (Fig. l). lane 10, Aaa 333-376. In the latter case, two successive helices were removed at a time in order to preserve the overall tertiary structure of the protein. Most of the deletions were44 residues in length, be- Aaa 333-376 mutant (Fig. 2, lane 10) compared to that of cause most of the apoA-IV helices are formed by 22-residue mutants with the same length of deletion (baa 117-160, Aaa the repeats (Fig. 1).The numberingof the deleted residuesin the 161-204, baa 206-248, and Aaa 289-332) probably reflecta this mutant was highly hydrofact that the deleted portion of mutant proteins corresponds to that of residues in the entire protein sequence, excluding the poly-histidine tag and the N€&- philic. SDS binds to proteins through hydrophobic interactions, terminal methionine (Table I).Purified proteins were analysedand this mutant was more hydrophobic than the other muSDS by SDS-PAGE (Fig.2). Protein purity, assessedby densitomet- tants. Thus, the Aaa 333-376 mutant probably fixed more ric scanning, was 63-98% (Table 1). The faster mobility of the than theothers, resulting in greater electrophoretic mobility.

140600

-

67000

-

FIG.3. Polyacrylamide gel electrophoresis of undeleted t-apoA-IV and mutants, performed under non-denaturing conditions. Prepacked gradient gels containing 4 2 0 % polyacrylamide were used. Proteins were revealed with Coomassie Blue. Lunes 1,6,7, and13, undeleted t-apoA-IV; lune 2. t-auoA-IV Aaa 1-12: lune 3. Aaa 13-61: lune 4. Aaa 62-116; lune 5,Aaa 117-160; lune 8,Aaa 161-204; lune 9, Aaa 205-248; lune 1O:Aaa 249-288; lune 11, Aaa 289-332; and’lune 12, Aaa 333-376.

Analysis of t-ApoA-N Mutantsby Non-denaturing Polyacryl- lipid- apolipoprotein complexes, the complexes were subjected amide Gradient Gel Electrophoresis-Following non-denatur- t o density gradient ultracentrifugation (Fig.4). The POPCing polyacrylamide gradient (4-20%) gel electrophoresis, unde- apolipoprotein complexes seemed to be more homogeneous leted t-apoA-IV appeared as two major bandsmigrating than those formed with egg lecithin.For these complexes, between the 67- and 140-kDa molecular mass references plus slight differences in density were observed, but all of the proa minor band migrating with the 232-kDa reference (Fig. 3). tein was recovered floating in three fractions co-eluting with the [14C]cholesterol activity. The egg lecithin-apolipoprotein The same pattern was found for human plasma apoA-IV by Weinberg and Spector (451, who attributed thetwo major bands complexes floated as a complex of broader density range (four to monomeric and dimeric forms, even though their relative fractions). In the complexes formed with mutants Aaa 13-61, molecular masses were higher than46,000 and 92,000, respec- Aaa117-160, and Aaa 161-204, some protein was either incomtively. One of the hypotheses proposed for the unexpectedly pletely incorporatedor spunoff during ultracentrifugation. The slow mobility was that human apoA-IV contains carbohydrate, small amountof unbound protein would not, however, account which would interfere withprotein migration under thesecon- for the significant differences in LCAT activity observed with ditions. The proteins we studied are not glycosylated, because mutant Aaa117-160, which was well incorporated into the they were expressed in E. coli, but their mobility was equally complexes, as demonstrated with POPC. Thus, the decreased slow. Therefore, i t seems more likely to us that thetwo major cofactor activity of this mutant cannotbe due to a dissociation bands corresponded to dimeric and trimeric forms of apoA-IV, of the complexes. while the minor band corresponded to a pentamer. BiophysicalCharacterisation of the t-ApoA-NMutantThe t-apoA-IV mutants migrated more slowly than unde- DMPC Complexes-The DMPC complexes were made by a difleted t-apoA-IV (Fig. 3), although the molecular weight was ferent procedure than thecholate dialysis method used for the lower. t-ApoA-IV Aaa 1-12, Aaa 13-61 and Aaa 289-332 mi- LCAT assays (see “Methods”). The elution volumes, composigrated as two bands between the 67 and 232 kDa molecular tion, percentage of bound protein and Trp maximal fluoresmass references. t-ApoA-IV Aaa 62-116, Aaa 249-288, and Aaa cence wavelength of the DMPC.t-apo complexes are summa333-376 had similar patterns, with one band migrating be- rized in Table 111. A major complex with an elution volume of tween the two major bands of the undeleted protein. The mu- around 24 ml was observed for all mutants, except Aaa 13-61, tants Aaa 117-160, Aaa 161-204, and Aaa 205-248 each had for which the elution volume of the major complex was 22.5 ml. one predominant bandwhich could correspond to theirdimeric For undeleted t-apoA-IV and the t-apoA-N Aaa 1-12, Aaa form. The electrophoretic mobility of the mutants might there- 117-160, and Aaa 333-376 mutants, homogeneous complexes fore reflect changes in the overallcharge and three-dimen- eluted as a symmetrical peak, and only 5% of the t-apoA-IV sional structure with respect to the undeleted protein. eluted as free protein. For the other mutants, the total amount LCAT Cofactor Activity-To determine the capacity of the of bound protein was decreased. The presence of free protein t-apoA-IV mutants to activate LCAT, the proteins were incor- could be explained, at least in part,by the decrease in protein porated into discoidal POPC-cholesterol or egg lecithin-choles- purity. However, this explanation cannot evidently be applied terol complexes. The apparentkinetic constants obtained upon to mutant Aaa 289-332, which was 94% pure and yielded 24% incubation of these complexes with LCAT are shown in Table 11. free protein. For mutants Aaa 13-61,Aaa62-116,Aaa161When the t-apoA-IV Aaa117-160 mutant was incorporated 204, baa 205-248, and Aaa 249-288, a second, larger complex into particles consisting of either POPC or egg lecithin, its eluted a t a volume of 15-17 ml. There was less of the larger cofactor activity was significantly lower than thatof the unde- complex than of the smaller complex, and the phospholipid/ leted protein. When incorporated into egg lecithin particles, protein (w/w) ratios ranged between 7 and 14 for the larger mutants t-apoA-IV Aaa 161-204 and t-apoA-IV baa 205-248 complex and between 2.7 and 4 for the smallercomplex (Table were also significantly less active than the undeleted t-apoA-IV, N). The DMPC-t-apoA-IVmutant molar ratio rangedfrom 155 although they were more active than t-apoA-IV Aaa 117-160. to 246 for the smaller complex and from 381 to 780 for the The POPC-cholesterol complexes prepared with these mutants larger complex. had the same activity as the complexes prepared with undeThe exposure of the Trp residue at position 12 to thesolvent leted t-apoA-IV and as those prepared with the other mutants. was monitored by maximal fluorescence wavelength measureThe apparent K,,, differed only by a factor of two to three be- ment. The Trp conformation in the t-apoA-IV Aaa 13-61 mutween t-apoA-IV Aaa 117-160 and most of the other mutants, tant differed from that in the undeleted protein and in the while the apparentV, was 10-fold lower for the t-apoA-IV Aaa other mutants,as the emission wavelength wasshorter and the 117-160 mutant-egg lecithin-cholesterol complexes. Trp was apparently betterprotected from the aqueous solvent. To determine the extent of incorporation of the t-apos into the The shift of the Trp wavelength upon lipid binding was, more-

ApoA-N Helical Domain (aa 11 7-160) and LCAT Activation

29887

TABLEI1 Apparent kinetic constants for the reaction of LCAT with different t-apoA-N-phospholipid-cholesterol complexes Parameters were derived fromLineweaver-Burk analysis of initial velocities versus apolipoprotein concentration. Protein

Apparent K,

Apparent V,, POPC Egg lecithin

reaction

Relative

POPC Egg lecithin

nmol CE"I h

Plasma apoA-IV 0.75 t-ApoA-IV7 1.05 0.8 0.149 Aaa0.068 117-160 Aaa0.5-1.25 7.2161-204 0.384 Aaa205-248 7 0.5-1.25 0.384 All others O&l

rates

POPC Egg lecithin mglml

1.1

6.8 x

8x 8x 2.7 x 10-3 x 10-3 5.5-9 x 10-3 90.9-140 75.5-105 x 10-3

10-3 5.5 x 10-3 5.5-9 x 10-3 x 10-3 7-8 5.5-9 x 10-3 X

0.5-1.25

-

100 98 t 4 10.4 1 44.4 112L 4.7 36.1 f 2.3

100 104 f 3 23.6 f 3 f 8.5 90.9 2

Cholesteryl ester,

TAEILE I11 Characterization of the t-apoA-N mutant-DMPC complexes

a) POPC

~

Protein aoo

aoo

b) egg lecithin

B 8 e

,.,.,.,.,.,

30

' I V

v:

Bound aoo

ml

%

Plasma apoA-IV' 97 t-ApoA-IV Aaa 1-12 Aaa 13-61 14.8 8 baa 62-116 15.2 57 24.48 Aaa 117-160 Aaa 74 25.0 161-204 915.2 Aaa 205-248 15.3 4 Aaa 249-288 15.3 14 Aaa 289-332 Aaa 333-376

FRACTION NUMBER

40

Minor complex

Major complex

v,

Bound

ml

%

24.0 24.0 24.0 22.5

95 95 80

24.3

95

24.5 62 24.8 24.7 24.6

65 76 95

Free protein Bound ml

%

nm

343 342.5 307.!jd 330.0 339.0 5 343.5 17 343.5 31 338.5 24 340.5 24 342.5 5 342.5 3 5 5 12 35

29.0 29.0 29.0 29.0 29.0 29.0 29.0 29.2 28.6 29.2 29.0

V,, elution volume. wavelength of max. Trp emission in the second complex. e Data from Lins et al. (44). Wavelength of max. Tyr emission in the second complex. a

' A,,

A m 1-12

20 10

TABLEIV

€4

Composition of the t-apoA-N mutant-DMPC Complexes

L $ m

Protein

IO

Minor complex lipid to protein ratio W/+

0

Plasma apoA-IV t-ApoA-IV Aaa 1-12 Aaa 13-61 223 Aaa 62-116 Aaa 117-160 Aaa 2.9 161-204 564 haa 205-248 Aaa249-288 Aaa289-332 Aaa 333-376

1 3 5 7 9 1 1 1 3 5 7 9 1 1 1 3 5 7 9 1 1 1 3 5 7 9 1 1 1 3 5 7 9 1 1

FRACTION NUMBER

FIG.4. Density gradient ultracentrifugation profilesof undeleted t-apoA-IV and mutants incorporated into POPC (a)or egg lecithin ( b ) particles containing [14Clcholesterol. Substrate complexeswereformed with [14C]cholesteroland phospholipid by the cholate dialysis procedure.ARer ultracentrifugation, the bottoms of the tubes were punctured, and 1-ml fractions were collected. Fraction 1 contained the highest density (1.21 dm]), decreasing approximately linearly to fraction 11 ( a = 1.006 drnl). Aliquots of the fractions were counted for ['4Clcholesterol activity and plotted. Inserted numbers indicate deleted amino acids of the respective mutant. Mutantst-apoA-IV Aaa 161-204 tended to form two distinct sets of particles with egg lecithin.

over, more pronounced for this mutant than for any of the others. This change inconformation is not surprising, because the deletion in this mutantconsisted of the two helices closest to the single Trp residue. For the t-apoA-IV Aaa 1-12 mutant, the %, instead of the Trp, fluorescence emission intensity was measured, as theonly Trp residue had been deleted from this protein. The size of the DMPC complexes was determinedby gradient gel electrophoresis of the topfraction of the majorelution peak of the chromatographic run (Table V). The diameters of the complexes formed with all the mutants were around 120-140 A. The electron micrographs of the complexes showed homogeneous discoidal particles (Fig. 5).The size distribution and the

14 4.0 7 10 11 9 2.7

Major complex lipid to protein ratio

dmb

w/w

d m

258

3.8 3.8 3.5

246 217

780 381 218 191 621 516 202

4.0 3.6

203 166

3.4 3.4 3.6

155 193

wfw, weight ratio. d m , molar ratio. e Data from Lins et al. (44). a

'

mean radius of the particles are presented in Table V. The average radius was larger than thatobtained by gradient gel electrophoresis, as previously reported (46, 47). The transition temperature of the phospholipid, measured by fluorescence polarization, was 2.5-3" higher for the complexes than for pure DMPC, as previously reported for apolipoproteinlipid complexes (46). DISCUSSION

To study the structure-function relationshipof apoA-IV, we generated t-apoA-IV variants by deleting successive domains, including the 22-residue repeats, along thesequence. ApoA-I and apoA-IV are the two most efficient cofactors for LCAT. Many attempts have been made to identify the LCATactivating region(s) of apoA-I by using synthetic peptides (19,

29888

A p o A - N Helical Domain (aa

TARLE V Molecular weight fMW) and Stokes' radius of the t-apoA-IV mutant: DMPC major complexes determined by gradient gel electrophoresis and size determined by electron microscopy (EM)

117-1601 and LCAT Activation

theother apolipoproteins. Using site-directed mutagenesis, Minnich et al. (52) disrupted the structure of this region by introducing point mutations at Pro-99 and Pro-121, and, in contrast, foundonlyaslightlyreduced LCAT activation by Protein MW Stokes' radius Radius EM apoA-I. However, these authors have observed a drastic deA kDn crease of LCAT activation by apoA-I variants havingdeletions 421 65 82 Plasma apoA-IV" spanning residues148-186,212-233, and 213-243, suggesting 77 t 20 t-ApoA-IV 42 1 60 t 5 that the carboxyl-terminal residues of apoA-I are crucial for h a 1-12 496 9 1 t 16 67.7 LCAT activation. Sorci-Thomas et al. (53) whose results are 9 1 f 20 466 Aaa 13-61 64.3 consistent with those of Minnich et al., found "that deletion of 1 1 6 k 20 Aaa 62-116 425 60.0 79 f 14 h a 117-160 390 59.0 any of the 22- or 11-mer repeats of apoA-I results in a t least a Aaa 161-204 78 k 13 423 60.0 40% reduction in LCAT activation" and that thiseffect is most Aaa 205-248 80 2 12 465 64.0 apparent when the deletion is located in the region spanning 82 2 11 421 59.9 h a 249-288 90 t 12 Aaa 289-332 419 59.9 residues 143-208. Aaa 333-376 110 f 25 505 69.0 In contrast to the extensive studies of apoA-I, only a few reports have appeared about apoA-IV, and no study has yet succeeded in assigninga specific function to a particular region of the latter protein. That the catalytic efficiency of LCAT activation of a naturally occurring mutant (apoA-N-2, Glu"" -> His) was greater than that of the wild type protein was attributed t o the increased hydrophobicity of the mutant and to its greater affinity for, and deeper penetration into, phospholipid surfaces (27).Although these results have notbeen confirmed by other authors (56), in vivo reassociation data argue for an increased binding of apoA-IV-2 to HDL, which might be why the in vivo catabolism of this mutant protein is slower (28). In this study we identified a particular region of apoA-IV that isspecifically involved in the LCAT activating function of this protein. We observed a significant decrease of the LCAT cofactor activity following the deletion of 44 residues in the apoA-IV sequence between residues 117 and 248; this effect was most pronounced for the variant lacking residues 117-160, as theLCAT cofactor activity decreasedby 90 and75% with egg lecithinand POPC complexes, respectively. When residues 161-204 or 205-248 were deleted, cofactor function also decreased, but only for complexes made with egg lecithin; this decrease was less marked than that observed with t-apoA-N Aaa117-160.As indicated in Fig. 4b, substrate particles formed with t-apoA-N Aaa 161-204 and Aaa 205-248 seemed heterogeneous in that while the bulk of the cholesterol was associatedwith particles of lower density (fractions 7-8), a subpopulation of particles with higher density (fractions 4-5) FIG.5. Electron micrographs at a 168,000~magnification of was formed consisting of less cholesterol (Fig. 4b) and more t-apoA-IV.DMPCcomplexes. a , undeletedt-apoA-IV h , t-apoA-IV protein (data not shown). There were two types of particles probably becausethe composition of phosphatidylcholine in egg Aaa 117-160; c, Aaa 161-204; d , Aaa 205-248. Complexes a t a protein concentration of 150 pg/ml were negatively stained with a 20 gfliter yolk lecithin is heterogeneous and because the various phossolution of potassiumphosphotungstate a t pH 7.4. 7-1.11 aliquots pholipids interact differently with a given apolipoprotein. As of the samples were applied to Formvar carbon-coated grids prior to substrate particle size and composition are determinants of examination. LCAT reactivity (i.e. the reactivity decreases with increasing particle size) (571, the difference in particle density seen with account for the slight 48, 49), apoA-I CNBr fragments (46), monoclonal antibodies these two mutants of apoA-IV may in part (211, recombinant HDL particles (20, 50, 511, site-directed mu- decrease in V,,, we found. At this point, we did not further tagenesis (52), and sequentialdeletions of 22- or ll-mer repeats pursue this issue, which will be the subject of a more detailed (53). It isnow clear that LCAT-activating proteins require am- investigation. The POPC particles (Fig. 4u) have a uniform phipathic helical sequencesable to associate with lipid-contain- phosphatidylcholine content and, a s such, constitute a better ing surfaces(54). The role of this domain is probably to disrupt defined system. With such a system, more homogeneous parthe waterlphospholipid interface and expose the buried subticles were formed,specifically with mutants haa161-204 and strate to LCAT ( 5 5 ) . This disruption isnecessary, but not suf- Aaa 205-248. The reactivity of these substrate complexes did ficient, for activation (19,481. Although specific apoA-I regions not differ significantly from that of those formed by the undemay be implicated in LCAT activation, results are inconsistent. leted protein. Thus, the discrepancy between the results obAnantharamaiah et al. (19) and Banka et al. (21) haveproposed tainedwith POPC and egg lecithinparticles for mutants that anapoA-I domain consistingof residues 66-120 is involved t-apoA-IV Aaa 161-204 and Aaa 205-248 may be attributed to in LCAT activation. They have suggested that the positioning the difference in complex formation rather thant o a difference in the specific interaction with LCAT. of a Glu residue on the non-polar face of each helix in this The reactivityof the t-apoA-NAaa 117-160 mutant protein, region (residues 66-87 and 99-120) is responsible for the higher LCAT-activating ability of apoA-I compared to that of however, was markedly diminished with both phospholipids,

ApoA-N Helical Domain (aa 117-160) and LCAT Activation even though homogeneous complexes were formed in each case. The part of the protein deleted from this mutant, therefore, is probably a functionally important domain of apoA-IV. Either the deleted region contains a pattern,a sequence or one or more amino acids specifically responsible for LCAT activation, or the deletion led to a drastic loss of the protein structure, resulting in much less cofactor activity. The latter possibility seems unlikely in light of the results of our biophysical studies of the complexes. The t-apoA-IV Aaa 117-160 mutant generated complexes with DMPC to the same extent as the undeleted protein: in both cases, only 5% of the protein was unbound while 95%was incorporated into the complexes. The reason the t-apoA-IV Aaa 117-160 mutant has a significantly different LCAT reactivity may be that apoA-IV mediates the binding ofLCAT to the lipid surface and consequently activates LCAT or renders thelipid accessible t o it (18). The amphipathic a-helical structure and surface activity of an apolipoprotein are necessary, but not sufficient, prerequisites for LCAT catalysis (51). The t-apoA-IV Aaa 117-160 mutant still bears helical form, and its surface activity and binding to lipids are indistinguishable from those of the undeleted protein. Major differencesin thelipid binding would, therefore, not be responsible for the grossly diminished cofactor activity seen here, although the degree ofLCAT activation also depends upon the depth to which an apolipoprotein penetrates a phospholipid monolayer (58). The results of our experiments on LCAT-reaction kinetics (significantly diminished V,,) indicate an overall diminished efficiency of cholesteryl ester formation by the enzyme, when t-apoA-IV Aaa 117-160 was present in the substrateparticles. This may reflect a reduced activation of the enzyme or a diminished efficiency of the LCAT-catalyzedreaction. Similarities in apparent K,,,indicate comparable binding affinities of LCAT for thesubstrate particles. The deletion Aaa 117-160 in the apoA-IV protein, however, doesnot disrupt the binding to phospholipid, nor apparently the binding of the enzyme to the substrate. According to hypothetical models for the activation of LCAT by a reconstituted apolipoproteidphospholipid substrate, thedeletion could affectthe process by still other mechanisms. Parts of the deleted sequence of the protein might be necessary for activating the enzyme or,less likely, the interaction of the enzyme with this region might be required for releasing the reaction products. Although we found here that a relatively small region of apoA-IVis necessary to activate LCAT, Sorci-Thomaset al. (531, using the same type of methodology, i.e. sequential deletion of helicoidal domains, have demonstrated thatthe region of apoA-I responsible for LCAT activation spans a large number of residues distributed all along the protein on the amphipathic helices and particularly on the COOH-terminal half. Thus, apoA-IV and apoA-I mayhave different ways to activate LCAT, which would beconsistent with their different degrees of association with HDL particles. Our biophysical experiments showed that mutant Aaa 289332 binds to unilamellar DMPC liposomes differently than the undeleted protein or the other mutants. Under these conditions, about 24% of the protein was not incorporated into the complexes, suggesting the involvement of the deleted region in spontaneous lipid binding. For mutants with deletions at positions 13-61,62-116,161-204,205-248, and 249-288, the presence of an additional larger complex suggests, too, that the deletions have resulted in a modification in the way these proteins bind to lipids. As part of our comparison between apoA-IV and apoA-I, we tried to determine whether therewere any similarities between the 117-160 region of apoA-IV and the apoA-I sequence. When

29889

the 117-160 sequence was aligned with the apoA-I sequence, 18%identity was found; this fell in the region of residues 154193 of apoA-I. When the two proteins were compared along their entire lengths, there was about 21%identity. The region of residues 154-193 of apoA-I corresponds to a part of this protein previously proposed(aa 148-186) by Minnich et al. (52) and by Sorci-Thomas et al. (aa 143-208) (53)to be involved in LCAT activation. Future experiments are planned to localize more precisely the specific region involved in LCAT activation and t o delineate the apoA-IV domains involved in lipid binding, by producing mutants with shorter deletions.In addition to these in vitro approaches, it will also perhaps be feasible to introduce into mice the DNA encoding one of the mutants generated in this study, as well as that encoding the non-deleted apoA-IV protein. Such studies may contribute to the understanding of the processes involved in atherosclerosis and might thereby suggest new ways to therapeutically control the efficiency of LCAT activation. Acknowledgments-We thank Dr. G. Jung and his team for high density bacterial fermentations studies, L. Bassinet, T. Ciora, and S. Motzny for their helpful technical assistance, and K. Pepper for critically reading the manuscript. REFERENCES 1. Green, P. H. R., Glickman, R. M., Riley, J. W., and Quinet, E. (1980) J. Clin. Znuest. 66, 911-919 2. Gordon, J. I., Bisgaier, C. L., Sims, H. E, Sachdev, 0. P., Glickman, R. M., and Straws, A. W. (1984) J. Biol. Chem. 269,468474 A. (1974) Biochem.Bwphys.Res. 3. Swaney, J. B., Reese, H., andEder,H. Commun. 59,513-519 4. Bisgaier, C. L., Sachdev, 0. P., Megna, L., and Glickman, R. M. (1985)J. Lipid Res. 26,ll-25 5. Dieplinger, H., Lobentanz, E.”., Ktining, P., Graf, H., Sandholzer, C., Matthvs. E.. Rosseneu.. M.,. andutermann.G. (1992)Eur. J.Clin. Znuest. 22,

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