Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein particles.

The mechanism of assembly of lipoprotein particles in the lumen of the endoplasmic reticulum is an important but poorly understood biological problem. A knowledge of this process is of great practical importance because possession of elevated levels of lipoproteins is one of the major risk factors for the development of atherosclerosis. This review describes a major advance in the delineation of the mechanisms involved in the assembly and secretion of apolipoprotein-B-containing lipoproteins: the demonstration of a requirement for microsomal triglyceride transfer protein.

Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.

Abetalipoproteinemia is a human genetic disease that is characterized by a defect in the assembly or secretion of plasma very low density lipoproteins and chylomicrons. The microsomal triglyceride transfer protein (MTP), which is located in the lumen of microsomes isolated from the liver and intestine, has been proposed to function in lipoprotein assembly. MTP activity and the 88-kilodalton component of MTP were present in intestinal biopsy samples from eight control individuals but were absent in four abetalipoproteinemic subjects. This finding suggests that a defect in MTP is the basis for abetalipoproteinemia and that MTP is indeed required for lipoprotein assembly.

An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues.

A triacylglycerol and cholesteryl ester transfer activity has been isolated from rat liver. After homogenization, the liver cells were subfractionated into the 10 000 X g pellet, microsomal fraction and postmicrosomal supernatant. Most of the transfer activity appeared to be associated with the microsomal fraction. Rough and smooth microsomes contained nearly equal transfer activities. When isolated microsomes were subject to proteolytic attack, the transfer activity was not inactivated, unless it had been released from the microsomes prior to proteolytic treatment. This indicates that the activity is probably located within the microsomal vesicles. Similar transfer activities were found in the intestinal mucosa of rats, whereas little or no activity was detected in the brain, heart, kidney, or plasma.

Crystallization and preliminary X-ray diffraction studies on the amino-terminal (receptor-binding) domain of human apolipoprotein E3 from serum very low density lipoproteins.

Human apolipoprotein E is a component of several classes of circulating plasma lipoproteins. In addition to binding lipids, this apolipoprotein, which is composed of two structural domains, mediates some lipoprotein-receptor interactions by binding to the low density lipoprotein receptor. The receptor-binding function, as well as some lipid-binding capability, is contained in the amino-terminal structural domain of apolipoprotein E. Thrombin-catalyzed hydrolysis of apolipoprotein E yields a fragment (residues 1 to 191) that has the same properties as, and seems to be a good model for, the amino-terminal domain. Crystals of this amino-terminal fragment suitable for high-resolution X-ray diffraction experiments have now been grown. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of a = 86.0 A, b = 40.9 A, and c = 53.3 A (1 A = 0.1 nm). This is the first human serum apolipoprotein to be crystallized.

A novel series of highly potent benzimidazole-based microsomal triglyceride transfer protein inhibitors.

A series of benzimidazole-based analogues of the potent MTP inhibitor BMS-201038 were discovered. Incorporation of an unsubstituted benzimidazole moiety in place of a piperidine group afforded potent inhibitors of MTP in vitro which were weakly active in vivo. Appropriate substitution on the benzimidazole ring, especially with small alkyl groups, led to dramatic increases in potency, both in a cellular assay of apoB secretion and especially in animal models of cholesterol lowering. The most potent in this series, 3g (BMS-212122), was significantly more potent than BMS-201038 in reducing plasma lipids (cholesterol, VLDL/LDL, TG) in both hamsters and cynomolgus monkeys.

Characterization of plasma lipoproteins in patients heterozygous for human plasma cholesteryl ester transfer protein (CETP) deficiency: plasma CETP regulates high-density lipoprotein concentration and composition.

To understand the role of human cholesteryl ester transfer protein (CETP) in plasma lipoprotein metabolism, CETP activity and mass levels, lipoprotein and apolipoprotein concentrations, and the size of high-density lipoprotein (HDL) were determined in 15 heterozygotes and compared with those of four homozygotes and 20 normolipidemic controls. Plasma CETP activity and mass were totally deficient in the four homozygotes for CETP deficiency, while heterozygotes had approximately half the level of normals. CETP activity positively correlated with CETP mass levels (r = .95, P less than .001). No significant difference was observed in the level of low-density lipoprotein (LDL)-cholesterol among the three groups. The concentration of HDL2-cholesterol in the heterozygotes was approximately twice as high as that in controls, while that of homozygotes was sixfold higher than that in controls. No significant difference in the HDL3-cholesterol level was observed among the three groups. The HDL2-cholesterol to HDL3-cholesterol ratio of homozygotes was sixfold higher than that of controls, while heterozygotes showed intermediate values between homozygotes and controls. Negative correlations were found between CETP activity and HDL2-cholesterol level (r = -.884, P less than .001) and between CETP mass and HDL2-cholesterol level (r = -.829, P less than .001). Plasma apolipoprotein (apo) A-I, C-III, and E were markedly increased in homozygotes, but the differences between normal and heterozygotes were not statistically significant. The HDL size of homozygotes, determined by high-performance liquid chromatography (HPLC), was large, whereas that of heterozygotes was intermediate between homozygotes and normals.(ABSTRACT TRUNCATED AT 250 WORDS)

A delayed-addition enzyme immunoassay for the relative cholesteryl ester transfer protein mass in patients with deficient plasma cholesteryl ester transfer activity.

The biochemical basis for the apparent deficiency of cholesteryl ester (CE) transfer activity was investigated in two unrelated subjects with markedly elevated high density lipoprotein-cholesterol (Atherosclerosis 1988; 70:7-12). Essentially no CE or triglyceride transfer activity was detected in the patients' plasma, utilizing four different lipid transfer assays. Using polyclonal antibodies raised against human plasma cholesteryl ester transfer protein (CETP), a delayed-addition enzyme immunoassay was developed to determine plasma CETP mass. CETP could not be detected with this assay in the plasma of the two subjects with transfer activity deficiency, indicating that the CE transfer activity deficiency in these subjects is due to the absence of plasma CETP. In addition, three hyperalphalipoproteinemic subjects with a partial deficiency of CE transfer activity had a reduced level of CETP mass. There was a good correlation between plasma CETP activity and mass levels. The principles of this immunoassay may be applicable to measure the mass levels of other proteins with catalytic activities.

Purification and characterization of microsomal triglyceride and cholesteryl ester transfer protein from bovine liver microsomes.

A lipid transfer protein was isolated from bovine liver. Following the release of soluble proteins from liver microsomes, the transfer protein was purified 75-fold to near homogeneity by a combination of DEAE-cellulose ion exchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. About 7% of the original activity was recovered. The purified fraction promoted the transfer of triglyceride, cholesteryl ester, phosphatidylcholine, and phosphatidylethanolamine. When the fractional rates of lipid transfer were compared, the transfer of apolar lipids was over 10 times faster than that of phospholipid. The purified transfer complex contained less than 5% lipid. No carbohydrate was detected. Electrophoresis of the purified protein on polyacrylamide gels under non-denaturing conditions showed a single band. Elution of protein from slices of unstained gels showed that lipid transfer activities coincided with the position of the protein band on the stained gel. When the purified protein was electrophoresed in the presence of SDS, two bands, accounting for more than 95% of the staining density, were observed with molecular weights at 58 000 and 88 000. The purified transfer protein eluted from a Sephadex G-200 column at a position corresponding to a protein with a molecular weight of 220 000, which probably represents a complex of two or more polypeptides. The purified transfer protein was activated by increasing NaCl concentrations up to about 100 mM. At higher NaCl concentrations the transfer activity decreased. Maximal transfer activities were observed at pH 7. The protein was inactivated by heating above 50 degrees C. The transfer rates were not greatly increased by changing the assay temperatures between 20 degrees C and 50 degrees C. These activity characteristics of the transfer protein were the same whether triglyceride or cholesteryl ester transfer activities were measured.

Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein.

The gene for the microsomal triglyceride transfer protein (MTP) is defective in subjects with the genetic disease abetalipoproteinemia, indicating that MTP is essential for the assembly of apolipoprotein B containing lipoproteins. In vitro, MTP is a lipid molecule binding protein that catalyzes lipid transport between membranes by a shuttle mechanism. In this study, the lipid binding properties of MTP were examined. MTP was incubated with donor phosphatidylcholine vesicles of varying neutral lipid composition. MTP was subsequently reisolated by ultracentrifugation, and MTP-bound lipid was quantitated. When the triolein content of the vesicles was increased up to 4 mol %, neutral lipid binding to MTP increased proportionately, while phosphatidylcholine binding appeared to remain constant around two molecules per MTP. Using phosphatidylcholine emulsions containing 60 mol % triolein as the donor particles resulted in only a slight increase in triolein binding to MTP. The highest triolein:MTP ratio observed was (0.20-0.25):1. Differences in the neutral and phospholipid binding properties of MTP were observed by measuring the transport of lipid from MTP to acceptor vesicles. Transport of triolein was rapid and complete, while phosphatidylcholine transport was biphasic, containing rapid and slow phases. These results indicated that MTP contains more than one class of lipid molecule binding site. Measurements of fluorescent lipid transport from donor vesicles to MTP supported this hypothesis. The transport of pyrene-labeled triglyceride from donor particles to MTP was rapid, while phosphatidylcholine transfer had fast and slow phases. From these data, we propose that MTP contains at least two distinct classes of lipid molecule binding sites that differ in function. The fast site or sites are responsible for lipid transport.

A triglyceride and cholesteryl ester transfer protein associated with liver microsomes.

An intracellular protein accelerates the transfer of triglyceride and cholesteryl ester. The fraction of phospholipid transferred was much less than for the less polar lipids. A rich source of this activity was obtained from low ionic strength washes of liver microsomes. The protein was partially purified by column chromatography on Bio-Gel A-5m and hydroxylapatite. The elution position of the transfer protein on gel filtration corresponds to a protein with a molecular weight of about 200,000. The isoelectric point of the partially purified protein is between pH 5.2 and 5.6. At each step of the purification the stimulation of triglyceride transfer was greater than that of cholesteryl ester.

Mechanism of microsomal triglyceride transfer protein catalyzed lipid transport.

The microsomal triglyceride transfer protein (MTP) is found in the lumen of microsomes isolated from liver and intestine. This protein, which catalyzes the transport of neutral lipids between membranes, appears to play an important role in the biogenesis of plasma very low density lipoproteins and chylomicrons. Enzyme kinetic studies were used to investigate the mechanism of MTP-catalyzed lipid transport. Initial rates of [14C]triolein and [14C]cholesteryl oleate transport from donor to acceptor small unilamellar vesicles were determined at varying donor and acceptor membrane concentrations. Results using two different kinetic analyses demonstrated lipid transport was best described by ping-pong bi-bi kinetics, indicating that MTP is a lipid binding protein which shuttles triglyceride and cholesteryl ester molecules between membranes. This model for lipid transport was supported by a fluorescent lipid binding assay in which MTP was able to extract pyrene-labeled cholesteryl ester from a vesicle. MTP-membrane interactions and lipid transport were regulated by membrane surface charge. Equilibrium gel filtration chromatography demonstrated MTP has a higher affinity for neutrally charged membranes than negatively charged membranes. In agreement with the membrane binding studies, MTP-mediated lipid transfer was inhibited by increasing the concentration of negatively charged phospholipid molecules in donor membranes.

Effect of dipalmitoylphosphatidylcholine vesicle curvature on the reaction with human apolipoprotein A-I.

Large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) were prepared by sonication and were fractionated by gel filtration on Sepharose Cl-2B in the size range from 180- to 380-A Stokes radii. Negatively stained electron micrographs of these preparations indicated the presence of unilamellar, spheroidal structures of the expected size. Fluorescence polarization of diphenylhexatriene, dissolved in the vesicles, revealed progressively broader phase transitions, shifted to lower temperatures for vesicles of decreasing sizes. The fractionated unilamellar vesicles and multilamellar vesicles of DPPC were reacted with human apolipoprotein A-I at 41 degrees C for periods from 1 to 120 h. The reaction mixtures were then passed through a Bio-Gel A-5m column to separate unreacted lipid vesicles and protein from micellar complexes of DPPC with apolipoprotein A-I. Smaller vesicles were much more reactive than larger vesicles or multilamellar vesicles with the apolipoprotein. This difference in reactivity was explained by the increasing bilayer curvature of smaller vesicles which changes the packing of DPPC molecules in the bilayer and facilitates its penetration by the apolipoprotein.

The molecular basis of abetalipoproteinemia.

Abetalipoproteinemia is a recessive genetic disease in humans characterized by the virtual absence of apolipoprotein (apo)B and apoB-containing lipoproteins in plasma. Microsomal triglyceride transfer protein (MTP), a resident lipid transfer protein within the endoplasmic reticulum of hepatocytes and enterocytes, has been shown to be absent in enterocytes from subjects with this disease. MTP is a heterodimer of a unique large subunit and protein disulfide isomerase. It has been demonstrated that the absence of MTP in abetalipoproteinemia is secondary to mutations in the gene for the large subunit of MTP. Thus, mutations in the gene for the large subunit of MTP are a cause of abetalipoproteinemia, which indicates that the MTP is a necessary component for the assembly and secretion of apoB-containing lipoproteins from the liver and intestine.

Microsomal triglyceride transfer protein (MTP) regulation in HepG2 cells: insulin negatively regulates MTP gene expression.

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apoB-containing lipoproteins. In this study, four factors that modulate lipid and lipoprotein metabolism were tested for their ability to regulate MTP levels in HepG2 cells. Of the factors tested, only insulin (> or = 10(-9) M), and high concentrations of glucose (> 30 mM) were found to decrease MTP large subunit mRNA levels. Oleate and glucagon had no effect on MTP mRNA levels. The insulin effect was dose- and time-dependent and was mediated through the insulin receptor. In addition, insulin also decreased protein disulfide isomerase (the small subunit of MTP) mRNA levels, although to a lesser extent. Due to the slow turnover rate of MTP (t1/2 = 4.4 days), short-term insulin treatment (24 h) did not change MTP activity levels, indicating that the regulation of MTP mRNA levels by insulin is unrelated to insulin's acute inhibition of apoB secretion in HepG2 cells. In summary, MTP mRNA levels are acutely regulated by insulin in HepG2 cells; however, sustained changes in MTP mRNA levels would be required to affect MTP protein levels.

Human microsomal triglyceride transfer protein large subunit gene structure.

Microsomal triglyceride transfer protein (MTP) is a heterodimer consisting of the multifunctional enzyme protein disulfide isomerase and a unique, large 97-kDa subunit. MTP is required for the assembly and secretion of very low density lipoproteins and chylomicrons by the liver and intestine, respectively. In vitro, MTP catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between phospholipid surfaces. We have characterized the gene encoding the large subunit of human MTP. It contains 18 exons and spans approximately 55-60 kb. Fluorescent in situ hybridization localized this gene to band 4q24 of chromosome 4. A (CA)n repeat polymorphic marker, which may be useful for investigating a link between the MTP gene and genetic defects in lipid metabolism, was identified in intron 10. Sequence analysis of the 5' flanking region of the gene revealed potential sites which may bind transcriptional factors and control MTP expression.

Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains.

The stability and structure of human apolipoprotein (apo) E3 in aqueous solution were investigated by guanidine HCl denaturation and limited proteolysis. The guanidine HCl denaturation curve, as monitored by circular dichroism spectroscopy, was biphasic; the two transition midpoints occurred at 0.7 and 2.5 M guanidine HCl, indicating that there are stable intermediate structures in the unfolding of apoE. Limited proteolysis of apoE with five enzymes demonstrated two proteolytically resistant regions, an amino-terminal domain (residues 20-165) and a carboxyl-terminal domain (residues 225-299). The region between them was highly susceptible to proteolytic cleavage. Because of their similarity to the proteolytically resistant regions, the amino-terminal (residues 1-191) and carboxyl-terminal (residues 216-299) thrombolytic fragments of apoE were used as models for the two domains. Guanidine HCl denaturation of the carboxyl- and amino-terminal fragments gave transition midpoints of 0.7 and 2.4 M guanidine HCl, respectively. The results establish that the two domains identified by limited proteolysis correspond to the two domains detected by protein denaturation experiments. Therefore, the thrombolytic fragments are useful models for the two domains. The free energies of denaturation calculated from the denaturation curves of intact apoE or the model domains were approximately 4 and 8-12 kcal/mol for the carboxyl- and amino-terminal domains, respectively. The value for the carboxyl-terminal domain is similar to those of previously characterized apolipoproteins, whereas the value for the amino-terminal domain is considerably higher and resembles those of soluble globular proteins. These studies suggest that, in aqueous solution, apoE is unlike other apolipoproteins in that it contains two independently folded structural domains of markedly different stabilities: an amino-terminal domain and a carboxyl-terminal domain, separated by residues that may act as a hinge region.

The role of the microsomal triglygeride transfer protein in abetalipoproteinemia.

The microsomal triglyceride transfer protein (MTP) is a dimeric lipid transfer protein consisting of protein disulfide isomerase and a unique 97-kDa subunit. In vitro, MTP accelerates the transport of triglyceride, cholesteryl ester, and phospholipid between membranes. It was recently demonstrated that abetalipoproteinemia, a hereditary disease characterized as an inability to produce chylomicrons and very low-density lipoproteins in the intestine and liver, respectively, results from mutations in the gene encoding the 97-kDa subunit of the microsomal triglyceride transfer protein. Downstream effects resulting from this defect include malnutrition, very low plasma cholesterol and triglyceride levels, altered lipid and protein compositions of membranes and lipoprotein particles, and vitamin deficiencies. Unless treated, abetalipoproteinemic subjects develop gastrointestinal, neurological, ophthalmological, and hematological abnormalities.