Organization of the human cholesteryl ester transfer protein gene

The plasma cholesteryl ester transfer protein (CETP) catalyzes the transfer of phospholipids and neutral lipids between the lipoproteins. Thus, this protein may be important in modulating lipoprotein levels in the plasma. We have determined the primary structure and organization of the human CETP gene. Southern blotting of cellular DNA indicated a single copy of the CETP gene exists per haploid genome. Analysis of three overlapping genomic clones showed that the gene spans approximately 25 kbp and contains 16 exons (size range 32-250 bp). Overall, the sequence and organization of the CETP gene do not resemble those of other lipid-metabolizing enzymes or apolipoproteins. However, comparison of the CETP sequence, one exon at a time, with the sequences in the sequence databases revealed a striking identity of a pentapeptide sequence (ValLeuThrLeuAla) within the hydrophobic core of the signal sequences of human CETP, apolipoproteins A-IV and A-I, and lipoprotein lipase. This pentapeptide sequence was not found in the signal sequences of other proteins, suggesting that it may mediate a specialized function related to lipid metabolism or transport.     

Simultaneous transfer of cholesteryl ester and phospholipid by protein(s) isolated from human lipoprotein-free plasma

Protein(s) catalyzing the transfer of [³H]cholesteryl ester and [¹⁴C]-phosphatidylcholine from high density lipoproteins to low density lipoproteins have been purified 4829-fold from human plasma by chromatography of the d > 1.21 g/ml infranatant fraction of human plasma on phenyl-Sepharose, CM-cellulose, concanavalin A-Sepharose, and finally by isoelectric focussing. At each step of the purification, both transfer activities coelute. The purified protein(s), molecular weight 150,000, transfer cholesteryl esters and phosphatidylcholine with a 1:1 stoichiometry and at equal rates of flux. Rat plasma contains a protein(s) which facilitates the transfer of phosphatidylcholine as effectively as human plasma. However, the rat plasma protein(s) does not facilitate the transfer of cholesteryl esters. These results suggest that human plasma contains one or more proteins which transfer both lipids, possibly as a 1:1 complex, whereas rat plasma lacks the cholesteryl ester transfer protein.     

Establishment of anti-human cholesteryl ester transfer protein monoclonal antibodies and radioimmunoassaying of the level of cholesteryl ester transfer protein in human plasma.

Plasma cholesteryl ester transfer protein (CETP) facilitates the net transfer and exchange of cholesteryl ester (CE), triglyceride (TG), and phospholipids between lipoproteins. A series of monoclonal antibodies (mAbs) against human CETP was obtained, comprising mAbs either inhibiting or not inhibiting these transfer activities. One mAb (LT-J1) inhibited the transfer activity of TG almost completely, but not that of CE, indicating that CE and TG binding sites on the CETP molecule may be distinct from each other, and that this mAb may specifically recognize the TG binding site. A radioimmunoassay system for determining the level of CETP was also established using these mAbs, and the plasma CETP levels in 20 normolipemic Japanese adults were found to range from 2.1 to 2.7 mg/liter.     

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.     

Structure-function studies of human cholesteryl ester transfer protein by linker insertion scanning mutagenesis

Human plasma cholesteryl ester transfer protein (CETP) enhances transfer and exchange of cholesteryl ester (CE) and triglyceride (TG) between high-density lipoprotein and other lipoproteins. To define regions responsible for the neutral lipid transfer activities at the molecular level, a total of 27 linker insertion mutants at 18 different sites along the CETP molecule were prepared and transiently expressed in a mammalian cell line (COS). The inserted linkers were small (usually 6 bp) and did not interrupt the translational reading frame of the CETP cDNA. Although secretion of each mutant protein was less than that of wild-type CETP, the majority of the mutants had normal cholesteryl ester transfer activity (transfer activity per nanogram of CETP in media). However, insertional alterations in three regions severely impaired CE transfer activity: (1) in the region of amino acids 48-53; (2) at amino acid 165; and (3) in the region of amino acids 373-379. Although the impaired activities could also be a result of globally incorrect folding of these CETP mutants, hydrophobicity analysis and secondary structure predictions tended to exclude this possibility for most of the insertion sites at which insertions resulted in inactivation. The insertion at amino acid 379 occurs immediately after a triplet of lysine residues, suggesting that this region might be involved in an essential step in the mechanism of CE and TG transfer, such as the binding of CETP to phosphatidylcholine molecules in the lipoprotein surface. Effects on TG transfer activity were generally similar to those on CE transfer activity, suggesting a similar structural requirement for both neutral lipid transfer activities.     

Human plasma cholesteryl ester transfer protein enhances the transfer of cholesteryl ester from high density lipoproteins into cultured HepG2 cells

The role of human plasma cholesteryl ester transfer protein (CETP) in the cellular uptake of high density lipoprotein (HDL) cholesteryl ester (CE) was studied in a liver tumor cell line (HepG2). When HepG2 cells were incubated with [3H]cholesteryl ester-labeled HDL3 in the presence of increasing concentrations of CETP there was a progressive increase in cell-associated radioactivity to levels that were 2.8 times control. The CETP-dependent uptake of HDL-CE was found to be saturated by increasing concentrations of both CETP and HDL. The CETP-dependent uptake of CE radioactivity increased continuously during an 18-h incubation. In contrast to the effect on cholesteryl ester, CETP failed to enhance HDL protein cell association or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d greater than 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d greater than 1.21 g/ml fraction of rat plasma which lacks cholesteryl ester transfer activity. HDL cholesteryl ester entering the cell under the influence of CETP was largely degraded to free cholesterol by a process inhibitable by chloroquine. CETP enhanced uptake of HDL [3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or J774 macrophages. These experiments show that, in addition to its known role in enhancing the exchange of CE between lipoproteins, plasma CETP can facilitate the in vitro selective transfer of CE from HDL into certain cells.     

Diphenylpyridylethanamine (DPPE)-based aminoheterocycles as cholesteryl ester transfer protein inhibitors

A series of diphenylpyridylethanamine-based inhibitors of cholesteryl ester transfer protein with aminoheterocycles appended onto the N-terminus of the chemotype were explored as urea mimetics. Potent compounds were discovered and were further optimized to improve metabolic stability and PXR transactivation profile.     

Purification and characterization of a human plasma cholesteryl ester transfer protein

The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and promotes transfer of cholesteryl esters between the lipoproteins. CETP has been purified 55,000-fold, with a 27% recovery of activity, from the d greater than 1.21 g/ml fraction of human plasma. In the final purification step, partially purified CETP is incubated with a synthetic lipid emulsion consisting of phosphatidylcholine, triglyceride, and fatty acid, and the bound activity, which elutes in the void volume, is separated from nonbound proteins by gel filtration on Sepharose 4B. Sodium dodecyl sulfate-gel analysis of fractions containing bound activity shows the presence of a single protein with an apparent Mr of 74,000. Inclusion of fatty acid in this emulsion was required to prevent the binding of a contaminant protein. However, incubation of CEPT with fatty acid emulsions containing lipid peroxides resulted in substantial inactivation and covalent degradation of the 74-kDa protein. This could be prevented by the inclusion of antioxidants during preparation of the emulsion. Solvent extraction of emulsion-bound CEPT gave a delipidated, active preparation. Purified IgG from a rabbit immunized with the 74-kDa protein completely removed activity from partially purified fractions. Amino acid analysis of the purified protein showed it to contain an unusually high content (45%) of nonpolar residues; the calculated hydrophobicity was greater than that of any other plasma apolipoprotein. These results show human CETP to be a unique plasma apolipoprotein with an apparent Mr of 74,000 which is hydrophobic, self-associating, and susceptible to covalent degradation by lipid peroxides.     

Genetic variants affecting alternative splicing of human cholesteryl ester transfer protein

Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (Δ9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T > C located in intron 8 in a putative splicing branch site and rs5883 C > T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that Δ9-CETP protein is expressed in the liver but fails to circulate in the blood.     

Penetration of an emulsion surface by cholesteryl ester transfer protein

Quenching of the intrinsic fluorescence of cholesteryl ester transfer protein (CETP) by spin labelled fatty acids (5-NS and 16-NS) was investigated to determine the degree to which the protein penetrated the phospholipid monolayer surface of a lipid emulsion. When bound to the phospholipid surface approximately 50% of the fluorophores of the transfer protein were accessible to quenching by 5-NS whose nitroxy group locates near the monolayer surface. On the other hand, only 22% of the fluorophores of CETP were accessible to quenching by 16-NS whose nitroxy group locates deeper in the surface monolayer. Quenching of the CETP fluorescence by an aqueous phase quencher (acrylamide) shows that the protein undergoes a conformational change on binding which increases the proportion of the tryptophan residues exposed to the aqueous phase. The results indicate that CETP does not penetrate the lipid surface to a significant degree. Received: 29 March 1996 / Accepted: 30 May 1996     

Mechanisms of enhancement of cholesteryl ester transfer protein activity by lipolysis

Lipoprotein lipase enhances the cholesteryl ester transfer protein (CETP)-mediated transfer of cholesteryl esters from plasma high density lipoproteins (HDL) to very low density lipoproteins (VLDL). In time course studies the stimulation of cholesteryl ester transfer by bovine milk lipase was correlated with accumulation of fatty acids in VLDL remnants. As the amount of fatty acid-poor albumin in the incubations was increased, there was decreased accumulation of fatty acids in VLDL remnants and a parallel decrease in the stimulation of cholesteryl ester transfer by lipolysis. Addition of sodium oleate to VLDL and albumin resulted in stimulation of the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. The stimulation of transfer of cholesteryl esters into previously lipolyzed VLDL was abolished by lowering the pH from 7.5 to 6.0, consistent with a role of lipoprotein ionized fatty acids. CETP-mediated cholesteryl ester transfer from HDL to VLDL was also augmented by phosholipase A2 and by a bacterial lipase which lacked phospholipase activity. When VLDL and HDL were re-isolated after a lipolysis experiment, both lipoproteins stimulated CETP activity. Postlipolysis VLDL and HDL bound much more CETP than native VLDL or HDL. Lipolysis of apoprotein-free phospholipid/triglyceride emulsions also resulted in enhanced binding of CETP to the emulsion particles. Incubation conditions which abolished the enhanced cholesteryl ester transfer into VLDL remnants reduced binding of CETP to remnants, emulsions, and HDL. In conclusion, the enhanced CETP-mediated transfer of cholesteryl esters from HDL to VLDL during lipolysis is related to the accumulation of products of lipolysis, especially fatty acids, in the lipoproteins. Lipids accumulating in VLDL remnants and HDL as a result of lipolysis may augment binding of CETP to these lipoproteins, leading to more efficient transfer of cholesteryl esters from HDL to VLDL.     

Removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo is not enhanced by plasma cholesteryl ester transfer protein

The putative role of cholesteryl ester transfer protein (CETP) in the removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo was studied in hamsters. The parameter tested was retention of [3H]cholesteryl linoleyl ether ([3H]CLE), a nonhydrolysable analog of cholesteryl ester, in the liver after injection of [3H]CLE labeled acetylated LDL, which is targetted to nonparenchymatous littoral cells. In hamsters fed laboratory chow, plasma cholesteryl ester transfer activity (CETA) was 10.6 +/- 0.9 units and the retention of [3H]CLE in the liver 28 days after injection was 86% of the 4 h value. It was about 55% in rats fed the same diet, in which CETA was not detectable. When the diet was supplemented with 2% cholesterol and 15% margarine, CETA activity in hamsters increased 2-fold, yet no change in retention of [3H]CLE in liver was seen after 28 days. In rats, the retention of [3H]CLE in the liver was also not changed by the dietary fat supplementation. These results do not support the role of CETP in vivo in removal of cholesteryl ester from intact reticuloendothelial cells.     

Pros and cons of inhibiting cholesteryl ester transfer protein

Whether or not it is desirable to inhibit cholesteryl ester transfer protein (CETP) has been an important question for over fifteen years since genetic CETP deficiency was found. Recently, some epidemiological studies which have been reported in Japan as well as Western countries help to clarify the atherogenicity of human subjects with mutations or polymorphisms in the CETP gene. In addition, some experimental atherosclerosis studies, in which CETP was inhibited in rabbits with different approaches, have been reported. There was a considerable difference in the atherogenicity of human CETP deficiency and CETP-inhibited rabbits. In this review, we summarize the recent advances in this field as well as discussing the significance of CETP in reverse cholesterol transport, a major protective system against atherosclerosis.     

Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice

The human cholesteryl ester transfer protein (CETP) facilitates the exchange of neutral lipids among lipoproteins. In order to evaluate the effects of increased plasma CETP on lipoprotein levels, a human CETP minigene was placed under the control of the mouse metallothionein-I promoter and used to develop transgenic mice. Integration of the human CETP transgene into the mouse genome resulted in the production of active plasma CETP. Zinc induction of CETP transgene expression caused depression of serum cholesterol due to a significant reduction of high density lipoprotein cholesterol. There was no change in total cholesterol content in very low and low density lipoproteins. However, there was a decrease in the free cholesterol/cholesteryl ester ratio in plasma and in all lipoprotein fractions of transgenic mouse plasma, suggesting stimulation of plasma cholesterol esterification. The results suggest that high levels of plasma CETP activity may be a cause of reduced high density lipoproteins in humans.     

The essential role of a free sulfhydryl group in blocking the cholesteryl site of cholesteryl ester transfer protein (CETP)

Cholesteryl ester transfer protein (CETP) has at least one unpaired sulfhydryl residue, which we have shown previously to be in or near the active site region. We investigated the location of this unpaired cysteine residue(s) of CETP using chemical modification with fluorescent sulfhydryl-specific reagents, limited proteolysis, and amino acid/sequence analysis. The kinetics of labeling CETP by either 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) or acrylodan were followed by observing the increase in fluorescence of the bound probes. Labeling was inhibited strongly by preincubation of the CETP with either PNU-617, a competitive inhibitor of cholesteryl ester (CE) transport, and TP2 antibody. In addition, the transfer activities of the substrate CE by the modified CETP's were also inhibited but not competitively. Finally, preincubation of the native protein with N-ethylmaleimide (NEM) resulted in inhibition of activity that was dependent upon the time of exposure of the protein to the alkylating agent. These results provide further evidence that there is a cysteine residue in the active site region of CETP and ligands that either react or bind to this residue produce steric hindrance to CE transfer activity. Finally, although not conclusive, results of the protein chemistry experiments with the modified CETP suggest that the cysteine residue at position 333 is unpaired.