Recombinant lecit

Fish-eye disease is a rare genetic disorder of high density lipoprotein (HDL) metabolism that is characterized biochemically by a partial deficiency of the enzyme lecithin:cholesterol acyltransferase (LCAT). One of the mutations that is causative for fish-eye disease occurs at codon 123 of the LCAT gene. This mutation results in the exchange of a threonine residue for an isoleucine in the LCAT protein (Thr123+Ile). In order to understand the functional significance of this exchange, we have used site-directed mutagenesis to reconstruct this mutation in an LCAT cDNA followed by expression of the mutant LCAT in COS-1 cells. The fish-eye disease mutation resulted in a 50% decrease in LCAT mass in the culture medium compared to wild type enzyme. The secreted mutant protein was incapable of esterifying cholesterol in HDL and HDL analogues. However, this protein retained the ability to esterify cholesterol in plasma and low density lipoprotein. These results support the hypothesis that this mutation is responsible for biochemical abnormalities of LCAT observed in fish-eye disease and the mutant LCAT protein has lost the potential to esterify cholesterol in the HDL pool but retains the ability to esterify cholesterol from other lipoproteins.-0, K., J. S. Hill, X. Wang, and P. H. Pritchard. Recombinant 1ecithin:cholesterol acyltransferase containing a Thr,z3+Ile mutation esterifies cholesterol in low density lipoprotein but not in high density lipoprotein. J: Lipid Res. 1993. 34: 81-88. Supplementary key words transient transfection site-directed mutagenesis recombinant LCAT cholesterol esterification Lecithin:cholesterol acyltransferase (LCAT, phosphatidylcho1ine:sterol 0-acyltransferase, E C 2.3.1.43) is the enzyme responsible for the formation of cholesteryl ester in plasma via transfer of the sn-2 fatty acid from phosphatidylcholine to the 3-hydroxyl group of cholesterol (1). A great deal of information on the biochemistry and pathophysiology of this enzyme is now available and it has been the topic of numerous reviews (2, 3). Furthermore, studies on patients with familial LCAT deficiency have clearly illustrated the central role that this enzyme plays in plasma cholesterol homeostasis (4). LCAT is a glycoprotein with a n apparent molecular mass of 67,000 daltons and the human enzyme has been purified to homogeneity (2, 5). The substrates of LCAT are primarily high density lipoproteins (HDL) (2, 6, 7) but several investigators have provided evidence that LCAT may act directly on the lower density lipoproteins (8, 9). The major structural protein of HDL, apolipoprotein A-I, is believed to be the principle activator of LCAT (3). The gene for the human LCAT has been sequenced and is made up of 6 exons and 5 introns. LCAT mRNA contains 1550 nucleotides and is expressed primarily in the liver (10, 11). Mature LCAT protein contains 416 amino acids and 25% of total LCAT mass is carbohydrate that is covalently linked to four potential Nglycosylation sites (3, 5). A number of mutations in the LCAT gene have been demonstrated to be causative for familial LCAT deficiency. These include mutations at codons 10, 141, 147, 228, and 293 (12-15). A variant of familial LCAT deficiency was recently reported by Funke et al. (16) in collaboration with our laboratory. It was found that a molecular defect at codon 123, which resulted in the exchange of a threonine (Thr) residue for an isoleucine (Ile) residue, was the defect underlying this familial partial LCAT deficiency (also known as fish-eye disease) in homozygotes from two unrelated families. Fish-eye disease is an unusual disorder because the apparent loss of LCAT activity does not result in a marked increase in the ratio of cholesterol to cholesteryl ester in plasma (16-19). It has been postulated that partial familial LCAT deficiency seen in fish-eye disease is caused by a defective Abbreviations: HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; LCAT, 1ecithin:cholesterol acyltransferase; PCR, polymerase chain reaction; TBS, Tris-buffered saline. Journal of Lipid Research Volume 34, 1993 81 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom LCAT that has lost the ability to esterify cholesterol in the HDL pool but retains its activity on cholesterol in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) (16, 18). Carlson and Holmquist (18) have suggested that there are two different LCAT activities present in normal plasma. One of these activities, denoted a-LCAT, is proposed to be specific for HDL and the other, /3-LCAT, is specific for LDL and VLDL. Thus, they have suggested that fish-eye disease is caused by the lack of a-LCAT activity in plasma (18). In order to further investigate this hypothesis, we have recreated the C to T substitution in codon 123 of an LCAT cDNA and expressed this mutant LCAT (Thr123-Ile) in COS-1 monkey kidney cells. The secreted mutant LCAT protein exhibited a loss of the activity against HDL as substrate which mimics the fish-eye disease phenotype. MATERIALS AND METHODS Mutagenesis of LCAT cDNA The natural mutation at codon 123 of LCAT is a substitution of thymidine for cytosine which causes an amino acid change from Thr to Ile. We introduced this mutation into the LCAT cDNA (a gift from Dr. J. McLean, Genentech, San Francisco, CA) using oligonucleotide-directed mutagenesis. The mutagenic oligonucleotide carried a mismatched base for substitution of ThrIz3 to Ile (5'-CCTGCACATACKGTGC-3'). The full-length wild type LCAT cDNA in pUC19 was used as a template for the polymerase chain reaction (PCR). First round amplification with PCR was carried out by using 10 pg of template DNA, 100 pmol of a mutagenic oligonucleotide as a forward primer, and 100 pmol of the reverse pUC primer which hybridizes to the vector downstream from the LCAT cDNA sequence. PCR was carried out for 30 cycles, with step cycles of 95OC for 30 sec, 55OC for 30 sec, and 74OC for 60 sec. After the thirtieth cycle, the reaction was extended at 74°C for 5 min. The PCR product was purified by electrophoresis in a 0.7% agarose gel. A second round of PCR contained 20 pg of the cDNA LCAT template in pUC19, 10 pmol of universal primer, and the purified first round amplification product as the other primer. PCR conditions were the same as that for the first round, except that the extension time for each cycle was increased to 90 sec. The amplified DNA was purified and digested with restriction enzymes Kpnl and Pstl to generate a 527 bp fragment encompassing the 123 mutation. This fragment was inserted as a cassette in the wild type LCAT cDNA in pUC19 vector. DNA sequencing was performed to identify a clone that contained the desired mutation at codon 123 but no other mutations. The LCAT cDNA from this clone was cut out with restriction enzymes Xhol and BamHl and subcloned into the mammalian expression vector pNUT (20, 21). Prior to the transfection experiments, the final construct was sequenced to confirm that it contained the 123 mutation. Transient transfection of COS cells COS-1 monkey kidney cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) (GIBCO BRL Canada). Subconfluent COS-1 cell monolayers were washed twice with transfection buffer (25 mM Tris-HC1, pH 7.4, 140 mM NaCl, 1 mM CaCI2, 3 mM KCI, 0.5 mM MgCl,, 0.9 mM Na2HP04). The expression vector pNUT containing either wild type or mutant LCAT cDNA was transfected into COS-1 cells by DEAE-dextran transfection. After incubation of the cells at 37OC for 30 min, the DNA solution was removed and replaced with DMEM containing 10% FBS and 80 pM chloroquine. After a 3-h incubation at 37OC, transfection was completed by a 3-min incubation with DMEM containing 10% DMSO at room temperature. After washing with transfection buffer, the cells were incubated in DMEM containing 10% FBS for 12 h and subsequently in serum-free medium (OptiMEM, GIBCO BRL Canada) for 48 h. Radiolabeling and immunoprecipitation of recombinant LCAT After 48 h of incubation in DMEM containing 10% FBS, transfected COS-1 cells were incubated in methionine-free DMEM (DMEM-Met) for 20 min at 37OC to deplete the methionine pool. The cells were then incubated for 30 min in DMEM-Met supplemented with 100-200 pCi/ml [35S]methionine (700 Ci/mmol, New England Nuclear) followed by incubating in DMEM with 10% FBS. After 4 h incubation, the medium was collected and the cellular protein was harvested in lysis buffer (50 mM Tris-HC1, pH 8.0, 62.5 mM EDTA, 1% Nonidet P40, 0.4% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride). Solid phase immunoprecipitation was performed to detect the presence of LCAT in the culture medium and the cellular lysate. Polyclonal goat anti-human LCAT antibodies (kindly provided by Dr. A. Lacko, Texas College of Osteopathic Medicine, Fort Worth) were pre-adsorbed onto agarose-immobilized protein G (GammaBind G Agarose, Genex Corporation, Gaithersburg, MD) for 30 min at 4OC. The LCAT antibodies were specific for LCAT protein in the culture medium as determined by Western blot analysis of SDS PAGE gels. An aliquot of medium or cell lysate was added to the antibody-protein G-agarose suspension and the mixture was rotated overnight at 4OC. Agarose beads were pelleted by centrifugation and then washed twice with 1 ml of Tris-buffered saline (TBS) containing 10 mM Tris-HC1, pH 7.4, 150 mM NaC1, 0.1% sodium azide. The pellet was resuspended in the buffer containing 0.1 M Tris-HC1, pH 6.8, 2% SDS, 40% 82 Journal of Lipid Research Volume 34, 1993 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom glycerol. The adsorbed materials were then eluted from the agarose beads by heating at 90°C for 10 min. The agarose beads were removed by centrifugation. The supernatant was subjected to electrophoresis on SDSpolyacrylamide gels (10%) with 14C-labeled methylated protein standards (Amersham Canada Ltd., Oakville, Ontario) as molecular weight markers. After electrophoresis, the gels were agitated in 25% methanol-5% acetic acid for 30 min, then in Amplify (Amersham Canada Ltd.) for 15 min. The gels were dried and autoradiography was performed with Kodak X-Omat AR film (Eastman Kodak) at -70°C for 12-24 h. Determination of LCAT mass The mass of secreted LCAT protein was determined by solid-phase immunoassay. The aliquots of culture media and the purified human LCAT standard (kindly provided by Dr. A. Lacko) were applied to nitrocellulose membrane (Schleicher & Schuell, Keene, NH) in a Bio-Dot SF apparatus (Bio-Rad, Richmond, CA). Culture medium from cells transfected with plasmid DNA that did not contain the LCAT cDNA insert was used as a control. The membrane was blocked with 5% non-fat dry milk in TBS for 30 min at 37°C. The bound protein was allowed to react with polyclonal goat anti-human LCAT antibodies by incubating the membrane overnight in antibody solution. After washing three times with TBS containing 0.02% Tween 20, the membrane was incubated with protein G conjugated to horseradish peroxidase (GammaBind-G-HRP, Genex Corp.) for 60 min. The color was developed in a solution containing 25 mg diaminobenzidine (Sigma Chemical Co.), 15 mg CoC12, and 0.010 ml of 30% H202 in 50 ml TBS. The quantitation of LCAT protein was carried out by scanning the membrane with a Bio-Rad Model 620 Video Densitometer. The LCAT mass was proportional to absorbance up to 80 ng of protein. The interassay coefficient of variation was 7.3% for a single mass measurement in nine separate assays. Preparation of plasma and lipoproteins Blood was collected from normal volunteers after 12 h fasting and plasma was prepared by low speed centrifugation (1,200 20 min). The different lipoproteins were isolated by preparative ultracentrifugation using lipoprotein fractions defined by their densities: 1.006 < d < 1.063 g/ml for LDL and 1.063 < d < 1.21 g/ml for HDL (22). Total lipoprotein-depleted plasma was prepared by ultracentrifugation at density 1.21 g/ml. In some experiments, the infranatant (inf. d > 1.21 g/ml) was recombined with the isolated LDL fraction. All lipoprotein preparations were dialyzed extensively at 4OC against 0.01 M Tris-HC1 (pH 7.4) containing 0.15 M NaCl and 0.005 M EDTA. The plasma, HDL fraction, and LDL + inf. d > 1.21 g/ml were heat-inactivated at 56°C for 30 min to eliminate LCAT activity associated with those preparations. The heat-inactivation procedure has been carried out in a number of laboratories to elucidate the catalytic defects in LCAT protein (9, 23) and has been recently shown not to disturb the gradient gel pattern of HDL compared to a native preparation (23). There was no measurable LCAT activity associated with the isolated LDL fraction. The concentration of unesterified cholesterol in plasma and lipoproteins was determined enzymatically by a reagent kit (Boehringer-Mannheim GmbH). Determination of LCAT activity and cholesterol esterification rates The enzyme activities of wild-type and mutant LCAT gene products were determined using a proteoliposome as a substrate. The egg yolk phosphatidylcho1ine:cholesterol liposome was prepared by ethanol injection according to Batzri and Korn (24). The substrate mixture containing 4.66 nmol [3H]cholesterol (0.03 pCi/nmol), 18.46 nmol phosphatidylcholine, and 7.5 pg purified human apoA-I in 10 mM Tris-HC1 (pH 7.4)-150 mM NaCI-5 mM EDTA was preincubated at 37OC for 30 min. Subsequently, 5 mM P-mecaptoethanol and 1.5 % bovine serum albumin (essentially fatty acid-free) were added to the substrate mixture. The reaction was initiated by the addition of 100-200 pl cell culture medium (containing 0.08-0.18 pg/ml LCAT protein) to a final volume of 0.3 ml. The reaction was carried out at 37OC for 2 h and was terminated by the addition of 4 ml of chloroform-methanol 2:l (v/v) and water was added to achieve phase separation. Unesterified cholesterol and cholesteryl ester in the organic phase were separated by thin-layer chromatography (Si1 G 60F-254, BDH Inc. B.C.) with the solvent system petroleum ether-diethyl ether-acetic acid 70:12:1 (v/v). The radioactivity associated with cholesterol and cholesteryl ester was determined by liquid scintillation counting. Radiolabeled plasma or lipoprotein was prepared by equilibration with [3H]cholesterol at 4°C as described by Dobiasova and Schutzova (25). An aliquot of heatinactivated substrate (plasma, HDL fraction, LDL + inf. d > 1.21 g/ml) or isolated LDL was added to a precooled test tube containing [3H]chole~ter01-labeled filter paper discs and the mixture was incubated at 4OC for 20 h. The labeled plasma or lipoprotein fractions were incubated with recombinant LCAT and the rates of [3H]cholesterol esterification in plasma and lipoproteins were determined. Briefly, an aliquot (0.2-0.3 ml) of cell culture medium containing LCAT protein (0.025 pg) was added to a mixture containing labeled plasma or HDL or LDL or LDL + inf. d > 1.21 g/ml, 5 mM 0-mercaptoethanol, and 1.5% bovine serum albumin (essentially fatty acidfree) to a final volume of 0.40 ml. The reaction mixture 0 et a/. Expression of a ThrlZ3+Ile mutation in LCAT 83 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom was incubated at 37OC for 1-6 h. The reaction was terminated by adding 2 ml of ethanol. After the mixture was centrifuged at 2000 rpm for 10 min, the cholesterol and cholesteryl ester in the supernatant were separated by thin-layer chromatography (Si1 G 60F-254, BDH Inc. B.C.). The results were expressed as nmoles of free cholesterol esterified per h per pg LCAT protein which refers to the rate of cholesteryl ester formation in plasma or isolated lipoprotein fraction catalyzed by the recombinant enzyme.