The Arg123-Tyr166 Central Domain of Human ApoAI Is Critical for Lecithin:Cholesterol Acyltransferase–Induced Hyperalphalipoproteinemia and HDL Remodeling in Transgenic Mice
Abstract—High density lipoprotein (HDL) metabolism and lecithin:cholesterol acyltransferase (LCAT)–induced HDL remodeling were investigated in transgenic mice expressing human apolipoprotein (apo) AI or an apoAI/apoAII chimera in which the Arg123-Tyr166 domain of apoAI was substituted with the Ser12-Ala75 domain of apoAII. Expression of apoAI and of the apoAI/apoAII chimera resulted in a respective 3.5-fold and 2.9-fold increase of HDL cholesterol. Human LCAT gene transfer into apoAI-transgenic mice resulted in a 5.1-fold increase of endogenous LCAT activity. This increase was associated with a 2.4-fold increase of the cholesterol ester–to–free cholesterol ratio of HDL, a shift from HDL3 to HDL2, and a 2.4-fold increase of HDL cholesterol levels. Agarose gel electrophoresis revealed that human LCAT gene transfer into human apoAI–transgenic mice resulted in an increase of pre-β-HDL and of pre-α-HDL. In contrast, human LCAT gene transfer did not affect cholesterol levels and HDL distribution profile in mice expressing the apoAI/apoAII chimera. Mouse LCAT did not “see” a difference between wild-type and mutant human apoAI, whereas human LCAT did, thus localizing the species-specific interaction in the central domain of apoAI. In conclusion, the Arg123-Tyr166 central domain of apoAI is not critical for in vivo lipoprotein association. It is, however, critical for LCAT-induced hyperalphalipoproteinemia and HDL remodeling independent of the lipid-binding properties of apoAI.
- Received March 5, 1999.
- Accepted June 8, 1999.
Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the metabolism of HDL cholesterol.1 2 Naturally occurring variants with point mutations in the central domain of apoAI, such as apoAIFin (Leu159→Arg),3 apoAIOita (Val 156→Glu),4 apoAIOslo (Arg160→Leu),5 apoAIPisa (Leu141→ Arg),6 or with the deleted Glu146→Arg160 central domain (apoAISeattle),7 are associated with impaired LCAT activation and reduced HDL cholesterol levels.
Expression of human LCAT in mice8 9 10 11 12 13 or rabbits14 15 16 17 18 resulted in a significant increase of HDL cholesterol levels. Expression of human LCAT resulted in a 4-fold increase of HDL cholesterol levels in human apoAI–transgenic mice but only a 2-fold increase in transgenic mice expressing both human apoAI and apoAII. These data indicated that human LCAT has a significant preference for HDLs containing human apoAI.
Deletion of the Leu122-Pro165, the Leu144-Gly186, or the Glu146-Arg160 domain of apoAI resulted in impaired LCAT activation with in vitro reconstituted HDL.19 20 21 Furthermore, the hydrophobic face orientation of the Pro143-Ala164 helix domain of apoAI was found to be critical for LCAT activation.22 Finally, substitution of the Arg123-Tyr166 domain of human apoAI with the Ser12-Ala75 helical domain of apoAII resulted in a >20-fold reduction of the in vitro LCAT activity in reconstituted HDL. This decrease in LCAT activity was independent of the size and of the apolipoprotein, phospholipid, and cholesterol contents of the reconstituted HDL.23
In the present study, transgenic mice that expressed human apoAI or the apoAI[(ΔArg123-Tyr166);∇apoAII(Ser12-Ala75)] chimera were used to study the role of the Arg123-Tyr166 central domain of apoAI in HDL metabolism both in the absence and presence of human LCAT expressed with a recombinant human LCAT adenovirus.23
Preparation of Human ApoAI DNA Constructs for Zygote Injection
The isolation of a genomic clone containing the human apoAI gene24 has been previously reported. A 2.2-kb EcoRI-XbaI fragment was obtained by courtesy of E.M. Rubin, Lawrence Berkeley Laboratory, Berkeley, Calif. The 2.2-kb fragment was inserted in the pBlueScript (pBS) vector resulting in the pBS-AI vector. The pBS–AI(1–122)–AII(12–75) DNA fragment was amplified from the pBS-AI vector by polymerase chain reaction (PCR) using the 5′-dCTCGAAATTAACCCTCACTAAA forward primer and the 5′-dCTCCACCTTCTGGCGG reverse primer. The apoAI[Δ(123–166);∇AII(12–75)] DNA fragment was amplified from the pMc-5–apoAI[Δ(123–166);∇AII(12–75)] vector14 by using the 5′-dGAAGTACTGAGACAGCGGCTCCACCTTCTGGCGG forward primer and the 5′-dCACTTTGGAAAGTTTATTCTGAGC reverse primer that contains an additional Psp site. The apoAI(1–122)–apoAII(12–75)–apoAI(166–243) DNA fragment was amplified from pBS–AI(1–122)–AII(12–75) and the apoAI[Δ(123–166);∇AII(12–75)] fragments by using the 5′-dCTCGAAATTAACCCTCACTAAA forward primer and the 5′-dCACTTTGGAAAGTTTATTCTGAGC reverse primer. The PCR product was digested with Psp and XbaI and ligated in the Psp and XbaI-treated pBS-AI vector. A 2.2-kb EcoRI-XbaI fragment was excised and purified for zygote injection. All DNA constructs were confirmed by DNA sequencing.
Generation of Transgenic Mice
Transgenic mice expressing human apoAI or the apoAI/apoAII chimera were generated by zygote injection into the C57BL/6 background according to previously published procedures.25 26 All experimental procedures in mice were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee.
Production of Recombinant LCAT Adenovirus
The pUC19 LCAT plasmid, containing the entire human LCAT cDNA, was a kind gift of Dr J. McLean (Department of Cell Biology, Genentech Inc, South San Francisco, Calif). The human LCAT cDNA fragment was obtained by digestion of pUC19 LCAT with EcoRI and HindIII and was subcloned by using identical restriction sites in the shuttle plasmid pACCMVpLpA, which contains the 760-bp cytomegalovirus (CMV) promoter/enhancer and the simian virus splice/polyadenylation site. The recombinant human LCAT virus was generated by cotransfection of pACCMVpLpA.LCAT and the rescue plasmid pJM17 in 293 cells. The LCAT adenovirus was amplified and purified as described previously.27
Tail tips were excised from 3- to 4-week-old mice and incubated overnight at 55°C in lysis buffer (2 mmol/L EDTA, 1% sarcosyl, 0.2 mol/L Tris-HCl [pH 8.0], 0.4 mol/L NaCl, and 8 mol/L urea) including proteinase K (3.6 mg/mL). The mixture was then incubated with RNase (13 μg/mL) at 37°C for 2 hours. After centrifugation, DNA was extracted with phenol/chloroform (1:1, vol/vol) and precipitated in isopropanol/ethanol. The DNA was resuspended in 700 μL of 10 mmol/L Tris-HCl, (pH 8.0), and 0.1 mmol/L EDTA. DNA (10 μg) was digested with PstI, fractionated in 0.7% agarose, transferred to a charge-modified nylon 66 binding matrix, and hybridized in 50% formamide with nick-translated human apoAI genomic DNA (specific activity 2 to 5×108 counts per minute per microgram). Filters were washed with a stringency of 0.1× NaCl/citrate (1× NaCl/citrate is 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.5) and 0.1% SDS at 65°C and autoradiographed. Liver-specific human LCAT gene transfer was demonstrated by the appearance of a 1.4-kb EcoRI-HindIII fragment on Southern blots developed with a human LCAT cDNA probe.
Total RNA was isolated from mouse liver by a single-step method (Trizol reagent, Life Technologies) based on the guanidinium isothiocyanate–acid–phenol RNA isolation method developed by Chomczynski and Sacchi. Twenty micrograms of total RNA was separated on a 1.25% agarose gel containing 0.21 mol/L formamide and 3.8 volume % formaldehyde. RNA was blotted overnight in 20× SSC (20× SSC is 3 mol/L NaCl and 0.3 mol/L trisodium citrate · 2H2O, pH 7.0). After UV cross-linking, the membrane was prehybridized in Quickhyb (Stratagene) containing 800 μL of herring sperm DNA for 6 hours. Human apoA-I cDNA and human GAPDH cDNA probes were labeled with [α-32P]dCTP by using the Rediprime labeling system (Amersham Life Sciences). Hybridization was performed for 18 hours by adding 3×107 cpm of denatured probe. The membrane was washed 2 times for 20 minutes at room temperature in a solution containing 2× SSC and 0.05% SDS and subsequently washed at 37°C for 20 minutes in a buffer containing 0.01% SSC and 0.1% SDS. RNA signals were quantified in a PhosphorImager. The human apo A-I mRNA signals were normalized for the GAPDH signal.
ApoAI Quantification by ELISA and Western Blotting
Blood was collected in heparinized microhematocrit capillary tubes from the tail or from the retro-orbital plexus. After centrifugation (2000g for 5 minutes), levels of human apoAI and of the apoAI/apoAII chimera were measured in a sandwich ELISA by using mouse monoclonal antibodies to human apoAI as described previously.25 The antibodies used in this assay had <0.01% cross-reactivity with mouse apoAI. Purified proteins23 were used as internal standards. The interassay variation coefficient of the ELISA is 12%. Alternatively, levels of human apolipoproteins were determined by quantitative scanning of Western blots of 10% to 15% gradient SDS-polyacrylamide gels that had been developed with the monoclonal antibodies to human apoAI. For quantification of mouse apoAI, Western blots were developed with polyclonal anti-mouse apoAI antibodies prepared in cynomolgus monkeys and generously supplied by Dr George Melchior (The Upjohn Co, Kalamazoo, Mich). These antibodies had <0.01% cross-reactivity with human apoAI. The interassay variation coefficient of the immunoblot assay is 20%.
Plasma Lipid and Lipoprotein Analyses
At ≈3 months of age, the mice were fasted overnight and blood was collected. Plasma was obtained by centrifugation, and lipoprotein fractions were separated by gel filtration on a Superdex 200 HR column equilibrated with 20 mmol/L Tris-HCl buffer, pH 8.1, containing 0.15 mol/L NaCl, 1 mmol/L EDTA, and 0.02 mg/mL NaN3 in a fast protein liquid chromatography system (Waters Associates). Samples (200 μL) were applied. Human apoAI distribution in HDL was assessed by ELISA. Mouse apoAI in HDL was assessed by Western blotting.25 26 HDL particle size distribution was either assessed by gel filtration after injection of radiolabeled purified apolipoproteins28 or estimated by comparison of their migration position on native 4% to 15% gradient polyacrylamide gels with that of standard proteins: thyroglobulin (Stokes’ radius of 8.5 nm), apoferritin (6.1 nm), catalase (5.2 nm), and lactate dehydrogenase (4.1 nm) (Pharmacia). Electrophoretic mobilities of plasma lipoproteins were analyzed by agarose gel electrophoresis, and blots were analyzed by quantitative scanning densitometry with the use of the NIH image analysis program.
Endogenous LCAT Activity
The endogenous LCAT activity of the plasma of transgenic mice was measured by determining the esterification rate of cholesterol in human HDL3. Aliquots containing 5 μg of cholesterol were incubated with 10 μL of plasma at 37°C for 30 minutes. Free cholesterol (FC) and cholesterol esters (CEs) were quantified by high-performance liquid chromatography. They were eluted isocratically at 45°C with a mixture of acetonitrile/isopropanol at ratios of 50:50 and 90:10 (vol/vol), respectively.29 Endogenous LCAT activity, expressed as nanomoles of CE per milliliter per hour, was quantified in samples collected before and at 6 days after intravenous injection of 109 plaque-forming units of the recombinant LCAT adenovirus.
In Vivo ApoAI Metabolism
Recombinant apoAI and apoAI[(ΔArg123-Tyr166);∇apoAII(Ser12-Ala75] were purified from the periplasmic fraction of Escherichia coli cells as previously described.23 Proteins were iodinated by the Iodo-Gen (Pierce Chemical Co) method. The specific activities were 3 μCi/μg apolipoprotein. The clearance rates (Cl) and the fractional catabolic rates (FCRs) of radiolabeled apolipoproteins were determined in mice as described previously.25 26 The endogenous production rates (PRs) of the apolipoproteins were calculated from the plasma steady-state concentrations (mg/dL), the clearance rates (mL · h−1), and the body weights (g) as described by Kleinberger et al.30
The significance of differences in lipoprotein levels, cholesterol-CE ratios, endogenous apolipoprotein PRs, and FCRs were tested by the Mann-Whitney nonparametric test. Human apoAI levels in mice before and after human LCAT gene transfer were compared in a paired t test. A P value of <0.05 was considered to be statistically significant.
Effects of ApoAI Genotype on Apolipoproteins and Lipoproteins
Total cholesterol, non-HDL cholesterol, and HDL cholesterol levels of C57BL/6 mice were (mean±SEM, n=8) 45±4.0, 12±1.2, and 31±12.5 mg/dL, respectively. HDL cholesterol levels were 3.5-fold increased in apoAI-transgenic mice (n=8) and 2.9-fold increased in mice expressing the apoAI/apoAII chimera (n=8, Figure 1⇓). The ratio of HDL to total cholesterol was 0.67±0.041 in C57BL/6 mice, 0.91±0.077 (P<0.001) in apoAI-transgenic mice, and 0.87±0.070 (P<0.001 versus C57BL/6 mice and P=NS versus apoAI-transgenic mice) in mice expressing the apoAI/apoAII chimera. Levels of human apoAI and of the apoAI/apoAII chimera in transgenic mice were similar: 220±11 and 180±5.8 mg/dL, respectively. Mouse apoAI levels were 70±6.7 mg/dL in C57BL/6 control mice and <1 mg/dL in mice expressing human apoAI and in mice expressing the apoAI/apoAII chimera (data not shown). Expression of human apoAI or of the apoAI/apoAII chimera on the C57BL/6 background did not change β-VLDL cholesterol levels (Figure 1⇓).
Effect of Human LCAT Gene Transfer on LCAT Activity, HDL Cholesterol Levels, and ApoAI Levels
Before human LCAT gene transfer, the endogenous LCAT activity in apoAI-transgenic mice and in mice expressing the chimera was very similar (Figure 2⇓). The FC-CE ratios of HDL in apoAI- and apoAI/apoAII-expressing mice were very similar (0.39±0.013 and 0.49±0.038, respectively). After human LCAT gene transfer, the endogenous LCAT activity in apoAI-transgenic mice was 5.1-fold higher than in mice expressing the apoAI/apoAII chimera (Figure 2⇓). This higher LCAT activity was associated with a 2.4-fold increase of the CE-cholesterol ratio in the HDLs of apoAI-transgenic mice (Figure 2⇓). Human LCAT gene transfer into transgenic mice expressing the apoAI/apoAII chimera did not increase the endogenous LCAT activity and did not alter the FC-CE ratio of their HDL (Figure 2⇓). The differences in LCAT activity in mice expressing apoAI or the apoAI/apoAII chimera could not be attributed to differences in human LCAT levels.
Adenovirus-mediated expression of human LCAT resulted in a 2.4-fold further increase of HDL cholesterol levels in apoAI-transgenic mice 6 days after intravenous injection of the recombinant LCAT adenovirus (Figure 1⇑). In contrast, human LCAT gene transfer did not induce a significant increase of HDL cholesterol levels in C57BL/6 mice or in transgenic mice expressing the apoAI/apoAII chimera (Figure 1⇑). Whereas HDL cholesterol levels in mice expressing human apoAI or the apoAI/apoAII chimera were similar before human LCAT gene transfer, HDL cholesterol levels were 2.8-fold higher in apoAI-transgenic mice than in mice expressing the apoAI/apoAII chimera after LCAT gene transfer (Figure 1⇑). Human LCAT gene transfer into apoAI-transgenic mice resulted in a 2.5-fold increase of non-HDL cholesterol levels, whereas non-HDL cholesterol levels in C57BL/6 mice or in transgenic mice expressing the apoAI/apoAII chimera were not affected by human LCAT gene transfer (Figure 1⇑).
Figure 3⇓ shows that at day 0, plasma levels of apoAI in the 2 groups of 8 apoAI transgenic mice were very similar: 220±11 and 200±9.8 mg/dL. At day 6, plasma levels of apoAI were very similar in the group that did not receive the human LCAT adenovirus, whereas plasma levels of apoAI in mice treated with the LCAT adenovirus were increased to 420±30 mg/dL. Human LCAT gene transfer did not alter plasma levels of the apoAI/apoAII chimera (Figure 3⇓ and Table 1⇓). Northern blotting showed that human LCAT gene transfer in apoAI-transgenic mice did not result in an increase of human apoAI mRNA levels (data not shown).
Endogenous Apolipoprotein Production and Clearance
The plasma clearance of radiolabeled apolipoproteins was analyzed in transgenic mice before and after human LCAT gene transfer. The apolipoprotein steady-state plasma concentration (Css), the FCR, the plasma clearance rate (Clp), and the endogenous PR of apoAI and of the apoAI/apoAII chimera were very similar (Table 1⇑). Human LCAT gene transfer did decrease the FCR of apoAI but not that of the apoAI/apoAII chimera (Table 1⇑). Human LCAT gene transfer also resulted in an increased endogenous PR of apoAI but not of the apoAI/apoAII chimera (Table 1⇑).
Lipoprotein Association of ApoAI and of the ApoAI/ApoAI Chimera Before and After Human LCAT Gene Transfer
Figure 4⇓ illustrates the distribution of apoAI and of the apoAI/apoAII chimera in HDL fractions isolated by gel filtration from the plasma of transgenic mice expressing apoAI or the apoAI/apoAII chimera before and after LCAT gene transfer. More than 85% of apoAI and of the apoAI/apoAII chimera were recovered in HDL fractions (Table 2⇓). As previously demonstrated,17 HDL particles of apoAI-transgenic mice were polydisperse, with major populations of particles with diameters of 8.4, 9.2, and 9.6 nm, respectively, thus corresponding to humanlike HDL3. Nondenaturing polyacrylamide gel electrophoresis revealed a similar pattern of HDL in transgenic mice expressing the apoAI/apoAII chimera. Human LCAT gene transfer into apoAI-transgenic mice resulted in a shift to larger HDL particles, with sizes ranging between 11 and 13 nm, thus corresponding to humanlike HDL2. In contrast, human LCAT gene transfer into transgenic mice expressing the apoAI/apoAII chimera did not affect the size of the HDL particles. Whereas human LCAT gene transfer into apoAI-transgenic mice thus resulted in a shift from HDL3 to HDL2, in agreement with the enhanced LCAT activity and HDL cholesterol esterification, HDL particles in LCAT adenovirus–treated apoAI/apoAII-transgenic mice migrated primarily as HDL3. In addition, human LCAT gene transfer into apoAI-transgenic mice resulted in a 2-fold increase of small, dense HDL (Table 2⇓). In contrast, human LCAT gene transfer into transgenic mice expressing the apoAI/apoAII chimera did not result in an increase of small, dense HDL (Table 2⇓).
The cholesterol to apoAI, FC to apoAI, and CE to apoAI molar ratios of HDLs from apoAI-transgenic mice were 51±5.4, 15±1.5, and 39±3.0, respectively. After human LCAT gene transfer, these values were 64±5.5 (P=NS), 9.5±0.81 (P=0.0080), and 65±5.9 (P=0.0013), respectively.
Plasma lipoproteins were also separated by nondenaturing agarose gel electrophoresis. As shown in Figure 5⇓, 2⇑ distinct HDL populations were observed in the plasma of apoAI-transgenic mice: 1 migrated in the α-position (≈90%) and 1 migrated in the pre-β position (≈10%). After human LCAT gene transfer, we observed 3 HDL populations: 1 in the pre-β position, 1 in the α-position, and 1 in the pre-α position. Human LCAT gene transfer into apoAI-transgenic mice resulted in an increase of pre-β-HDL and of pre-α-HDL and a decrease of α-HDL (Figure 5⇓). In contrast, human LCAT gene transfer into transgenic mice expressing the apoAI/apoAII chimera did not alter the HDL distribution profile (Figure 5⇓). Both before and after LCAT gene transfer, HDL particles of transgenic mice expressing the apoAI/apoAII chimera showed α-electrophoretic mobility.
The present study in transgenic mice demonstrates that (1) the Arg123-Tyr166 domain of human apoAI is not critical for its in vivo lipoprotein association, as evidenced by the similar increases of HDL cholesterol levels in transgenic mice expressing the central domain apoAI/apoAII chimera as in apoAI-transgenic mice; (2) human LCAT gene transfer into apoAI-transgenic mice is associated with hyperalphalipoproteinemia and HDL remodeling; and (3) the Arg123-Tyr166 domain of human apoAI is critical for in vivo LCAT activation and thereby for inducing hyperalphalipoproteinemia and HDL remodeling.
A first very important observation was that substitution of the Arg123-Tyr166 domain of apoAI with the apoAII segment did not affect in vivo apolipoprotein synthesis and lipoprotein association. Indeed, the in vivo PRs of human apoAI and of the apoAI/apoAII chimera were very similar, as well as the HDL cholesterol levels and the HDL distribution profiles of mice expressing apoAI or the apoAI/apoAII chimera. Thus, these transgenic mice were indeed suitable to investigate the role of LCAT activation in HDL metabolism independent of lipid binding and thus, independent of baseline levels of HDL cholesterol as well as of the baseline distribution profiles of HDL.
Adenovirus-mediated expression of human LCAT in apoAI-transgenic mice was associated with an increase of HDL cholesterol levels as has previously been shown in human apoAI/LCAT-transgenic mice8 and in human apoAI-transgenic mice that were treated with a similar human LCAT adenovirus.9 Increased LCAT activity in LCAT adenovirus–treated apoAI-transgenic mice was associated with an increase of both pre-β-HDL and of pre-α-HDL. Davidson et al31 demonstrated that the increased electronegative charge on α-HDL and pre-α-HDL, compared with that on pre-β-HDL, is mostly due to the presence of a neutral lipid ester core and only in a small way to a direct effect of negatively charged phospholipids. Because neutral lipids do not possess a net negative charge, they cannot directly contribute a charge to a lipoprotein particle. Therefore, the change in charge is most probably due to a conformational change in the apolipoprotein molecules. It has been suggested that this electronegative HDL fraction may be the preferential substrate for cholesterol ester transfer protein (CETP)–mediated lipid exchange. This electronegative HDL fraction is similar to an HDL fraction in the plasma of CETP-deficient patients.32 Thus, our results suggest that “recycling” of the large HDL that is generated as a consequence of increased LCAT activity is impaired in the absence of CETP and that the increase in HDL cholesterol is due to impaired HDL cholesterol flux. This hypothesis will be investigated in mice that overexpress human apoAI, human LCAT, and human CETP.
At this moment, we can only speculate about the mechanisms that lead to the increase of pre-β-HDL in apoAI-transgenic mice, especially in those mice treated with the LCAT adenovirus. Jiang et al33 have demonstrated that the phospholipid transfer protein (PLTP) increases the influx of phospholipid and secondary cholesterol into HDL, leading to an increase in potentially antiatherogenic pre-β particles. Increased PLTP activity and PLTP-mediated HDL remodeling in human apoAI-transgenic mice may explain to some extent the increase of pre-β-HDL in human apoAI-transgenic mice.34 Another molecule that may play a key role in HDL remodeling is the scavenger receptor BI.35 This molecule mediates the selective uptake of HDL CEs in the liver and may thus contribute to the generation of pre-β-HDL.35 It is thus possible that by the action of human LCAT in LCAT adenovirus–treated apoAI-transgenic mice, α-HDL is converted into pre-α-HDL by the accumulation of CEs and that the scavenger receptor BI–mediated selective uptake of these CEs results in the generation of pre-β-HDL. Finally, the increase of pre-β-HDL may partly be due to an increased production of nascent HDL, as evidenced by the increased apoAI PR in apoAI-transgenic mice after LCAT gene transfer. These data are not necessarily in conflict with the lack of an increase in human apoAI mRNA levels in these mice. Northern blotting may not be sensitive enough to detect a 50% increase in apoAI production.
Baseline levels of HDL cholesterol and baseline HDL distribution profiles of mice expressing the central domain chimera were very similar to these of apoAI-transgenic mice, demonstrating similar lipid-binding properties of apoAI and of the apoAI/apoAII chimera. Human LCAT gene transfer in mice expressing the central domain chimera did, however, not increase the endogenous LCAT activity and HDL cholesterol levels and did not induce HDL remodeling in these mice. We did not observe differences in size and lipid composition between HDLs of apoAI-transgenic mice and of mice expressing the apoAI/apoAII chimera before human LCAT gene transfer. It is therefore very unlikely that the observed differences in LCAT activity after LCAT gene transfer were due to differences in apolipoprotein/lipid content. Most likely differences in LCAT activity are due to the presence in apoAI, but not in the apoAI/apoAII chimera, of an amino acid sequence that may allow a conformation in apoAI that is critical for LCAT activation. Sparks et al36 demonstrated that LCAT can interact with lipid-poor apoAI, suggesting that LCAT does not need to bind to the lipid interface on an HDL particle but may directly interact with apoAI; thus, the conformation of apoAI may be critical for interaction with LCAT. Mouse LCAT did not see a difference between wild-type human apoAI and mutant apoAI, whereas human LCAT did so, thus localizing the species-specific interaction to the central domain of apoAI.
Serum cholesterol levels of carriers of the apoAIFin mutation,3 a Leu159→Arg point mutation, are decreased to 20% to 30% of normal. Whereas the ability of this mutant to activate LCAT is defective, its phospholipid-binding and cholesterol efflux properties are normal. Serum cholesterol levels of carriers of the apoAIOita mutation,4 a Val156→Glu point mutation; of the apoAIOslo mutation,5 an Arg160→Leu point mutation; or of the apoAISeattle mutation, a Glu146→Arg160 deletion7 19 are <10% of normal. All of these mutations, in contrast with the apoAIFin mutation, are associated with both defective lipid binding and defective LCAT activation. The latter mutants are thus not well suited to study the effect of defective LCAT activity on HDL cholesterol levels independently of defective phospholipid binding and lipoprotein association. In the present study, an apoAI variant with normal phospholipid binding properties but defective LCAT was used to study the effect of reduced LCAT activity on HDL cholesterol levels in the absence of reduced capacity to interact with phospholipids and thus to produce HDL particles. In the presence of human LCAT, HDL cholesterol levels in transgenic mice expressing the apoAI/apoAII chimera were only 30% of those of mice expressing wild-type human apoAI. As discussed above, the similar levels of HDL cholesterol in mice expressing human apoAI or the apoAI/apoAII chimera in the absence of human LCAT may be explained by a lack of interaction between human apoAI-containing HDL and mouse LCAT.
In conclusion, the present study demonstrates clear evidence for the critical role of the central domain of apoAI for in vivo LCAT activation and associated hyperalphalipoproteinemia and HDL remodeling, independently of its capacity to associate with lipids.
This work was supported by the Interuniversitaire Attractiepolen Program (P4/34) and by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (program G.0110.98). Bart De Geest is a postdoctoral Fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. Sophie Van Linthout is a Research Assistant of the Instituut voor Wetenschappelijk Onderzoek-Vlaanderen. We thank Eddy Demarsin, Els Deridder, and Michèle Landeloos for excellent technical assistance.
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