Proc. Natl. Acad. Sci. USAVol. 88, pp. 1716-1720, March 1991Biochemistry

Effects of site-directed mutagenesis at residues cysteine-31 andcysteine-184 on lecithin-cholesterol acyltransferase activity

(lipid metabolism/cholesterol/acyltransferase)

OMAR L. FRANCONE* AND CHRISTOPHER J. FIELDINGCardiovascular Research Institute and Department of Physiology, University of California Medical Center, San Francisco, CA 94143

Communicated by Richard J. Havel, November 28, 1990 (received for review September 7, 1990)

ABSTRACT Native lecithin-cholesterol acyltransferase(LCAT; phosphatidylcholine-sterol acyltransferase; phospha-tidylcholine:sterol O-acyltransferase, EC 2.3.1.43) protein,and LCAT in which either or both of the enzyme free cysteineshad been replaced with glycine residues by site-directed mu-tagenesis, has been expressed in cultured Chinese hamsterovary cells stably transfected with the human LCAT gene. Themass of LCAT secreted, determined by immunoassay, did notdiffer in the native and mutant species. LCAT specific activitywas also unchanged in the mutant species. In particular, thecysteine-free double mutant, in which Cys-31 and Cys-184 hadboth been replaced, was fully active in the synthesis of cho-lesteryl esters. This result is not consistent with a catalytic rolefor LCAT free cysteine residues. The classical inhibitor ofLCAT activity, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB),which strongly (89%) inhibited the native enzyme, had partial(45%) inhibitory activity with mutant enzyme species contain-ing a single -SH residue, while the double mutant was notsignificantly inhibited by DTNB. These data are interpreted tosuggest that Cys-31 and Cys-184 are vicinal both to each otherand to the "interfacial binding site" at residues 177-182, andthat DTNB exerts its effect by steric inhibition.

The majority of cholesteryl esters in normal blood plasma areformed by lecithin-cholesterol acyltransferase (LCAT; phos-phatidylcholine-sterol acyltransferase; phosphatidylcho-line:sterol O-acyltransferase, EC 2.3.1.43). The enzyme ispresent as a complex with lipids and apoproteins in the highdensity lipoprotein fraction (1). In vivo LCAT catalyzes thetransacylation of the sn-2 position fatty acid of lecithin to the3-hydroxyl group of cholesterol, but in the absence of cho-lesterol LCAT effectively acts in the hydrolysis of lecithinand the transacylation of lecithin and lysolecithin (2, 3). It hasusually been considered that LCAT, in a reaction analogousto that of classical serine-dependent esterases, acts via aninitial deacylation of lecithin, with formation of an acyl-LCAT intermediate, followed by transfer of this acyl groupto acceptors with a free hydroxyl function, including choles-terol and other sterols, water, or lysolecithin, with regener-ation of the free LCAT protein (4).LCAT contains two free cysteine residues at positions 31

and 184 of the mature protein (5, 6). Unlike most esterases,LCAT is inhibited by sulfhydryl reagents such as p-hy-droxymercuribenzoate (7) and 5,5'-dithiobis(2-nitrobenzoicacid) (DTNB) (8). Recently, a more complex mechanism hasbeen proposed, in which transacylation to cholesterol in-volved an obligatory LCAT-S-acyl intermediate formed inreaction with one or both of the free cysteine residues (9, 10).To test this hypothesis, LCAT molecules modified by theremoval of one or both cysteine residues by site-directed

mutagenesis have been synthesized, and their biochemicalproperties have been determined.

EXPERIMENTAL PROCEDURESIsolation of LCAT cDNA. LCAT-specific cDNA was syn-

thesized via the polymerase chain reaction from the mRNAofhuman hepatoblastoma (HepG2) cells. The mRNA of cellsgrown in 175-cm2 flasks was prepared by oligo(dT)-cellulosechromatography (K-1593-02; In Vitrogen, San Diego). Thepoly(A)+ mRNA was precipitated at -70'C with 0.1 vol of 2M sodium acetate and 2 vol of 100% ethanol.A cDNA reaction mixture was prepared to contain 10 pmol

of 23-mer primer with an internal Bgl II restriction site,antisense to the 3' end ofLCATmRNA (5'-AGCTAGATCTTTA TTC AGG AGG-3') (Operon Technologies, Alameda,CA). mRNA from one flask of cells, 200 units of Moloneymurine leukemia virus reverse transcriptase (BRL), 10 unitsof RNase inhibitor (Promega Biotec), and 0.5 mM eachdeoxynucleotide in 50 mM Tris HCI, pH 8.3/75 mM KCI/10mM dithiothreitol/3 mM MgCl2. After incubation for 1 hr at37°C, 3 ,ul of 1Ox buffer was added (100 mM Tris HCI, pH8.3/500 mM KCI/15 mM MgCl2/0.1% gelatin) together witha further 40 pmol of the 3'-end primer and 50 pmol of a5'-sense primer (5'-CC AAG CTT GGA ATG GGG CCGCCC-3') containing a HindlIl restriction site correspondingto the beginning ofthe coding region ofLCATcDNA and 1.25units of Thermus aquaticus DNA polymerase (Perkin-Elmer/Cetus) in a final aqueous vol of 50 ,u. The polymerasechain reaction was carried out in a Perkin-Elmer/Cetusthermocycler for 40 cycles (94°C for 1 min; 55°C for 2 min;72°C for 3 min).

Synthesis of the expected full-length (1.4 kilobases) cDNAwas confirmed by electrophoresis in 1.5% agarose/ethidiumbromide gel.cDNA Cloning and Sequencing. The cDNA was inserted

into the Sma I site of a pUC18 vector (Pharmacia LKB). Theligation mixture was transformed into DH5-a Escherichiacoli and clones containing the insert subcloned into theEcoRI/BamHI sites ofM13mpl8 and M13mpl9 vectors. Theinsert was sequenced via the dideoxynucleotide chain-termination reaction (11) using adenosine 5'-[-[35S]thio]-triphosphate. The entire cDNA was then sequenced, but nodifferences were found from that sequence ofthe LCAT genepreviously reported (5).

Site-Directed Mutagenesis. The full-length human LCATcDNA sequence was ligated into the EcoRP/BamHI site ofpTZ18 phagemid vector (Bio-Rad). Synthetic oligonucleo-tides 24-30 bases long carrying one mismatched base wereused to mutagenize Cys-31 to Gly (5'-CTGATTCCCCAG-GCCGCCGGGCACGAG, complementary to GGC insteadof TGC in the LCAT cDNA sequence) or Cys-184 to Gly

Abbreviations: LCAT, lecithin-cholesterol acyltransferase; DTNB,5,5'-dithiobis(2-nitrobenzoic acid); apoA-I, apolipoprotein A-I.*To whom reprint requests should be addressed.

1716

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Proc. Natl. Acad. Sci. USA 88 (1991) 1717

(5'-GAGCAAGTGTAGACCGCCGAGGCTGTGGCC,complementary to GGT in place ofTGT). The double mutantLCAT-Gly-31, Gly-184 was prepared in the same way bymutagenizing LCAT-Gly-31 with the second oligonucleo-tide. In this protocol, first described by Kunkel (12), coloniestransformed with the mutated DNA were grown to producesingle-stranded pTZ18 human LCAT DNA. In each case, thepredicted base sequence of the mutated LCAT cDNAs wasconfirmed by dideoxynucleotide sequencing as describedabove.In Vitro Transcription and Translation. pTZ18 plasmids

containing full-length human LCAT cDNA were purified byalkaline hydrolysis (13) and via a cesium chloride gradientcontaining ethidium bromide; then they were linearized withBamHI and transcribed with T7 RNA polymerase (Pharma-cia LKB). The transcripts were capped by addition ofm7G(5')ppp(5')G in the transcription reaction. TemplateDNA was removed with RQ1 DNase and portions of thetranscription reaction were run in 1% agarose/formaldehydegels to verify the size and integrity of the transcripts. mRNAsynthesized by T7 RNA polymerase was translated in acell-free system containing nuclease-treated rabbit reticulo-cyte lysate, [35S]methionine, and RNasin in the presence orabsence of canine pancreatic microsomal membranes underconditions described by the manufacturer (Promega Biotec).Primary translation products were further analyzed on aSDS/8% polyacrylamide gel. The gels were fixed, dried, andexposed to Kodak XAR-5 film at -70'C with an intensifyingscreen.Construction of an Expression Plasmid for Human LCAT.

An expression plasma pSV2dhfr (American Type CultureCollection ATCC37146) containing the cDNA sequence of amouse dihydrofolate reductase (Dhfr) was digested withHindIII and Bgl II to remove the Dhfr sequence. Thewild-type and mutant full-length LCAT cDNA cloned inpTZ18 plasmids were digested with Bgl II and HindIII and afull-length LCAT cDNA clone extending from 8 bases beforethe ATG start codon to 5 bases after the TAA stop codon wasthen ligated to the unique HindIII/Bgl II sites of the pSV2plasmid.

Culture and Transfection of CHO Cells. CHO (Dhfr-) cells(DXB 11 line), deficient in the Dhfr gene, were grown in F-12medium supplemented with 10% fetal calf serum and genta-mycin. The expression vector pSV2hLCAT was cotrans-fected with pSV2dhfr in a 20:1 ratio into CHO cells bycalcium phosphate-mediated transfection (14). The calciumphosphate-DNA coprecipitate was allowed to form in thetissue culture medium during prolonged incubation (15-24 hr)under controlled conditions of pH (6.96) and CO2 tension(3%) (15). Transfected cells were selected by their ability togrow in modified Eagle's (MEM) alpha medium withoutnucleosides, containing 10% dialyzed fetal calf serum andindividual colonies were propagated for assay. Clones ex-pressing LCAT were identified by solid-phase immunoassayand LCAT functional activity.Immunoassay of Expressed LCAT. The mass of secreted

LCAT was determined by solid-phase immunoassay. Me-dium from CHO untransfected (control) and transfected cellswas concentrated 20- to 40-fold in Millipore filters. Protein inthe concentrated samples was bound to nitrocellulosescreens (Sartorius, West Coast Scientific, Hayward, CA) ina Bio-Rad dot-blot apparatus. Bound medium protein, andpure antigen standard, underwent reaction as described firstwith site-directed antibody to the mature human LCAT (16)and then with 125I-labeled goat antibody to rabbit IgG.Screens were then assayed in a Searle 1185 y spectrometer.Assay of LCAT Activity. Wild-type and mutant clones

expressing both Dhfr and human LCAT genes were grown inT25 flasks in MEM alpha medium without nucleosides sup-plemented with 10% dialyzed fetal calf serum and gentamycin

to =90% confluency. The cells were then extensively washedwith MEM alpha medium containing gentamycin and wereincubated 24-48 hr with MEM alpha medium without nucle-osides supplemented with 5% Ultroser G (GIBCO). LCATactivity was determined as the rate of synthesis of 3H-labeledcholesteryl esters from [1,2-3H]cholesterol (New EnglandNuclear) and unlabeled egg lecithin and apolipoprotein A-I(apoA-I) (Sigma). Single walled vesicles containing choles-terol and lecithin (1:8, wt/wt; cholesterol specific activity,1.2 x 105 dpm/,ug) were prepared with a French press (17),activated with apoA-I (18), and assayed in the presence ofrecrystallized human serum albumin (2.5%, wt/vol), 10 mMphosphate buffer (pH 7.5) in 0.15 M NaCI, together withprotease inhibitors (19). The labeled cholesteryl ester wasseparated from free cholesterol by thin-layer chromatogra-phy on silica gel layers on plastic sheets (Merck) developedin hexane/diethyl ether/acetic acid (83:16:1) (vol/vol). Someincubations were done in the presence of 1.5 mM DTNB (8).

RESULTSTranslation of Human LCAT mRNA. LCAT cDNA tran-

scribed in vitro with T7 RNA polymerase generated a single1.4-kilobase RNA with a complete absence of detectableshorter transcripts, consistent with previous findings (5, 20).The capped LCAT mRNA was then translated in vitro witha rabbit reticulocyte lysate, which (in the absence of mi-crosomes) generates only nonglycosylated translated pro-teins (21). Following incorporation of [35S]methionine intothe translated protein, LCAT with an apparent molecularmass of -45 kDa was obtained (Fig. 1), similar to theexpected molecular mass of the protein moiety of the full-length 416-amino acid enzyme (5, 6). When the same mRNAwas translated in the presence of microsomal vesicles ob-tained from canine pancreas, the 45-kDa LCAT band wasalmost completely replaced with a band of 67 kDa apparentmolecular mass, consistent with the size of mature plasmaLCAT (2, 3). These data indicate the addition of substantialN-linked carbohydrate to the LCAT polypeptide catalyzedby enzymes of the endoplasmic reticulum in the early secre-tory pathway, to the extent of -25% of total molecular mass,consistent with direct analysis of the plasma LCAT protein(1).

Construction and Expression of Glycine Mutant LCATGenes. Three mutant genes were constructed in which eitheror both cysteine codons were replaced with glycine codons.As shown in Fig. 2, LCAT-Gly-31 and LCAT-Gly-184 con-tained a single base mismatch at positions 183 and 622,respectively, while the double-mutant LCAT-Gly-31, Gly-184 contained both modifications.

MW(KDa) A92.5

B

69.0_46.0

30.0_

FIG. 1. In vitro translation of wild-type LCAT mRNA. Full-length human LCAT cDNA was cloned in a pTZ18 phagemid vector.The recombinant plasmid was digested with BamHI, and an in vitrotranscription was performed. The mRNA was then translated in areticulocyte lysate system in the absence (A) or presence (B) ofcanine pancreatic microsomes as a source of glycosylating enzymes.

Biochemistry: Francone and Fielding

1718 Biochemistry: Francone and Fielding

Hind III

ATG Cys-31c tCcy

GAxC )cysG/GxA leu

C/g

163

Cys-1 84

622

Bglll

AATAAA

1328

\1 fn an LCAr

B

leu A

\G \G A T C G A Tgly c -w

/C* - _ _:

gly(_ S§

leu gA\T/G / Ply 184 mutant, wild type

his (T I

XG

A leu

GxC )glyG/A

_a" C )CySA-

G\A )leuT/

\)

FIG. 2. Comparison of the wild-type and mutant LCAT cDNAspecies. The sequences of wild-type and either Gly-31 (A) or Gly-184(B) mutant LCAT cDNAs were determined by the dideoxynucleotidechain-termination method. The double-mutant Gly-31, Gly-184 was

obtained by mutagenizing the Gly-31 single mutant as described forthe preparation of Gly-184. The gel shows the complementary basesequence of the codons indicated.

The structure of the expression vector used in this study isshown in Fig. 3. As described in Experimental Procedures,cells were cotransfected with this vector together with thevector pSV2dhfr, which includes the Dhfr gene, permittingtransfected colonies to grow in nucleoside-free medium. Ofthe colonies surviving in nucleoside-free medium, 30-40%oexpressed both LCAT and Dhfr genes asjudged by the abilityof these colonies both to grow in the absence of nucleotidesand to secrete LCAT protein into the culture medium. Ofthese colonies, three of the wild type and three ofeach mutantwere selected for enzymatic analysis.Enzymatic Properties of Wild-Type and Mutant LCAT

Species. LCAT activity in the medium of cells transfectedwith wild-type LCAT was 9.3 ± 2.1 ng of cholesterol ester-ified per hr in the complete assay medium described inExperimental Procedures. Nontransfected cells secreted no

detectable LCAT activity under the same conditions. Verysimilar rates of secretion were obtained for each single

AmpR

FIG. 3. Human LCAT expression vector. The pSV2dhfr plasmidwas digested with HindlII/Bgl II to remove the Dhfr sequence anda 1336-base-pair fragment of cloned human LCAT cDNA was

inserted at the unique HindIII/Bgl lI site in the vector. pSV2hLCATwas cotransfected with pSV2dhfr as described. The expression ofhuman LCAT and mouse Dhfr genes was driven by the simian virus40 (SV40) early promoter. Cysteine residues selected for site-specificmutagenesis are shown beneath the LCAT cDNA. AmpR, ampicillinresistance.

mutant and the double-mutant species (Table 1). In the caseof both wild-type and mutant enzymes, the production ofcholesteryl esters was linear over at least 6 hr at 37°C.The secretion of LCAT protein by cells transfected with

wild-type and mutant LCAT DNA is shown in Fig. 4. Ratesof secretion of LCAT protein by cells transfected with eitherwild-type or mutant genes were similar (Table 1). This findingindicates that the secretion of LCAT was also unaffected bythe Cys-to-Gly substitutions in the mutant enzyme species.As a result, the specific activity of LCAT was similar in eachcase, whether or not the protein contained free cysteineresidues.The activity ofLCAT is characteristically stimulated in the

presence of apoA-I. Wild-type and mutant enzymes were

assayed in the presence or absence of apoA-I in the assaymedium. There was no significant difference in the -foldactivation obtained with the two LCAT species.The wild-type enzyme was strongly inhibited in the pres-

ence of 1.5 mM DTNB (Table 1). This inhibition is similar to

Table 1. Catalytic rate of wild-type and mutant LCAT speciesActivity, ng of ApoA-Icholesteryl Specific Inhibition by activation,ester per hr Mass, ng activity DTNB, % -fold

Wild type 9.3 ± 2.1 34.5 ± 11.2 0.3 ± 0.1 89 ± 2 7.3 ± 3.2Cys-31 to Gly 7.5 ± 0.7 39.1 ± 3.1 0.2 + 0.1 46 ± 4 -Cys-184 to Gly 10.1 ± 1.9 31.6 + 14.1 0.3 ± 0.1 45 ± 14Cys-31, Cys-184 to Gly 8.9 ± 2.0 30.7 ± 7.0 0.3 ± 0.1 8 ± 3 5.8 ± 2.0

Enzyme activity was determined by incubation (6 hr, 370C) with [3H]cholesterol (5 Aug/ml; 1.2 x 105cpm/gg) and egg lecithin (40 ,jg/ml) in the presence of apoA-I (5 ,tg/ml) as described. Specific activityis expressed as ng of cholesterol esterified per ng of protein. Inhibition was determined from the ratioof LCAT activities obtained from assays carried out in the presence or absence of 1.5 mM DTNB, underthe same assay conditions. In studies carried out at the same time with LCAT protein isolated fromhuman plasma, cholesteryl ester synthesis was inhibited 92% ± 1% by 1.5 mM DTNB. Values shownare means ± 1 SEM for five to seven different experiments. Activity and mass measurements areexpressed per T25 flask. The -fold activation by apoA-I is expressed as the ratio between LCAT rateswith lecithin-cholesterol vesicles in the presence and absence of apoA-I (1.0 tkg/8 ug of lecithin).

A

9@(c\ G A T C G A TXC* -M.

gty c~Gy3 uan idtp

XGsG

leu A z"

\c .:I.../c

MYG/l Gly 31 mutant wild type

Proc. Natl. Acad. Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 1719

FIG. 4. Immunoblots of cell culture medium from untransfected (control) and pSV2hLCAT wild-type and mutant transfected cells. Mediumfrom CHO Dhfr- cells (untransfected) and transfected cells was concentrated and the samples were applied in duplicate to nitrocellulosemembranes as described. The LCAT protein was detected by reaction with a rabbit site-directed antibody to the mature LCAT protein and thenwith 1251-labeled goat antibody to rabbit IgG.

that obtained with the human plasma protein (8). The mutantLCAT species carrying a single -SH residue were onlypartially (45-46%) inhibited by DTNB under the same con-ditions. DTNB did not significantly inhibit the double-mutantLCAT, which contained no free sulfhydryl residues.These results indicate that substitution of glycine for

cysteine residues in LCAT had little or no effect on secretionor catalytic rates, or activation by apoA-I, but reduced thedependence of activity on free -SH residues.

DISCUSSIONThe data in this study provide strong evidence that the freesulfhydryl residues of LCAT are not directly involved in thecatalytic mechanism leading to the synthesis of cholesterylesters. In particular, the double mutant containing no freecysteine residues has full catalytic activity in the esterifica-tion of cholesterol. For this reason, the earlier proposal (9,10) that an S-acyl covalent intermediate was part of thecatalytic mechanism of LCAT appears to be incorrect. Re-cent studies of several other enzyme reactions, such as thosecatalyzed by RNase and hydroxymethylglutaryl CoA reduc-tase (21, 22) (in which an essential thiol function had beenpreviously proposed) have also shown, by mutagenesis, thatthe cysteine-free enzyme retained its activity. It will beimportant to modify the free cysteines in other lipases relatedto LCAT that have reported functional sulfbydryl residues(e.g., gastric lipase) (23) to determine whether in these, as inLCAT, the free cysteine residue has only a steric effect.As it is clear that sulfhydryl reagents such as DTNB

strongly inhibit native LCAT activity, some other explana-tion must be sought for its effect, and for other evidenceinterpreted to support the S-acyl intermediate hypothesis.DTNB may act to sterically hinder one or more of the triadof active site residues implicated in the activity of manyserine esterases (24). It is of interest that one of the twocysteine residues of LCAT (at position 184) is located closeto the serine residue at position 181, which has been proposedas part of an interfacial substrate binding site (1). The sameresidue is also one of two serines (the other at position 216),which in different studies were modified by the antiesterasediisopropyl fluorophosphate (25, 26). The reactivity ofLCATwith the bifunctional reagent aminophenylarsene dichlorideclearly indicated that Cys-31 and Cys-184 are closely apposedin LCAT (10). For this reason, substitution by DTNB ateither Cys-31 or Cys-184 would be likely to inhibit stericallya reaction mechanism involving this region of the protein.This concept is quite consistent with the additive effect ofDTNB (Table 1) as it reacts with one or two -SH groups inLCAT.

As part of the S-acyl hypothesis (9), it has also beenreported that the phospholipase activity of LCAT was notDTNB dependent; it was proposed that the S-acyl interme-diate was required for cholesteryl ester synthesis but not therelease of unesterified fatty acid from lecithin. Several otherobservations in the literature are not consistent with this,however. In comparative studies with lecithin and lecithin-cholesterol vesicles, Aron et al. (2) found that both phos-pholipase and transacylase activities ofLCAT were similarlyinhibited by DTNB. Swaney et al. (27) found LCAT-mediated phospholipase and transacylase activities with rathigh density lipoprotein to be similarly inhibited by DTNB.Subbhaiah et al. (3) found the lysolecithin-lecithin exchangereaction of LCAT to be as much inhibited by sulfhydrylreagents as was the generation of cholesteryl esters. Thesedata, like those obtained in the present research, argueagainst a unique catalytic role for LCAT -SH residues incholesterol esterification.

Finally, Jauhiainen and Dolphin (9) described the interac-tion of LCAT sulfhydryl groups with long-chain acyl CoA.However, the extent of reaction (1 mol of acyl CoA perreactive residue) provides no evidence for catalytic turnoverof acyl CoA at these sites. It seems more likely that thereaction of acyl CoA is simply a derivatization of LCATprotein driven by the high-energy acyl-S-CoA bond.

In summary, the present study indicates that the freecysteine residues of LCAT are not required for cholesterylester synthesis. The inhibition of LCAT by sulfhydryl inhib-itors may best be explained by the proximity of both residuesto a functionally important region centered around Ser-181.The general mechanism of LCAT would then be similar tothat of other lipases (including pancreatic, lipoprotein, andhepatic triglyceride lipases) with comparable structure in thisregion (1).

The expert technical assistance of Lolita Evangelista is acknowl-edged. O.L.F. is an American Heart Association, California Affili-ate, Research Fellow. This work was done during the tenure of aresearch fellowship from the American Heart Association, CaliforniaAffiliate, and with funds contributed by the Alameda County chap-ter. It was also supported by the National Institutes of Healththrough Arteriosclerosis Grant SCOR HL 14237.

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