Purification of Recombinant Lecithin:Cholesterol Acyltransferase

PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

10, 38–41 (1997)

PT960711

Purification of Recombinant Lecithin:Cholesterol Acyltransferase Maya P. Nair,* Bhalchandra J. Kudchodkar,* P. Haydn Pritchard,† and Andras G. Lacko* *Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, Texas; and †Atherosclerosis Specialty Laboratory, St. Paul’s Hospital, University of British Columbia, Vancouver, British Columbia, Canada

Received August 15, 1996, and in revised form December 9, 1996

Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetalcalf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis. q 1997 Academic Press

Lecithin:cholesterol acyltransferase (LCAT)1 catalyzes the esterification of plasma cholesterol, a ratelimiting step in mammalian reverse cholesterol transport (1). The reaction mechanism is unique, involving highly water-insoluble substrates. Under physiological conditions, the delivery of lipids to the active site is facilitated by a high-molecular-weight lipoprotein complex (2). Consequently, special thermodynamic and kinetic considerations must be considered regarding the enzyme/substrate interactions. The LCAT enzyme/substrate complex thus may be considered as a model for 1 Abbreviations used: LCAT, lecithin:cholesterol acyltransferase; BHK, baby hamster kidney; CHO, chinese hamster ovary; FBS, fetal bovine serum; DMEM, Dulbecco’s minimal essential medium; PAGE, polyacrylamide gel electrophoresis.

the interactions between enzymes and lipid/membrane surfaces (3). Studies on the molecular structure of LCAT have been difficult due to the limited availability of the highly purified enzyme (4–6). In a review article, Jonas suggested that the elucidation of the three-dimensional structure of LCAT was the next key step in characterizing the enzyme (2). To achieve this goal, a stably transfected cell line that secretes large amounts of recombinant LCAT would be required. In 1994, we reported the development of a baby hamster kidney (BHK) cell line which has been stably transfected with the human LCAT gene to constitutively express the enzyme (7). A purification procedure was also described that allowed the isolation of several milligrams of LCAT preparation in high yield (7). However, during subsequent studies it was found that the LCAT produced by this method may become desialylated and occasionally contaminated by lower-molecular-weight components (8). The extent of these complications depended on the length of time allowed for the secretion of the enzyme. In the present communication, we describe an improved procedure that retains the efficiency of the original scheme while producing large amounts of intact, homogeneous enzyme. MATERIALS AND METHODS

Culture Conditions for the Secretion of LCAT by the Transfected BHK Cell Line The development of a stably transfected cell line that secretes human LCAT has been described previously (7). Briefly, human LCAT cDNA was initially transfected into chinese hamster ovary (CHO) cells and subsequently into BHK cells to produce a stable line which secreted recombinant enzyme into a serum-free medium. The expression vector is composed of the LCAT

38

AID

1046-5928/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

PEP 0711

/

6q0f$$$381

04-21-97 16:28:20

pepa

AP: PEP

PURIFICATION OF RECOMBINANT LCAT

39

TABLE 1

Secretion of LCAT by Transfected BHK Cells Conditioning cycle

Protein (mg)

LCAT units (nM CE/h)

Specific activity LCAT units/mg protein

1 (24 h) 2 (48 h) 3 (72 h)

118 64 64

155 146 37

1.31 2.28 0.58

cDNA fused to a metallothionien promoter and the dihydrofolate reductase gene. The culture conditions for the production of LCAT involved seeding the transfected BHK cells (t-BHK) in 25 ml of Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) into a 150cm2 flask (TC 150, Corning, Inc., Corning, NY). The TC 150 flasks were placed in a CO2 incubator (5% CO2 , 377C and at 96% humidity) and were allowed to reach near confluency (5 1 107/flask) in 2–3 days. At this point the cells were harvested and split into three-layer flasks (Tc 500 cm2, Nunclon, NUNC A/S Denmark) for large-scale culturing. Approximately 900 1 104 cells were seeded in each flask with 75 ml of DMEM containing 10% PBS. The cells were grown to 80–90% confluency and washed with FBS. Subsequently, 50–60 ml of reduced serum medium (Opti-MEM, GibcoBRL, Grand Island, NY) was added to each flask. The LCAT activity was assessed in the conditioned medium at 24h intervals.

FIG. 2. Acrylamide gel (10%) electrophoresis of LCAT (A) following AcA 44 gel chromatography and molecular weight markers (B) (SDS, 0.1%; 0.025 M Tris, 0.192 M glycine).

Assay of LCAT Activity This assay system is similar to those previously described (9) utilizing a synthetic substrate. Briefly, liposomes (consisting of dimyristoyl phosphatidylcholine, egg lysophosphatidylcholine, and [3H]cholesterol in a 108:41:50 molar ratio) are prepared by injecting the lipid concentrate (0.5 ml) into 80 ml of 0.01 M TrisrHCl, 0.005 M EDTA, 0.15 M NaCl, pH 7.4, containing 7.5 mg of ApoAI. Three milligrams of BSA is added followed by 50 ml of b-mercaptoethanol (14 M) and the assay mix is diluted to 150 ml with the Tris buffer. The assay involves the incubation of 100 ml of substrate (containing [3H]cholesterol), with 15-ml aliquots of the enzyme in duplicates. The enzyme activity is expressed as LCAT units representing nmole of cholesterol esterified/h. Other Methods Protein determinations were carried out according to Markwell et al. (10) using bovine serum albumin as standard. Polyacrylamide gel electrophoresis (PAGE) was performed in the presence of 0.1% (w/v) SDS as described previously using Coomassie blue (7), silver (11), and Schiff reagent staining (12). RESULTS AND DISCUSSION

Production of Recombinant LCAT

FIG. 1. Gel chromatography of LCAT from phenyl-Sepharose on ACA-44. The chromatography was carried out on a 90 1 1.5-cm ACA44 column at 47C in 1 mM PO4 , 1 mM EDTA, 1 mM mercaptoethanol, 0.05% sodium azide, pH 7.4, with a flow rate of 20 ml/h.

AID

PEP 0711

/

6q0f$$$382

04-21-97 16:28:20

A major parameter which influences LCAT secretion is the number of t-BHK cells in culture. We have previously utilized a stirred culture of Collaspex Cultisphere-G microcarrier beads (Hyclone Laboratories, Inc. Logan, UT) in this process, where the t-BHK cells

pepa

AP: PEP

40

NAIR ET AL. TABLE 2

Purification Data of Recombinant LCAT

Source

Volume (ml)

Protein (mg)

LCAT activity (units)

Specific activity (units/mg protein)

Recovery of LCAT activity (%)

Conditioned medium Phenyl-Sepharose effluent ACA-44 effluent

800 130 17.5

64 3.2 0.7

146 144 57

2.3 81.6 81.6

100 99 39

secreted r-LCAT following their attachment and proliferation (7). While these studies yielded an impressive amount of enzyme (1.2 mg of LCAT/150 ml), the conditioned medium recovered from the stirred culture contained a species of LCAT that was essentially devoid of sialic acid (8) and appeared to have occasional contaminants (13). In order to overcome these difficulties, an alternate culturing procedure was developed based on less than maximal cell density and reduced exposure of the cells to the conditioned medium. These measures were taken to avoid autolysis and the secretion of degradative enzymes in order to preserve the structural integrity of the secreted LCAT. The secretion pattern of LCAT was established by removing the conditioned medium every 24 h and replacing it with fresh Opti-MEM. This procedure could be repeated three times; however, the fourth sample of the conditioned Opti-MEM generally became cloudy and turbid generally due to the increasing number of dying and autolysed cells. The results of these studies are summarized in Table 1. According to the data shown, the specific activity increased while the total the amount of LCAT activity remained steady up to 48 h. On the third day, however, both the enzyme activity and the specific activity declined precipitously. Simultaneously, a slight turbidity appeared in the conditioned medium, indicating the deterioration of the conditions for the production of purified LCAT. For these reasons only the conditioned media following 24- and 48-h incubations were used to subsequently purify LCAT by phenyl-Sepharose chromatography. Purification of r-LCAT The conditioned medium (48 h) collected from the BHK cell cultures was subjected to phenyl-Sepharose chromatography as described previously (7). Approximately 800 ml culture medium containing the r-LCAT was loaded on a phenyl-Sepharose column (2.5 1 18 cm) which had previously been equilibrated with 0.005 M PO4 , 0.3 M NaCl, pH 7.4. The column was washed with the same buffer until the A280 decreased below 0.01. Subsequently, the LCAT was eluted with deionized water.

AID

PEP 0711

/

6q0f$$$382

04-21-97 16:28:20

As mentioned before, concerns have been raised about potential lower-molecular-weight contaminants as well as by transferrin in the purified LCAT preparations (13), following phenyl-Sepharose chromatography. Because the molecular weight of transferrin is somewhat higher (Ç80,000) than that of LCAT (Ç62,000), its presence can be assessed by gel electrophoresis. We have performed studies on samples of conditioned media and purified LCAT preparations. Antibodies against LCAT and transferrin were used to prepare Western blots to identify both proteins. While the conditioned media have showed a faint band corresponding to the pure transferrin control, the purified LCAT samples (following phenyl-Sepharose chromatography) have been consistently free of transferrin, as evidenced by Coomassie blue staining and Western blotting. In order to eliminate potential lower-molecularweight contaminants, we have subjected the LCAT preparations obtained from the phenyl-Sepharose step to gel chromatography on ACA-44 (Spectrum Medical Ind., Los Angeles, CA). The LCAT-containing fractions obtained from phenyl-Sepharose were concentrated by dialysis against a saturated ammonium sulfate solution and applied to a 90 1 1.5-cm ACA-44 column. The chromatography was carried out at 47C in buffer containing 1 mM PO4 , 1 mM EDTA, 1 mM mercaptoethanol, 0.05% sodium azide, pH 7.4, with a flow rate of 20 ml/h. Approximately 4-ml fractions were collected. The results of this purification step are shown in Fig. 1, revealing a small, lower-molecular-weight contaminant (MW 35,000–40,000) that was eliminated by ACA-44 gel chromatography. We consider this upgrade of the recombinant LCAT purification very important as even minor contaminants (õ5%) of the total protein present are likely to interfere with the efforts to crystallize the enzyme. The chromatography on ACA 44 (Fig. 1) yielded one major and one minor peak where all the LCAT activity was associated with the first (highermolecular-weight) component. The LCAT activity closely corresponded with the absorbance throughout this peak and was homogeneous by acrylamide gel electrophoresis. The second, lower-molecular-weight component contained both protein (20%) and carbohydrate (80%), perhaps representing a cell surface proteoglycan

pepa

AP: PEP

PURIFICATION OF RECOMBINANT LCAT

that is released from the BHK cells into the culture medium. The efficiency of the overall purification procedure is shown in Table 1. As before (7), nearly quantitative recovery of the enzyme was observed from the phenylSepharose step. The AcA-44 chromatography substantially reduced the yield of the enzyme but resulted in a preparation of high purity (Fig. 2) and increased specific activity (Table 2). ACKNOWLEDGMENT

6.

7.

8.

These studies were supported by funds from the Texas Advanced Research Program, Project 009768-003. 9.

REFERENCES 1. Glomset, J. A. (1968) The plasma lecithin:cholesterol acyltransferase reaction. J. Lipid Res. 9, 155–167. 2. Jonas, A. (1991) Lecithin:cholesterol acyltransferase in the metabolism of high density lipoproteins. Biochim. Biophys. Acta 1084, 205–220. 3. Fielding, C. J. (1990) Lecithin:cholesterol acyltransferase, in ‘‘Advances in Cholesterol Research’’ (M. Estafahani and J. B. Swaney, Eds.), pp. 271–314, Telford & Telford, NJ. 4. Holmquist, L. (1987) Purification of plasma lecithin:cholesterol acyl transferase by covalent chromatography. J. Biochem. Biophys. Methods 14, 323–333. 5. Zhou, G. Y., Jauhiainen, M., Stevenson, K., and Dolphin, P. J.

AID

PEP 0711

/

6q0f$$$382

04-21-97 16:28:20

10.

11.

12.

13.

41

(1991) Human plasma lecithin:cholesterol acyltransferase. Preparation and use of immobilized p-aminophenylarsenoxide as a catalytic site-directed covalent ligand in enzyme purification. J. Chromatogr. 568, 69–83. Collet, X., and Fielding, C. J. (1991) Effects of inhibitors of Nlinked oligosaccharide processing on the secretion, stability and activity of lecithin:cholesterol acyltransferase. Biochemistry 30, 3228–3234. Hill, J., O. K., Wang, X., Paranjape, S., Dimitrijevich, D., Lacko, G., and Pritchard, P. H. (1993) Expression and characterization of recombinant human lecithin:cholesterol acyltransferase. J. Lipid Res. 34, 1245–1251. Lacko, A. G., Reason, A. J., Nuckols, C., Kudchodkar, B. J., Nair, M. P., Sundarrajan, G., Pritchard, P. H., Morris, H. R., and Dell, A. Characterization of recombinant human plasma LCAT: Nlinked carbohydrate structure and catalytic properties (unpublished observations). Jahani, M., and Lacko, A. G. (1982) Study of lecithin:cholesterol acyltransferase reaction with liposome and high density lipoprotein substrates. Biochim. Biophys. Acta 113, 504–511. Markwell, M. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 206–210. Merrill, C. R., Goldman, D., and Van Keuren, M. L. (1983) Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol. 96, 230–239. Mantle, M., and Allen, A. (1978) A colorimetric assay for glycoprotein based on the periodic acid Schiff stain. Biochem. Soc. Trans. 6, 607–608. Rosseneau, M., and Pritchard, P. H. (personal communication).

pepa

AP: PEP