THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 28, Issue of October 5, pp. 16801-16806,199O C0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Identification of the Active Site Serine in Pancreatic Cholesterol Esterase by Chemical Modification and Site-specific Mutagenesis*

(Received for publication, March 29, 1990)

Linda P. DiPersio, Robert N. Fontaine, and David Y. Hui$ From the Demvtment of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, dhio 45267-h529 I-

Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol es- terase. In the first approach, purified porcine pan- creatic cholesterol e&erase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol e&erase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-con- taining peptide was obtained for sequence determina- tion. The results revealed the binding of DFP to a serine residue within the serine e&erase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol e&erase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol e&erase cDNA was then mutagenized to ACT or GCT to yield muta- genized cholesterol e&erase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or ala- nine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [‘%]oleate and [‘“Cl lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol e&erase.

Bile salt-stimulated cholesterol esterase, also called carbox- ylester lipase, is synthesized primarily in the pancreas and then transported to the intestine where it catalyzes fat and vitamin absorption (see Ref. 1 for review). Although the physiological role and enzymology of cholesterol esterase have been characterized extensively, the relationship between the structure of the protein and its enzymatic activity is not well understood. Previous studies showed that cholesterol esterase is quite different from other lipases, such as the pancreatic lipase and phospholipase AZ. In contrast to the activities of these lipases, cholesterol esterase activity could be inhibited

* This work was supported in part by Grant DK40917 from the National Institutes of Health and by Grant-in-Aid SW-89-12 from the Ohio Affiliates of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of an Established Investigator Award from the Amer- ican Heart Association. To whom correspondence and reprint re- quests should be addressed: Dept. of Pathology and Laboratory Med- icine, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0529. Tel.: 513-558-9152.

by low concentrations of diisopropylfluorophosphate (DFP)’ (2-5). Additional chemical modification studies also demon- strated the importance of a hi&dine and a carboxylate residue in the catalytic activity of cholesterol e&erase (4,6). Chemical modifications of these residues abolished cholesterol esterase hydrolysis of both water- and micellar-soluble substrates sug- gesting that these residues are important for the catalytic function of the protein. Although the precise location of the key amino acid residues has not been identified, these results suggest that cholesterol esterase may be structurally and functionally similar to other serine e&erases (1).

Recent research in our laboratory has resulted in the iso- lation of the full-length cDNA for rat pancreatic cholesterol esterase (7). The primary structure of the protein, as deduced from nucleotide sequencing of the cDNA, revealed a 63-amino acid domain which is similar to the active site domain of other serine esterases (8-10). The active site serine in these proteins was localized to the serine residue within a common motif with the sequence G-E-S-A-G. This sequence is conserved in cholesterol e&erase between residues 192 to 196 suggesting that Ser-194 may be the active site serine of cholesterol e&erase (7). In the current study, we have used [3H]DFP labeling technique to identify Ser-194 as the site of DFP modification. Furthermore, using site-specific mutagenesis approach, we verified that Ser-194 is the active site serine of rat pancreatic cholesterol esterase.

EXPERIMENTAL PROCEDURES

Materials-The [“H]DFP and cholesteryl [%]oleate were obtained from Du Pont-New England Nuclear. The l-[l-‘4C]palmitoyl L-lyso- 3-phosphatidylcholine and I” I-labeled anti-rabbit IgG were products of Amersham Corp. Mutagenesis reagents, the corresponding bacte- rial cells, nitrocellulose paper, and electrophoresis reagents were obtained from Bio-Rad. Restriction enzymes and T4 DNA ligase were obtained from New England BioLabs. The expression plasmid vector PSVL and DEAE-dextran were products of Pharmacia LKB Biotech- nology Inc. All other reagents were of the highest grade obtainable from either Fisher or from Sigma.

Chemical Modification of Pancreatic Cholesterol E&erase-Porcine pancreatic cholesterol esterase, purified by the method of Rudd et al. (5), was a generous gift from Dr. Howard L. Brockman (Hormel Institute, University of Minnesota, Austin, MN). The purified cho- lesterol esterase was modified by incubating 4 mg of purified enzyme with 250 &i of [“HIDFP (specific activity, 3 Ci/mmol) in 4 ml of barbital buffer for 48 h at 4 “C. At the end of this incubation period, cholesterol esterase activity was assessed by the ability of the modified enzyme to hydrolyze the water-soluble substrate p-nitrophenyl butyr- ate as described below. Complete inhibition of cholesterol esterase activity was achieved using this procedure. Moreover, the prolonged incubation of cholesterol esterase with I”HlDFP resulted in the aeing of the modified enzyme which was ieqiired for the loss of-the isopropoxy groups and the specific labeling of serine residues (11). After removal of unreacted [“H]DFP by extensive dialysis against 0.1

’ The abbreviations used are: DFP, diisopropylfluorophosphate; pNPB, p-nitrophenyl butyrate.

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16802 Active Site Serine in Pancreatic Cholesterol Esterase

M ammonium bicarbonate, the sample was lyophilized and stored at -20 “C until use.

The [“HIDFP-labeled cholesterol esterase was subiected to cvano- gen bromide cleavage and sequence analysis as described previously (7). Brieflv, 1 ma of the f3H1DFP-labeled cholesterol esterase was dissolved in 1 mi of 7O%‘formic acid and treated with 300 pg of cyanogen bromide for 24 h at ambient temperature. The digested peptides were separated by reverse-phase high performance liquid chromatography using a Vydex C-18 column. One-ml fractions were collected and the sample containing the radiolabeled DFP was iden- tified by liquid scintillation counting. One hundred pmol of the 3H- labeled peptide was subjected to sequence analysis using a Model 470A gas-phase microsequenator equipped with a Model 120A on- line phenylthiohydantoin-derivative analyzer (Applied Biosystems, Foster City, CA).

Site-specific Mutagenesis-Oligonucleotide-directed, site-specific mutagenesis was performed according to the Kunkel et al. (12) mod- ification of the method originally described by Zoller and Smith (13). The full-length rat cholesterol esterase cDNA (7), containing the SmaI restriction sites at both ends of the cDNA, was subcloned into the SmaI site of bacteriophage M13mp18 (14). The recombinant DNA was used to transfect the dut- and ung- Escherichiu coli CJ236 cells. This method allows the incorporation of uracil into the DNA in place of thymine. Single-stranded DNA was then isolated from these cells and used as template for the mutagenesis experiments.

Two different approaches were used to generate the desirable mutations in the cholesterol esterase cDNA (Table I). The first procedure used a 53-base synthetic oligonucleotide as mismatched primer to introduce a serine-to-threonine mutation at serine 194. In addition, this oligonucleotide contained another mismatch in which the codon for leucine 202 would be altered from CTG to CTC. The latter mutation would not result in alteration of the amino acid sequence. However, the mutation would abolish the unique PstI site within the cholesterol esterase cDNA (7) and would facilitate the isolation and identification of the mutant clones. The second ap- proach to create mutant forms of cholesterol esterase cDNA was the use of a mixed primer in which all three nucleotide positions corre- sponding to serine 194 were randomly altered. This oligonucleotide mixture was synthesized by substituting all four deoxynucleotides at each position for the serine codon TCT.

The mutagenic oligonucleotide primers from either procedure were used to hybridize with the single-stranded Ml3 DNA template. In each experiment, heteroduplex plasmid was formed by the addition of the Klenow fragment of DNA polymerase I and T4 DNA ligase. The incubation was carried out for 2 h at 37 “C. Aliquots of the reaction were then used to transform competent E. coli DH5aF’ cells. The presence of uracil in the parent strand resulted in its specific hydrolysis and the selection of the non-uracil-containing, mutagen- ized daughter strand. The resultant clones were further screened by Pat1 digestion of the Ml3 replicative form DNA and by nucleotide sequencing using the dideoxy chain termination method (15). Ap- proximately 10% of the cDNA tested contained the desired mutations. The cDNA were further analyzed by detailed restriction mapping and complete sequence analysis. The latter experiments revealed no ad- ditional mutations within the cholesterol esterase cDNA.

Trunsfection and Expression of Cholesterol Esteruse cDNA-The native or mutant forms of cholesterol esterase cDNA was isolated from the replicative form of M13mp18 by SmuI digestion. The cDNA was then ligated to the SmuI site of the mammalian cell expression vector pSVL and used t.o transfect E. coli DH5crF’ cells. Alternatively,

a 1900-base pair fragment containing the entire coding sequence of the cholesterol ester&e cDNA was isolated by digestion with SmaI and Sac1 (7). This DNA fragment was subcloned into a similarly digested pSVL plasmid for propagation. Recombinant plasmid con- taining the cholesterol esterase cDNA was identified by hybridization. The orientation of the cDNA in the plasmid was determined bv restriction mapping analysis as described elsewhere (7). The plasmids containing the cholesterol esterase cDNA in the proper orientation were purified by CsCl centrifugation and were used to transfect COS- 1 cells by the DEAE-dextran method in the presence of chloroquine as previously described (16). The cells were transferred to serum-free medium 48 h after transfection and were incubated for an additional 24 h at 37 “C. At the end of this incubation period, the cell culture media were removed and assayed for cholesterol esterase activity. The cells were washed with saline and harvested by scraping of the tissue culture plates. Cell lysates were prepared by sonication and centrifugation at 10,000 X g for 10 min at 4 “C. Cholesterol esterase activity in the supernatant fractions was assayed immediately.

Characterization of Native and Mutant Cholesterol Esterase in Transfected Cells-Cholesterol esterase activity in cell culture media and in the lysates were determined by using either p-nitrophenyl butyrate (pNPB), cholesteryl [Wloleate, or [‘4C]lysophosphatidy1- choline as substrates for the cholesterol esterase.

Cholesterol esterase activity against the water-soluble substrate pNPB was determined by spectrophotometric method. In this pro- cedure, the samples were diluted 5-fold with 0.5 M Tris-HCl (pH 7.4), in the presence or absence of 6 mM taurocholate. The assay was initiated by adding 1 volume of a freshly prepared pNPB solution (100 pg/ml in sodium acetate, pH 5.0) to 2 volumes of sample. Cholesterol esterase activity was then monitored and compared with control autohydrolysis of the substrate using a Beckman Model 25 Spectrophotometer set at 405 nm.

In experiments using cholesterol [W]oleate as substrate, 150 ~1 of media containing 20-25 fig/ml of secreted proteins were used for each experiment. Each sample was incubated for 2 h at 37 “C with 2 nmol of cholesteryl [i4C]oleate in a total volume of 200 ~1 containing 50 mM Tris-HCl (pH 7.5), and 3 mM taurocholate. At the end of incubation, the reaction was terminated by adding 1.05 ml of 50 mM sodium carbonate and 50 mM sodium borate (pH 10). The reaction product, [‘4C]oleate, was extracted as the free fatty acid with 3.25 ml of methanol/chloroform/heptane (1.41:1.25:1.0) and was quantitated by liquid scintillation.

Lysophospholipase activity was determined by the hydrolysis of l- [l-‘4C]palmitoyl L-lyso-3-phosphatidylcholine in the absence of tau- rocholate as described previously (17). The incubation was performed at 37 “C! for 2 h in 50 mM potassium phosphate (pH 7.0), containing 20 pg of proteins secreted from the transfected cells. At the end of incubation, the samples in 200 ~1 final volume were extracted by addition of 2.5 ml Dole’s mixture (isopropyl alcohol/heptane/O.l N H,SO,, 40:10:1), 100 mg of silica gel, and 1.5 ml of water. The samples were vortexed and centrifuged to separate the phases. The top aqueous phase, containing the hydrolyzed product, was isolated and used to quantitate the amount of [Wlpalmitate by scintillation counting.

Gel Electrophoresis and Immunoblotting Procedures-Proteins se- creted by the transfected cells were analyzed on 10% sodium dodecyl sulfate-polyacrylamide gels (18). One ml of cell cultured media, con- taining 25 pg of protein, was precipitated by the addition of 30 ~1 of 1% sodium deoxycholate and 80 ~1 of 100% trichloroacetic acid. The samples were centrifuged at 5000 x g for 30 min at 4 “C. The

TABLE I Design and use of oligonucleotide primers used for mutagenesis

Residues 189-206 ThrIlePheGlyGluSerAlaGlyAlaAlaSerValSerLeuGlnThrLeuSer Coding strand 5’-ACCATCTTTGGGGAATCTGCTGGAGCTGCCAGTGTCTCTCTGCAGACCCTCTCC-3’ Complement 3’-TGGTAGAAACCCCTTAGACGACCTCGACGGTCACAGAGAGACGTCTGGGAGAGG-5’

Oligo-1 3’- GGTAGAAACCCCTTTGA CGACCTCGACGGTGACAGAGAGAGGTCTGGGAGAGG-5’ Coding strand 5’- CCATCTTTGGGGAAACT GCTGGAGCTGCCAGTGTCTCTCTCCAGACCCTCTCC-3’ Amino acids ThrIlePheGlyGluThreAlaGlyAlaAlaSerValSerLeuGlnThrLeuSer -

Oligo-2 3’- GGTAGAAACCCCTTNNNbCGACCTCGACGGTCACAGAGAGAGGTCTGGGAGAGG-5’ Coding strand’ 5’- CCATCTTTGGGGAAGCT GCTGGAGCTGCCAGTGTCTCTCTCCAGACCCTCTCC-3’ Amino acids ThrIlePheGlyGluAla AlaGlyAlaAlaSerValSerLeuGlnThrLeuSer -

a The amino acid substitutions resulting from the mutagenesis experiments are underlined. b N indicates a mixture of all four deoxynucleotides. ’ Only the coding strand from the mutant described in this study is shown here.

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Active Site Serine in Pancreatic Cholesterol E&erase 16803

supernatants were discarded and the precipitated proteins were re- suspended in 1 N NaOH and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels as described (18). The electrophoresed samples were transferred from the gels onto nitrocellulose paper as described elsewhere (19). Electrotransfer was performed at 150 mA for 16 h at 4 “C. Protein standards transferred onto the nitrocellulose were identified by staining with Ponceau S.

The nitrocellulose paper containing the samples was incubated for 1 h at ambient temperature with buffer made of 50 mM Tris-HCl (pH 7.5), 2 mM CaCh, 80 mM NaCl, 5% Carnation nonfat dry milk, 0.2% Nonidet P-40, and 0.01% Antifoam A. The paper was then incubated for 2 h with the same buffer containing a 1:500 dilution of rabbit anti-cholesterol esterase antiserum (20). At the end of incubation, the paper was washed four times with the above buffer and was then transferred to a second antibody solution containing “Wabeled anti- rabbit IgG at a specific activity of 1 x 10fi cpm/ml. The nitrocellulose paper was incubated for 2 h at room temperature and washed as described above. Immunoreactive proteins on the nitrocellulose paper were visualized by exposure to Kodak XAR-2 films for 18 h at -70 “C. The radioactivity associated with the proteins were quantitated by excising the immunoreactive bands from the nitrocellulose and count- ing in a gamma counter.

RESULTS

The incubation of pancreatic cholesterol esterase with the serine-modifying reagent DFP resulted in the complete loss of enzyme activity (5). In the current study, porcine pancreatic cholesterol esterase was modified with [3H]DFP to identify the key serine residue required for the catalytic activity of the protein. The incubation resulted in incorporation of 0.93 mol of [“HIDFP per mol of cholesterol esterase (Mr = 74,000). The 1:l stoichiometry of DFP interaction with cholesterol esterase was in good agreement with results reported by other investigators (5). The [3H]DFP-labeledprotein was then sub- jected to CNBr hydrolysis and the digested peptides were separated by reverse phase high performance liquid chroma- tography. The elution profile of the digested peptides showed the “H radioactivity migrating as a single peak (Fig. 1). When the fraction containing the peak radioactivity was isolated for

25 30 35 40 45 50 55 64 65 70

FRACTION NUMBER

FIG. 1. Chromatography of CNBr-digested peptides of [3H] DFP-labeled cholesterol esterase. Four mg of porcine pancreatic cholesterol esterase were incubated with 250 &i of [aH]DFP (3 Ci/ mmol) in 4 ml of barbital buffer for 48 h at 4 “C. At the end of incubation, the sample was dialyzed exhaustively. One mg of the labeled protein was treated with 300 pg of CNBr in 70% formic acid for 24 h at 23 “C. An aliquot of the sample was injected into a C-18 reverse phase column equilibrated with 0.1% trifluoroacetic acid. The flow rate was 1 ml/min. The peptides were eluted using a gradient of 0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in acetonitrile. The eluent was monitored at 218 nm and 1 ml fractions were collected. An aliquot of each fraction was added to 10 ml of ScintiVerse BD for determination of “H radioactivity (U). The arrow indicates the fraction used for sequence determinations.

analysis, one major sequence could be identified by protein sequencing technique. Comparison of the protein sequence with the amino acid sequence deduced from rat cholesterol esterase cDNA (7) revealed its similarities to the rat protein at residues 170-197 (Table II).

The position of the [3H]DFP-labeled amino acid in the CNBr-digested peptide was then determined by sequential degradation of the protein coupled to a solid phase. In this procedure, 20 nmol of the peptide was coupled to 30 mg of phenyl diisothiocyanate-glass beads in a buffer containing 0.1 M sodium bicarbonate and 10% 1-propanol (pH 9.3). The sample was then subjected to stepwise manual Edman deg- radation. The radioactivity appeared as a single peak during the 25th cycle indicating that the serine corresponding to residue 194 in the rat cholesterol esterase was labeled with [3H]DFP (Table II). This result supports previous hypothesis that Ser-194 is the catalytic active serine of cholesterol ester- ase (7).

The definitive proof that Ser-194 is the active site serine of cholesterol esterase requires site-specific mutagenesis and expression of the cDNA. Unfortunately, the cDNA for porcine pancreatic cholesterol esterase is not available for this pur- pose. In view of previous studies showing a high degree of homology between the porcine and rat cholesterol esterases (7,21), the mutagenesis experiments were performed with the rat cholesterol esterase cDNA. We have shown that transient

TABLE II

Sequence comparison of the [3H]DFP-labeled peptide with rat cholesterol esterase

The [3H]DFP-labeled peptide from CNBr-digested porcine choles- terol esterase was obtained as described in Fig. 1. The sequence of the radiolabeled peptide was determined by gas-phase microsequenc- ing technique. The radiolabeled peptide was also subjected to manual Edman degradation. The degradation products of each Edman cycle were monitored for radioactivity by liquid scintillation counting. The rat pancreatic cholesterol esterase sequence was deduced from se- quencing of the cDNA (7).

Cholesterol [3H]DFP-labeled peptide

Residue esterase (rat sequence

170-197) Amino acid Radioactivity

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Ala Ile Ala Trp Val LYS Arg Asn Ile Ala Ala Phe

g Asp Pro Asp Asn Ile Thr Ile Phe QY Glu Ser Ala Gb Ala

Ala Ile Ala Leu Val Ile Thr Asn Ile Ala Ala Phe GUY GUY Asp Pro LYS Asn Ile Thr Leu Phe GUY Glu Ser Ala GUY Ala

cpm 289 115

77 65 59 62 58 55 12 57 68 71 50 83 66 62 98 49 74 63 88 93 67 61

8721 322 109

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16804 Active Site Serine in Pancreatic Cholesterol E&erase

expression of the rat cholesterol esterase cDNA in COS cells resulted in significant bile salt-dependent cholesterol esterase activity in the cell lysate (7). The current study showed that COS cells transfected with the same cholesterol esterase cDNA were also active in the hydrolysis of the water-soluble substrate pNPB (Fig. 2).

Significant cholesterol esterase activity was also detectable in the cultured media of the transfected cells suggesting the secretion of cholesterol esterase (Fig. 3). Although the data in Fig. 3 shows higher level of cholesterol esterase activity in the media of cells transfected with the full length cholesterol esterase cDNA, in comparison with cells transfected with the SmaI-SacI-digested cDNA, the specific activity in both sam- ples was similar when the activities were normalized by the amount of immunoreactive protein produced by these cells (Table III). The differences observed in total pNPB hydrolysis were most likely due to differences in transfection efficiency in the two experiments. The result was expected since a unique Sac1 restriction site was identified in the 3’-untrans- lated region of the cholesterol esterase cDNA. The SmuI- S&J-digested cDNA would contain the entire coding region of the cholesterol esterase cDNA. Because the SmaI-SacI- digested cDNA could be subcloned into pSVL plasmid unidi- rectionally, and oriented properly with regard to the SV40 late promoter, this method of subcloning was chosen for subsequent studies.

Mutant forms of cholesterol esterase with amino acid sub- stitutions at residue 194 were produced by site-specific mu- tagenesis of the cDNA. In the initial study, a specific oligo- nucleotide probe was designed in which the Ser-194 codon TCT would be replaced by a threonine codon ACT (Table I).

PSV-CEH

/ /

/

/ : ~

PSVL

_._... .._..--

____.--I- _.__..--

._...--

2 4 6

TIME (min) FIG. 2. Spectrophotometric tracings of p-nitrophenyl bu-

tyrate hydrolysis by extracts of transfected COS cells. The COS cells were transfected with either pSVL plasmid (- - - - -) or with the pSVL plasmid containing the rat cholesterol esterase cDNA (-) using the DEAE-dextran method in the presence of chloro- quine. Cell lysates were obtained after 72 h by sonicating the cells in buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM glutathione, and 250 mM sucrose. After centrifugation at 10,000 x g for 10 min at 4 “C, the supernatant fraction from each sample was obtained and was diluted with 5-fold volume of buffer containing 0.5 M Tris-HCl (pH 7.4) and 6 mM taurocholate. The assay for pNPB hydrolysis was determined by adding 2 volumes of sample to 1 volume of substrate (100 fig/ml of pNPB in sodium acetate, pH 5.0) in a cuvette and the activity of each sample was determined by monitoring changes in absorbance at 405 nm. The data represent a direct tracing of a representative experiment on the recorder. Results of duplicate de- terminations were similar to this experiment.

0.6

0.2

I I I I 2 4 6 8

TIME (min)

FIG. 3. Spectrophotometric tracings of p-nitrophenyl bu- tyrate hydrolysis by proteins secreted from transfected COS cells. The COS cells were transfected with either control pSVL plasmid (. . . . ), plasmids containing either the full-length SmaI insert of native cholesterol esterase cDNA (PSV-CEH, 2,000) (-), the SmaI-SacI-digested cholesterol esterase cDNA (PSV-CEH, 1,900) (- - - - -), or with mutagenized cDNAs containing the mutation Th? or Alalg4. Results of the mutant cholesterol esterases overlapped a single line (-.-.). Culture media harvested between 48 and 72 h post- transfection were used for determination of pNPB hydrolysis.

TABLE III

Cholesterol esterase activity in media of transfected COS cells Culture media from COS cells transfected with native or mutagen-

ized cholesterol esterase were isolated and tested for immunoreactiv- ity with anti-cholesterol esterase on immunoblots as described in the legend to Fig. 4. The bands corresponding to cholesterol esterase were excised and counted for lZ51 activity to quantitate the amount of cholesterol esterase in each sample. An aliquot of the sample was used for pNPB hydrolysis as described under “Experimental Proce- dures” and in the legend to Fig. 3. The pNPB hydrolytic activity was reported as the change in absorbance unit (A Abs) per min per ml of cultured media. Specific activity was determined by dividing the pNPB activity by the “‘1 radioactivity obtained from the immunoblot experiment. The results were representatives of four different trans- fection exneriments.

Immunoreactivity pNPB Specific hvdrolvsis activi’v ” . ‘“J

cpm/ml P Abs/ml Xl@ Natives

SmaI insert’ 1975 0.46 23 SmaI-Sac1 insert” 704 0.13 18

Mutants Thr19’ 937 o.03b 3 Alalg4 1176 o.02b

pSVL control NA o.02b Ni

a Two control cholesterol esterase cDNA were prepared for trans- fection into COS cells. The first plasmid contained the full length cDNA in a SmaI insert and the second plasmid contained a SmaI- SacI-digested fragment of the cDNA. Both cDNA contained a SmaI- SucI-digested fragment of the cDNA. Both cDNA contained the entire coding sequence for cholesterol esterase expression.

*The values for pNPB hydrolysis observed with cells transfected with the mutant cDNAs or with the control pSVL plasmid were identical to values observed for autohydrolysis of pNPB in aqueous buffer.

’ NA, not applicable.

Subsequent experiments were performed with oligonucleo- tides in which all three nucleotides for the Ser-194 codon TCT were replaced at each position with all four deoxynu- cleotides (Table I). One of the mutant cDNA identified in the latter study contained an alanine codon GCT instead of TCT. The two mutant cDNAs were then subcloned into SmaI-SucI-

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digested pSVL vector and were expressed in COS cells as described above. Results showed that the pNPB hydrolysis by cholesterol esterase in the media of cells transfected with either mutant cDNA were identical to nontransfected cells and from cells transfected with the pSVL vector without the cholesterol esterase cDNA (Fig. 3). Moreover, the observed hydrolysis of the substrate was similar to autohydrolysis of the substrate when incubated under identical conditions with fresh media. Therefore, these results showed that substitution of Ser-194 with threonine or alanine abolished the enzyme activity of cholesterol esterase.

Consideration was given to the possibility that the observed results may be related to problems with expression of the mutant cDNA or with the stability of the mutant proteins. Therefore, the media from the transfected cells were subjected to immunoblotting analysis with antibodies raised against purified cholesterol esterase (20). Results, as shown in Fig. 4, indicated that the antibodies reacted with a single protein, with M, of 67,000, in the media of cells transfected with the full-length cholesterol esterase cDNA or with the SmaI-SacI- digested cholesterol esterase cDNA. A similar protein could be identified in the media of cells transfected with the cDNA containing either the threonine or alanine mutation at residue 194 (Fig. 4). As a control experiment, media from cells trans- fected with pSVL vector without the cholesterol esterase

1 2 3 4 5

67 kDa _

1 I 91

FIG. 4. Anti-cholesterol esterase blotting of media from transfected cells. COS cells were transfected with cholesterol ester- ase cDNA and cell culture media were isolated as described in Fig. 2. Lane 1 contained sample transfected with the full-length cholesterol esterase cDNA; lanes 2 and 3 contained samples transfected with the Thr”” or Ala’“’ mutant cDNAs, respectively; lane 4 contained sample transfected with the SmaI-SacI-digested native cDNA; and lane 5 was the sample transfected with the control pSVL plasmid. One ml of the media from each sample was concentrated by precipitation with 10% trichloroacetic acid in the presence of deoxycholate before loading onto the gel. After electrophoresis, the samples were trans- ferred to nitrocellulose paper and incubated with a 1:500 dilution of anti-cholesterol esterase antiserum. The immunoreactive bands were identified by incubation with ““I-labeled anti-rabbit IgG and were visualized by exposure to Kodak films for 18 h at -70 “C.

cDNA did not contain the M, 67,000 immunoreactive protein (Fig. 4). Results normalized to the level of immunoreactive cholesterol esterase in each sample revealed that serine resi- due 194 in cholesterol esterase is required for catalyzing pNPB hydrolysis (Table III).

The ability of the mutagenized cholesterol esterases in hydrolyzing physiological substrates was investigated by their ability to hydrolyze cholesterol [Wloleate. In this study, the proteins secreted from cells transfected with either native cholesterol esterase or with the mutagenized cDNA were used for comparison. Results showed that, while the COS cells transfected with native cholesterol esterase cDNA expressed and secreted high level of cholesterol esterase to the media, the media from cells transfected with the mutagenized cDNAs failed to hydrolyze cholesterol [“‘Cloleate to [W]oleate (Table IV).

Previous studies have shown that the same protein in the pancreas contained both cholesterol esterase and lysophos- pholipase activities (1, 22). Furthermore, the expression of the cholesterol esterase cDNA resulted in both cholesteryl ester hydrolysis (7) and lysophospholipase activity (23). To determine if serine 194 was also the catalytic site for hydro- lyzing lysophospholipids, the transfected cell media contain- ing either the native or the mutagenized cholesterol esterase were used for incubation with the radiolabeled lysophospho- lipid, 1-[1-‘“Clpalmitoyl L-lyso-3-phosphatidylcholine. Re- sults, as shown in Table IV, indicated that the mutant forms of cholesterol esterase containing either threonine or alanine at position 194 were not active in hydrolyzing the radioactive lysophospholipid. In contrast, media containing the native enzyme, with serine at position 194, displayed significant hydrolytic activity in converting the substrate to [ “Clpalmi- tate.

DISCUSSION

The role of serine 194 in cholesterol esterase activity was determined in this study using both chemical modification and site specific mutagenesis approaches. Results indicated the specific interaction of DFP, a potent inhibitor of choles- terol esterase, with serine 194 of cholesterol esterase. Oligo- nucleotide-directed mutagenesis changing serine 194 in cho- lesterol esterase to threonine and alanine abolished the ability of the enzyme to hydrolyze both water-soluble substrate p- nitrophenyl butyrate and lipid micellar substrates cholesteryl oleate and lysophosphatidylcholine. These results provided direct experimental verification to sequence comparison data indicating serine 194 as the active site serine of this protein (7).

TABLE IV Cholesterol esterase and lysophospholipase actioities

in media of transfected COS cells

Samples Cholesteryl ester hvdrolvsis

Lysophospholipid hydrolysis

Natives SmaI insert” SmaI-Sac1 insert”

Mutants Thr’“” A,alS4

DSVL control

pmol/h pmol/h

45.3 57.5 32.9 52.5

0.1 7.0 0 5.0 0 0

“Two cholesterol esterase cDNA samples containing the native coding sequence were used to transfect COS cells. One sample con- tained the full leneth cDNA within a SmaI insert while the second cDNA was a SmaI-Z&I-digested cDNA. The latter cDNA was trun- cated at the 3’untranslated region and had no effect on the coding sequence of the cDNA.

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16806 Active Site Serine in Pancreatic Cholesterol E&erase

Previous studies with human pancreatic carboxyl ester lip- ase (cholesterol esterase) as a model system have shown the importance of a serine residue for the catalytic activity of this protein (4, 24). Additionally, Lombard0 (24) has shown that modification of a histidyl residue or a carboxyl group also inhibited the catalytic activity of cholesterol esterase. Lom- bardo and Guy (25) have used nucleophilic methanol and butanol to show the partitioning of an acetylated enzyme intermediate between alcoholysis and hydrolysis. The pres- ence of an acyl-enzyme intermediate in substrate hydrolysis by cholesterol esterase was confirmed by the results of Stout et al. (26). These investigators used nucleophilic trapping approaches to show that hydrolysis of pNPB catalyzed by porcine pancreatic cholesterol esterase proceeded via an acyl- enzyme mechanism involving a tetrahedral transition state. These studies led to the hypothesis that cholesterol esterase catalysis follows a serine hydrolase mechanism similar to the catalytic activity of other serine esterases such as acetylcho- linesterase and butyrylcholinesterase.

Although the possible role of a serine residue in catalytic activity of cholesterol esterase had been implicated by chem- ical modification and enzyme mechanism studies, the identi- fication of the precise serine residue involved with this process remained unknown. Moreover, the specificity of DFP modi- fication of cholesterol esterase has not been determined pre- viously. Since cholesterol esterase and other serine esterases share a high degree of sequence homology between residues 159 and 221 (7) and that serine 194 is located within the active site motif (G-E-S-A-G) of the cholinesterases, we have speculated that serine 194 may be the acylable serine in cholesterol esterase and participates in the catalytic activity of cholesterol esterase (7). This hypothesis is confirmed di- rectly in this study by identifying serine 194 as the site of DFP modification.

The requirement for serine at residue 194 was also tested by site specific mutagenesis approach. One mutant form of cholesterol esterase produced in this study contained a thre- onine instead of serine at position 194. This alteration pre- served the hydroxyl group and thereby preserved the possi- bility of an acyl acceptor group in this position of the protein. However, as shown by results in this study, replacement of serine with threonine at position 194 abolished the enzymatic activity of cholesterol esterase. Furthermore, this study also showed that replacement of serine 194 with an amino acid of similar size, such as alanine, also resulted in an inactive cholesterol esterase. Thus, the serine residue at this position appears to be critically important for the catalytic activity of cholesterol esterase.

Consideration was also given to the possibility that amino acid substitution at residue 194 abolished enzyme activity by inhibiting the lipid interfacial recognition site or the bile salt binding site of the protein. However, experiments in this study have eliminated these possibilities. First, the mutagen- ized cholesterol esterases were inactive in hydrolyzing the water-soluble substrate p-nitrophenyl butyrate. Since enzy- matic hydrolysis of this substrate did not require lipid binding, the inactive enzymes due to threonine or alanine substitutions at residue 194 were not due to their inability to bind to lipid interface. Second, the hydrolytic activity of the enzyme for lysophospholipids was also abolished by the mutations gen- erated in this study. Since lysophospholipid hydrolysis was

determined in the absence of bile salt, effects of serine 194 mutation on cholesterol esterase activity against this sub- strate cannot be attributed to inhibition of bile salt activation. Taken together, these results showed that serine 194 is the active site serine required for the catalytic activity of choles- terol esterase.

Acknowledgments-We thank Dr. Howard L. Brockman, Hormel Institute, University of Minnesota, for the purified porcine pancreatic cholesterol esterase and for DFP Iabelingtechnique advice. We also thank Dr. Christonh W. Turck. Howard Huehes Medical Research Institute, University of California, San Francisco, for the initial study in characterization and sequencing of the DFP-labeling peptide. We also acknowledge Mike Hughes in the Department of Molecular Genetics, Biochemistry, and Microbiology for the synthesis of oligo- nucleotides used in this study. James Kissel provided excellent tech- nical assistance throughout the course of this study.

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L P DiPersio, R N Fontaine and D Y Huichemical modification and site-specific mutagenesis.

Identification of the active site serine in pancreatic cholesterol esterase by

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