Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation.

BACKGROUND AND METHODS: The plasma cholesteryl-ester transfer protein (CETP) catalyzes the transfer of cholesteryl esters from high-density lipoprotein (HDL) to other lipoproteins. We recently described a Japanese family with increased HDL levels and CETP deficiency due to a splicing defect of the CETP gene. To assess the frequency and phenotype of this condition, we screened 11 additional families with high HDL levels by means of a radioimmunoassay for CETP and DNA analysis. RESULTS: We found the same CETP gene mutation in four families from three different regions of Japan. Analysis of restriction-fragment-length polymorphisms of the mutant CETP allele showed that all probands were homozygous for the identical haplotype. Family members homozygous for CETP deficiency (n = 10) had moderate hypercholesterolemia (mean total cholesterol level [+/- SD], 7.01 +/- 0.83 mmol per liter), markedly increased levels of HDL cholesterol (4.24 +/- 1.01 mmol per liter) and apolipoprotein A-I, and decreased levels of low-density lipoprotein cholesterol (1.99 +/- 0.80 mmol per liter) and apolipoprotein B. Members heterozygous for the deficiency (n = 20), whose CETP levels were in the lower part of the normal range, had moderately increased levels of HDL cholesterol and apolipoprotein A-I and an increased ratio of HDL subclass 2 to HDL subclass 3, as compared with unaffected family members (1.5 +/- 0.8 vs. 0.7 +/- 0.4). CETP deficiency was not found in six unrelated subjects with elevated HDL cholesterol levels who were from different parts of the United States. CONCLUSIONS: CETP deficiency appears to be a frequent cause of increased HDL levels in the population of Japan, possibly because of a founder effect. The results that we observed in heterozygotes suggest that CETP normally plays a part in the regulation of levels of HDL subclass 2. There was no evidence of premature atherosclerosis in the families with CETP deficiency. In fact, the lipoprotein profile of persons with CETP deficiency is potentially antiatherogenic and may be associated with an increased life span.

Transverse-plane topography of long-chain acyl-CoA synthetase in the mitochondrial outer membrane.

Transverse-plane topography of mitochondrial outer-membrane long-chain acyl-CoA synthetase was investigated using proteases as probes for exposure of crucial domains, i.e. domains containing the active site or otherwise required for enzymatic activity. Incubation of intact mitochondria with the nonspecific proteases proteinase K and subtilisin resulted in a time-dependent loss of 90% or more of the long-chain acyl-CoA synthetase activity compared to control incubations. The integrity of the outer membrane before and during this treatment was shown by cytochrome c oxidase latency as well as the stability of adenylate kinase activity in the presence of protease. After a 15-min incubation in these conditions, site-specific proteases such as trypsin and chymotrypsin had only a limited inhibitory effect (29 and 58% loss of activity, respectively); however, treatment of hypotonically disrupted mitochondria with these proteases resulted in increased (71 and 77%, respectively) loss of activity. Exposure of trypsin-sensitive crucial domains on the inner surface of the membrane was directly demonstrated by incubation of trypsin-loaded outer-membrane vesicles. Together, these results suggest that mitochondrial long-chain acyl-CoA synthetase is a transmembrane enzyme, possessing crucial domains on both sides of the outer membrane. However, the cytosolic exposure of the enzyme does not appear to be affected by a change in the medium ionic strength as seen previously for other outer-membrane enzymes. In an experiment investigating the topography of the active site of the enzyme, an immobilized substrate analog, desulfo-CoA-agarose, was preincubated with intact mitochondria. This resulted in up to a 42% loss of the activity of long-chain acyl-CoA synthetase, consistent with a cytosolic exposure for at least the CoA-binding domain of the active site.

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane.

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.

Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.

Plasma high density lipoproteins (HDL) are a negative risk factor for atherosclerosis. Increased HDL is sometimes clustered in families, but a genetic basis has never been clearly documented. The plasma cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from HDL to other lipoproteins and therefore might influence HDL levels. Using monoclonal antibodies, we show that CETP is absent in two Japanese siblings who have markedly increased and enlarged HDL. Furthermore, they are homozygous for a point mutation in the 5'-splice donor site of intron 14 of the gene for CETP, a change that is incompatible with normal splicing of pre-messenger RNA. The results indicate that the family has an inherited deficiency of CETP due to a gene splicing defect, and illustrate the key role that CETP has in human HDL metabolism.

Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope.

The plasma cholesteryl ester transfer protein (CETP, Mr 74,000) has a binding site for neutral lipid which can readily equilibrate with lipoprotein cholesteryl esters or triglycerides. Recently, a monoclonal antibody (TP2) was obtained which neutralizes the cholesteryl ester (CE) and triglyceride (TG) transfer activities of the CETP. In this report, the epitope of the inhibitory monoclonal antibody has been localized to a hydrophobic 26-amino acid sequence at the COOH terminus of CETP. The Fab fragments of TP2 caused partial (50%) inhibition of CE transfer and complete inhibition of TG transfer by the CETP. Similarly, the Fab fragments inhibited (37%) the binding of CE to the CETP and abolished the binding of TG to the CETP. Surprisingly, the TP2 Fab was also found to enhance the binding of CETP to plasma lipoproteins and to phospholipid vesicles. In conclusion, the TP2 monoclonal antibody inhibits lipid transfer by blocking the uptake of lipid by CETP. The COOH-terminal epitope may be in or near the neutral lipid binding site. Occupancy of this site by TP2 Fab fragments or by neutral lipid may result in a conformational change of CETP causing enhanced binding to lipoproteins or vesicles.

Structure-function analysis of plasma cholesteryl ester transfer protein by protease digestion and expression of cDNA fragments in Escherichia coli.

In an attempt to define an active domain of the protein, fragments of cholesteryl ester transfer protein (CETP) were obtained by limited digestion of the native, plasma-derived protein with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease or by expression of CETP cDNA restriction fragments in Escherichia coli. Although digestion of native CETP with these proteases resulted in extensive fragmentation of the protein and loss of the intact 74-kDa molecule as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CE transfer activity was unaffected (trypsin or chymotrypsin treatment) or only partially lost (V8 protease treatment). Analysis by molecular sieve chromatography showed that the CE transfer-active product of this proteolysis consisted of polypeptide fragments which remained associated, retaining the native molecular weight of CETP. These proteolyzed complexes were resistant to dissociation by dithiothreitol, 8 M urea, or delipidating agents. As shown by CE transfer activity, native CETP was found to possess a stable conformation which remained unchanged in buffers containing up to 4.5 M urea, or following exposure to even higher (8 M) urea concentrations. CETP polypeptides from bacterially expressed cDNA fragments were found to be catalytically inactive although they contained the epitope for an inhibitory anti-CETP monoclonal antibody and had emulsion binding properties similar to native CETP. Selected synthetic CETP peptides (including the peptide containing the inhibitory monoclonal antibody epitope) were also devoid of CE transfer activity. Thus, no evidence was found for an independently active subunit of the CETP. Together, the results indicate that the CETP possesses a distinct and highly stable tertiary structure which is required for CE transfer catalytic activity.

Effect of a neutralizing monoclonal antibody to cholesteryl ester transfer protein on the redistribution of apolipoproteins A-IV and E among human lipoproteins.

The effect of inhibiting cholesteryl ester transfer protein (CETP) on the in vitro redistribution of apolipoproteins(apo) A-IV and apoE among lipoproteins in whole plasma was studied in seven normal male subjects. Plasmas were incubated in the presence of a purified monoclonal antibody TP2 (Mab TP2) that neutralizes the activity of CETP. Mab TP2 had no effect on lecithin:cholesterol acyltransferase (LCAT) activity. Prior to and following a 6-h incubation at 37 degrees C in the presence of Mab TP2 or a control mouse myeloma immunoglobulin (IgG), plasmas were gel-filtered on Sephacryl S-300 and the distribution of apoA-IV and apoE among lipoproteins was determined by radioimmunoassay. Incubation (i.e., with active LCAT and CETP) increased the amount of apoA-IV associated with lipoproteins by 240%. When CETP activity was inhibited during incubation, the amount of apoA-IV that became lipoprotein-associated was significantly increased (315% of basal). Plasma incubation also caused a redistribution of apoE from high density lipoproteins (HDL) to larger lipoproteins (131% of basal); however, when CETP was inhibited, significantly greater amounts of apoE became associated with the larger particles (155% of basal). These effects were observed in all seven subjects. Increased movement of apoE from HDL to triglyceride-rich particles was not due to displacement by apoA-IV since loss of apoE from HDL was still observed when no movement of apoA-IV onto HDL occurred, such as during LCAT or combined LCAT and CETP inhibition. We speculate that low CETP activity (e.g., in species such as rats) may lead to an increased content of HDL apoA-IV and also to apoE enrichment of triglyceride-rich lipoproteins, augmenting their clearance.

Monoclonal antibodies to the Mr 74,000 cholesteryl ester transfer protein neutralize all of the cholesteryl ester and triglyceride transfer activities in human plasma.

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Three monoclonal neutralizing antibodies to the CETP were obtained by immunizing mice with purified CETP. The antibodies, each recognizing a similar epitope on CETP, caused parallel and complete immunotitration of plasma cholesteryl ester and triglyceride transfer activities but only partial inhibition of phospholipid transfer activity. Monoclonal immunoaffinity chromatography of plasma or its fractions showed complete removal of cholesteryl ester and triglyceride transfer activities but incomplete removal of phospholipid transfer activity. Sodium dodecyl sulfate gel electrophoresis and immunoblotting of the immunoaffinity-retained fractions showed that only the Mr 74,000 protein was immunoreactive. The results suggest that the previously characterized CETP accounts for all of the cholesteryl ester and triglyceride transfer activity in human plasma but only part of the phospholipid transfer activity.

Cholesteryl ester transfer protein is secreted by Hep G2 cells and contains asparagine-linked carbohydrate and sialic acid.

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Cholesteryl ester transfer activity was found to accumulate in the medium of cultured Hep G2 cells. The transfer activity was removed by immunoprecipitation with specific antibodies to the plasma CETP. Sodium dodecyl sulfate gel electrophoresis of immunoprecipitates prepared from the medium of cells pulsed with [35S]methionine revealed a broad specific band of protein of Mr 72,000 to 76,000; by contrast, immunoprecipitates of cellular homogenates showed a sharp specific band of Mr 58,000. The Mr 72,000 to 76,000 band disappears, concomitant with the appearance of lower Mr products, upon neuraminidase or glycopeptidase F treatment of medium immunoprecipitates or of purified CETP. The results indicate that liver cells have the capacity to synthesize and secrete CETP. The CETP peptide acquires asparagine-linked carbohydrate and sialic acid during intracellular processing.

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.

The topography of glycerophosphate acyltransferase in the transverse plane of the mitochondrial outer membrane.

In low ionic media, mitochondrial glycerophosphate acyltransferase was inhibited virtually completely within 15 min by the nonspecific proteases, proteinase K and subtilisin. In high ionic media, the mitochondrial enzyme was either not inhibited or was marginally inhibited by these proteases. Chymotrypsin and trypsin, regardless of the ionic strength of the medium, did not inhibit the acyltransferase. Substantial inhibition by proteinase K and subtilisin was observed in the high ionic media when the incubation was continued for 30 or 45 min. Adenylate kinase, an intermembrane enzyme, was not inhibited under any of the above conditions. These results demonstrate a cytosolic exposure of the mitochondrial acyltransferase. In a low ionic environment, when the outer membrane integrity was damaged either by gradually decreasing the tonicity of the medium or by stepwise addition of Triton X-100, either chymotrypsin or trypsin caused virtually parallel inhibition of glycerophosphate acyltransferase and adenylate kinase. A more direct approach in establishing the existence of protease-susceptible sites on the inner side of the outer membrane was taken by observing the inhibition of mitochondrial glycerophosphate acyltransferase and adenylate kinase in trypsinloaded right-side-out outer membrane vesicles incubated in the presence of externally located soybean trypsin inhibitor. The above results, taken together, suggest that mitochondrial glycerophosphate acyltransferase spans the transverse plane of the outer membrane.