Interaction of Rat Lecithin-Cholesterol Acyltransferase with Rat Apolipoprotein A-I and with Lecithin-Cholesterol Vesicles

J. Biochem. 111, 413-418 (1992) Interaction of Rat Lecithin-Cholesterol Acyltransferase with Rat Apolipoprotein A-I and with Lecithin-Cholesterol Vesicles Yuji Furukawa,1 Takashi Urano,2 Yoshifumi Hida,1 Harumi Itch,1 Chizuko Takahashi,3 and Shuichi Kimura1 Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Aoba-ku, Sendai, Miyagi 981 Received for publication, October 16, 1991 The interaction of rat plasma lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles and with rat apo-a-i was studied in comparison with that of human plasma lecithin-cholesterol acyltransferase to clarify the reaction mechanism of rat plasma lecithin-cholesterol acyltransferase. The interaction of both human and rat lecithin-cho lesterol acyltransferase with lecithin-cholesterol vesicles was investigated by gel per meation chromatography on Superose 12. Both enzymes had almost the same affinity to the vesicles,,the affinity of rat enzyme to rat apo-a-i was stronger than that of human enzyme to human apo-a-i when estimated on the apo-a-i-sepharose 4B column. When human apo-a-i was added to the human enzyme/vesicle mixture which contained the enzymevesicle complex; the enzyme was effectively dissociated from the complex. But when rat apo-a-i was added to the rat enzyme/vesicle mixture, apo-a-i-enzyme-vesicle complex was still recognized by its elution pattern on gel permeation chromatography. This suggests that the mixture of rat enzyme, rat apo-a-i, and vesicles, which are the major components in the rat lecithin-cholesterol acyltransferase reaction, forms a stronger complex than do the components of the human reaction. Lecithin-cholesterol acyltransferase (LCAT) [EC 2.3.1.43] primarily catalyzes the transfer of an acyl group from the carbon-2 position of phosphatidylcholine to cholesterol (1-7). The enzyme is produced by the liver and secreted into the circulation (8). It is well known that the enzyme in plasma mostly associates with high-density lipoprotein (HDL) (9-11) and produces lysolecithin and cholesterol ester by the transfer of the fatty acid of lecithin to unesterified cholesterol. The cofactor activity of apolipo proteins, especially apolipoprotein A-I (apo-a-i), for this cholesterol esterification has been established (12-14). Only limited information is currently available concerning the role of apo-a-i in the mechanism of the enzyme reaction (15). We previously found several pieces of evidence for the reaction mechanism of purified human LCAT, as follows (16). First, when lecithin-cholesterol vesicles were used as substrates in the presence of human apo-a-i, human enzyme required an ionic strength of over 0.05 for its maximal activity. Second, when the enzyme incubated together with the vesicles was applied to a Sepharose CL 4B column, the enzyme was coeluted with the vesicles by the media of both high (0.1) and low (0.01) ionic strength. These results indicated that the association of the enzyme with the vesicles was independent of the ionic strength of the medium. Third, in media of low ionic strength, the enzyme bound to the vesicles was effectively displaced by 1 To whom correspondence should be addressed. Present addresses: 2 Central Laboratory of Key Technology, Kirin Brewe_??_ Company, Ltd., Takasaki, Gunma 370-12; 3 Mitsubishi _??_ oration Research Center, Yokohama, Kanagawa 227._??_,ions: apo-a-i, apolipoprotein A-I; HDL3, high density _??_em 3; LCAT, lecithin-cholesterol acyltransferase. apo-a-i. This displacement was reduced in the medium of high ionic strength. Therefore, apo-a-i may be required to promote the dissociation and the transfer of the enzyme to other substrate particles in human plasma LCAT reaction. Although the enzymatic reaction may require the simulta neous presence of apo-a-i and the enzyme on the substrate particles, there appeared to be no strong interaction between apo-a-i and the enzyme by affinity chromatog raphy on apo-a-i coupled with Sepharose CL 4B (16). We demonstrated recently that purified rat enzyme has a strong affinity to HDL and the enzyme was more easily inactivated, even in the media of low ionic strength, than the purified human enzyme (17). It is supposed that the rat enzyme interacts more strongly with rat apo-a-i than does the human enzyme. These considerations led us to investi gate the association of rat LCAT with rat apo-a-i. Considerable research has been carried out in recent years on the characterization and the reaction mechanism of this enzyme; but most of it has dealt with the enzyme in human plasma (18-24). Although an understanding of the lipoprotein metabolism of rats has been useful in the development of suitable nonhuman models for the study of atherosclerosis and other lipid-related disorders, there is relatively little information regarding the properties or the reaction mechanism of plasma LCAT in experimental animal such as rat (25). This report describes the interac tion of purified rat LCAT with rat apo-a-i and with lecithin-cholesterol vesicles, which was examined in order to study the role of apo-a-i in the reaction mechanism of the enzyme. Vol. 111, No. 3, 1992 413

414 Y. Furukawa et al. MATERIALS AND METHODS Buffer-Experiments were performed primarily in 39 mm sodium phosphate buffer (ionic strength of 0.1) con taining 0.025% EDTA. The ph of the phosphate buffer used was 7.4 unless specified otherwise. Materials-Cholesterol (99+%), crystallized, lyophi lized bovine serum albumin, and L-ƒ -phosphatidylcholine, Type III-E were obtained from Sigma Chemical.[7(N)-3H]- cholesterol (specific activity, 592 GBq/mmol) was pur chased from Amersham and purified by thin layer chromatography (26). 2-Mercaptoethanol (99+%) was purchased from Wako Pure Chemical (Tokyo); Sephadex G-150, DEAE-cellulose, DEAE-Sephadex A-50, and CNBr-activated-Sepharose 4B were from Pharmacia Fine Chemicals; Silicic acid-impregnated plastic sheets, Type 13181 were from Eastman Kodak (Rochester, N.Y.). All other chemicals were of reagent grade and used without further purification. Glass-distilled water was used to prepare all aqueous solutions. Preparation of apo-a-i-human and rat apo-a-i were prepared from human and rat HDL, respectively, by delipidation with ethanol/ether (27) followed by gel per meation chromatography on Sephadex G-150 and DEAEcellulose column chromatography at 4 Ž (28, 29). The EDTA, 2.5mg of bovine serum albumin, and the enzyme in a final volume of 250ƒÊl of the phosphate buffer. The incubation, lipid extraction, separation of [3H]cholesterol and [3H] cholesterolester, and determination of the radio activity were done as described previously (16, 17). The rate of cholesterol esterification is given as nmol of choles terol esterified/h, and 1 unit of enzyme designates the esterification of 1nmol of unesterified cholesterol/h at 37 Ž under the standard assay conditions in the presence of human apo-a-i. Gel Permeation Chromatography-Gel permeation chro matographic experiments were performed with a Fast Protein Liquid Chromatography System (FPLC), consist ing of a Superose 12 HR (cross-linked, agarose-based gel) 10/30 column (1.0x30cm), a GP-250 gradient programmer, a P-500 pump, a V-7 injection valve, a solvent mixer and a prefilter, all supplied by Pharmacia. Coupling of apo-a-i to Sepharose 4B-Human and rat apo-a-i were each coupled to CNBr-activated-Sepharose 4B by the method of Porath et al. (32). The unreacted groups of the activated Sepharose were blocked with 0.5M monoethanolamine (ph 9.0). Approximately 3.0mg of both human and rat apo-a-i were coupled per ml of packed Sepharose. preparations gave a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis. Both purified human and rat apo-a-i were stored in 6M urea at 4 Ž under nitrogen and were dialyzed against the phosphate buffer to remove urea prior to use. Purification of Human and Rat LCAT-Human plasma LCAT was purified according to the method developed by Doi and Nishida (30). The method involved treatment with dextran sulfate in the presence of Ca2+, and with 1-butanol in the presence of ammonium sulfate, DEAE-Sephadex chromatography, and hydroxyapatite chromatography. The final preparation of human plasma LCAT was purified approximately 5,900-fold with 14% yield. Unlike human plasma LCAT, rat plasma LCAT prepared under the same conditions was not adsorbed on the hydroxyapatite column. The final purification of rat LCAT was achieved by anion exchange column chromatography with a Mono Q column from Pharmacia. In this step, each fraction tube contained 10ƒÊg of rat apo-a-i in order to prevent the enzyme's inactivation by the high ionic strength of the elution buffer. The final rat LCAT preparation was purified approximately 7,100-fold with 6% yield (17). Because the final enzyme preparation contained a small amount of apo-a-i, the active fraction eluted from the hydroxyapatite column was used in the experiments for the interaction of the enzyme with apo-a-i and lecithin-cholesterol vesicles. Preparation of Lecithin-Cholesterol Vesicles-Lecithincholesterol vesicles prepared by the method of Batzri and Korn (31) were used for enzyme assay as substrates and for affinity experiments between the enzyme and the apo-a-i with substrates. A typical preparation contained 900 nmol of L-ƒ -phosphatidylcholine and 150nmol of [7(N)-3H]- cholesterol (specific activity; 196 MBq/mmol)/ml. Enzyme Assay-The enzyme activity was determined in a similar manner as described previously (16, 17, 30). The assay mixture consisted of 100ƒÊl of vesicle solution, 15ƒÊg of human apo-a-i, 4mM 2-mercaptoethanol, 0.7mM Fig. 1. Gel permeation chromatography of human apo-a-i (A) and its mixture with lecithin-cholesterol vesicles (B). Superose 12 column (1x30cm) was used for gel permeation chromatography. A shows the elution profile of human apo-a-i alone (10ƒÊg) ; B shows that of the apo-a-i/vesicle mixture with 39mM phosphate buffer. The apo-a-i/vesicle mixture contained 10ƒÊg of apo-a-i, 90nmol of lecithin, and 15nmol of [7(N)-3H]cholesterol in 200ƒÊl of the buffer. The mixture was incubated for 2 h at 4 Ž prior to application to the column equilibrated with the same buffer. The elution profiles of the apo-a-i and the vesicles were determined respectively by measuring protein and radioactivity in alternate fractions. J. Biochem

Rat Lecithin-Cholesterol Acyltransferase 415 RESULTS Binding Affinity of apo-a-i with Lecithin-Cholesterol Vesicles-Because of the complexity of the enzymatic reaction occurring in plasma or with natural substrates such as isolated plasma lipoproteins, clarification of the mecha nism is expected to be greatly aided by the use of well defined model systems including apo-a-i and lecithincholesterol vesicles as substitutes for HDL3. The affinities of both human and rat apo-a-i to lecithin-cholesterol vesicles in 39mM phosphate buffer were studied by gel permeation chromatography on Superose 12 (Fig. 1). Comparison of the elution pattern of human apo-a-i alone with that of apo-a-i/vesicle mixture showed that a part of apo-a-i was co-eluted with the vesicles in the high molecu- Fig. 2. Gel permeation chro matography of human and rat lecithin-cholesterol acyltransfer ases and their mixtures with leci thin-cholesterol vesicles. Super ose 12 column was used for gel permeation chromatography. A shows the elution profile of human enzyme (7.5 units) alone; B, that of the enzyme/vesicle mixture; C, that of rat enzyme (6.5 units) alone; and D, that of the enzyme/vesicle mix ture; all with 39mM phosphate buffer. The enzyme/vesicle mix tures contained 7.5 (human) and 6.5 (rat) units of the enzyme, respec tively, 90nmol of lecithin and 15 nmol of [7(N)-3H] cholesterol in 200ƒÊ 1 of the buffer. These mixtures were incubated for 2 h at 4 Ž prior to application to columns equilibrated with the same buffer. The elution profiles of the enzyme were deter mined by standard enzyme assay; those of the vesicles were from their radioactivities in alternate fractions. Fig. 3. Elution profiles of human and rat lecithin-cholesterol acyltransferases obtained from untreated Sepharose 4B and apo-a-icoupled Sepharose 4B columns. Human enzyme (9.4 units) and rat enzyme (7.8 units) were each applied to untreated Sepharose 4B and human and rat apo-a-i-sepharose 4B columns (1.0x1.5cm) equilibrated with 39mM phosphate buffer, ph 7.4. A and B are the elution profiles of human enzymes from untreated Sepharose and from human apo-a- I-Sepharose columns, respectively. The corre sponding profiles obtained with rat enzyme are shown in C and D. In both apo-a-i-sepharose columns, after washing the columns with the same buffer, the enzymes were eluted from the columns with 0.1mM phosphate buffer from fraction 17 (arrow a), then with the same buffer containing 0.5m M sodium taurocolate from fraction 30 (arrow b). The flow rate of each column was adjusted to 1.8 ml/h and fractions of 200ƒÊl were collected. Vol. 11, 1 No. 3, 1992

416 Y. Furukawa et al. Fig. 4. Effect of human apo-a-i on the interaction of human lecithin-cholesterol acyltransferase with the vesicles. Gel per meation chromatography on a Sepharose 12 column with 39mM phosphate buffer was used to determine the interaction. The enzyme/ vesicle mixtures contained 7.5 units of human enzyme, 90nmol of lecithin, and 15nmol of [7(N)-3H]cholesterol in 200ƒÊl of 39mM phosphate buffer. The mixtures were incubated at 4 Ž for 2 h under nitrogen. After addition of 10ƒÊg of human apo-a-i, the mixtures were incubated for six additional hours at 4 Ž under nitrogen prior to application to columns equilibrated with the same buffer. lar weight region (Fig. 1). The apo-a-i peak, which corre sponded to 62% of the amount of apo-a-i applied to the column, coincided completely with the peak of the vesicles. The elution patterns of rat apo-a-i alone and the apo-a-i/ vesicle mixture were exactly the same as those of their human equivalents (data not shown). Interaction of the Enzyme with the Vesicles-The inter actions of human and rat enzymes with the vesicles were also investigated by gel permeation chromatography on Superose 12. Figure 2 shows the elution patterns of human (A) and rat (C) enzyme alone and their mixtures with the vesicles (B and D) in 39mM phosphate buffer. Approxi mately 67 and 60% of the human and rat enzyme, respec tively, were co-eluted with the vesicles in the case of the enzyme/vesicles mixtures (Fig. 2, B and D). These results indicate that approximately 5.0 and 3.9 units of human and rat enzyme, respectively, bound to the vesicles in the concentration used in this experiment. The excess enzymes which did not associate were eluted as free enzymes. The elution volume of free enzymes corresponded to fraction 30 in all cases. Interaction of the Enzyme with apo-a-i-unlike the affinity of the vesicles to rat LCAT, rat apo-a-i did not interact significantly with the LCAT on gel permeation chromatography with Superose 12. When rat LCAT/rat apo-a-i mixture was applied to the Superose 12 column, the independently eluted LCAT and apo-a-i each had the same elution volume as when LCAT and apo-a-i were applied singly (data not shown). This agreed closely with the previous results of the gel permeation chromatography of human enzyme/human apo-a-i mixture on Sepharose CL 4B (16). We attempted to compare the affinity of human and rat enzymes to the respective apo-a-i-coupled Sepharose 4B column and the untreated Sepharose 4B column equilibrated with 39mM phosphate buffer (Fig. 3). Approximately 78% of the human enzyme applied to the apo-a-i-sepharose column was eluted from the column by Fig. 5. Effect of rat apo-a-i on the interaction of rat lecithincholesterol acyltransferase with the vesicles. Rat enzyme (6.5 units) and rat apo-a-i (10 u g) were used under the same conditions as described in Fig. 4. TABLE I. Activation of human and rat lecithin-cholesterol acyltransferase by human and rat apo-a-i. Human and rat enzyme (1.27 and 1.68 units, respectively) was added to the assay mixture containing human or rat apo-a-i (17 or 18ƒÊg, respectively) as the cofactor. The mixture was incubated under the standard assay conditions. Each value is the average of three independent experi ments. the same buffer, but it yielded a broader enzyme peak with a significantly larger elution volume than that obtained with the untreated column (Fig. 3, A and B), in agreement with previous results (16). In the case of rat enzyme, however, only 15% of the enzyme applied to the apo-a-i-sepharose was eluted by the same buffer, indicating almost complete retention of the enzyme on the column. When the buffer concentration for the elution was reduced to a low ionic strength (0.1mM phosphate buffer), approximately 23% of the applied enzyme was found in the eluate. The remaining enzyme was eluted with the buffer containing 0.5mM taurocholate (Fig. 3D). These results indicate that the association between rat LCAT and rat apo-a-i is stronger than that between human LCAT and human apo-a-i. Effect of apo-a-i on the Interaction of the Enzyme with the Vesicles-To obtain further information on the role of apo-a-i in the enzyme/vesicle interaction, apo-a-i was added to the enzyme/vesicle mixture in 39mM phosphate buffer, which was then subjected to gel permeation chro matography on Superose 12. In the case of human enzyme, the addition of human apo-a-i caused a significant displace ment of the enzyme from the vesicles (Fig. 4); the elution pattern of the enzyme was considerably different from that obtained in the absence of apo-a-i (Fig. 2, pattern B). Approximately 25% of enzyme activity was observed in the elution volume of the vesicles, but 15 and 60% of the activity was found respectively in a larger elution volume (fraction 27) and in the region of free enzyme (fraction 30). It was calculated from the elution profile of interaction of J. Biochem.

Rat Lecithin-Cholesterol Acyltransferase 417 the enzyme with the vesicles (Fig. 2, pattern B) that 1.88 units of the enzyme was co-eluted with the vesicles, and thus approximately 62% (3.12 units) of the enzyme which associated with the vesicles in the absence of apo-a-i was displaced by the addition of apo-a-i. The distribution of apo-a-i in the elution pattern was also different from the pattern of apo-a-i/vesicle mixture in Fig. 1. The decrease in the amount of apo-a-i eluted together with the vesicles from 62 to 36% of the elution pattern in the absence of enzyme (Fig. 1) suggested that the interaction of apo-a-i with the vesicles might also be affected by the presence of the enzyme. Furthermore, the enzyme peak eluted in the region of the vesicles was not superimposed on the peak of the vesicles. On the other hand, when rat apo-a-i was added to the rat enzyme/vesicle mixture in 39mM phosphate buffer, approximately 65% of the enzyme was observed in elution volume of the vesicles (Fig. 5), and the elution pattern and the recovery of the enzyme were similar to those obtained in the absence of apo-a-i (Fig. 2, pattern D). Similarly, in this case, the amount of apo-a-i eluted together with the vesicles was 64% of the amount of apo-a- I applied to the column. This recovery was similar to those obtained in the absence of enzyme. This indicated that the interaction of rat apo-a-i with the vesicles was not affected by the presence of rat enzyme. Moreover, the enzyme peak coincided completely with the peak of the vesicle-apo-a-i complex (Fig. 5B). The Activation of Human and Rat LCAT by Human and Rat apo-a-i-to determine the possible relationship between the enzyme activity and the enzyme's affinity with apo-a-i, the activities of human and rat enzyme were measured with the standard assay system in the presence of human or rat apo-a-i, respectively (Table I). When human apo-a-i was added to the assay mixture, the activation of both human and rat LCAT was greatly enhanced. On the other hand, the activities of human and rat enzymes in the presence of rat apo-a-i decreased to 18 and 43 % of that the produced by human apo-a-i, respectively. DISCUSSION The affinity of rat LCAT to rat apo-a-i was found to be greater than that of human LCAT to human apo-a-i on the apo-a-i-sepharose 4B column. Furthermore, when rat apo-a-i was added to the rat enzyme/vesicles mixture, an apo-a-i-enzyme-vesicles complex was recognized in the elution pattern of gel permeation chromatography. Clarification of the respective affinities among the com ponents of the LCAT reaction, such as LCAT, apo-a-i, and substrate vesicles, is very important for clarifying the reaction mechanism of human plasma LCAT. We have studied the reaction mechanism of human LCAT reaction by using purified human LCAT and human apo-a-i and substrate vesicles (16, 33). When human apo-a-i was added to the human enzyme/vesicles mixture, the enzyme peak eluted in the region of the vesicles on the Sepharose CL 4B column did not completely coincide with the peak of vesicles. This indicates that the human enzyme bound to single bilayer lecithin-cholesterol vesicles was effectively displaced by the addition of human apo-a-i (16). Although the _??_ may require the simultaneous presence of the _??_ and the enzyme on the substrate particles, no _??_ interaction appeared to exist between the apo-a-i and the enzyme by affinity chromatography on apo-a-i coupled with Sepharose CL 4B (16). We also demonstrated that an effective transfer of the enzyme from HDL3 to lecithin-cholesterol vesicles as the enzyme acceptor occur red in the presence of human apo-a-i (33). This indicates that the affinity of the enzyme to the HDL3 may vary with the condition of the HDL3 to which vesicle lipids are attached and with the contents of apo-a-i (34). These results indicate that human apo-a-i may not only serve as a cofactor in the LCAT reaction (12-14), but may also be needed to promote the enzyme transfer (32, 34). On the other hand, unlike human LCAT, rat LCAT showed a low level of dissociation from the vesicles on addition of apo-a-i (Fig. 5). The results of the present study agree with previous results on the affinity of rat LCAT to rat HDL3 (17); namely, this strong affinity may account for the affinity of the enzyme to the apo-a-i which is present on the surface of rat plasma HDL3 (17). Under normal conditions, such as in plasma, rat LCAT, unlike human LCAT, may not easily make the transfer to other substrates. The reasons for the differences in the mecha nisms between human and rat LCAT reactions are not sufficiently clear; but the difference in the reactivities of rat and human LCAT may arise from two main factors. One is a difference in properties between human and rat apo-a-i, such as binding affinity to the substrate. In the human LCAT reaction, we found that the addition of human LCAT to the vesicles containing human apo-a-i brought about complete coincidence of the elution profiles of these three components on a Sepharose C1 4B column chromatography (33). These findings demonstrate that the contents and the state of the apo-a-i present on the substrate affects the interaction of the enzyme with the substrate. They suggest that the stronger the binding affinity of apo-a-i to the substrate, the less the displacement of LCAT from the substrate may progress. 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