Nonspecific lipid transfer protein (sterol carrier protein-2) is located in rat liver peroxisomes

Intracellular localization of nonspecific lipid transfer protein (nsLTP) in rat hepatocytes was investigated by immunoblot analysis of the subcellular fractions and immunoelectron microscopy, using affinity-purified antibody against nsLTP. Immunoblot analysis showed that the protein exists in the peroxisomal and cytosolic fractions. Further study indicated that nsLTP exists in the soluble subfraction of the peroxisomes. Immunoelectron microscopic observation revealed that nsLTP is highly concentrated in the matrices of the peroxisomes. From these results, we concluded that nsLTP mainly exists in the matrix of the peroxisomes. The role of nsLTP is discussed.     

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 micrograms/mg) than normal liver peroxisomes (8 micrograms/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.     

A mass spectrometric study of the structure of sterol carrier protein SCP2 from rat liver

The amino acid sequence of Sterol Carrier Protein2 (SCP2) isolated from rat has been investigated. Using a novel mass spectrometric mapping approach, the C-terminus was found to be extended beyond the previously published sequence. Carbohydrate analysis of SCP2 samples suggest the presence of tightly bound mannose oligosaccharide of 5-10 residues, although probably not in a glycoprotein linkage.     

The non-specific lipid transfer protein (sterol carrier protein 2) from rat and bovine liver

The non-specific lipid transfer protein (nsL-TP) purified from rat and bovine liver accelerates the transfer of all common diacylglycerophospholipids, cholesterol as well as glycosphingolipids and gangliosides between membranes. These proteins have molecular weights in the order of 14 500 and are highly basic (isoelectric points between 8.5 and 9.5). The primary structure of nsL-TP from bovine liver has been elucidated yielding a single polypeptide chain of 121 aminoacid residues. The protein contains one cysteine residue, essential for transfer activity, a single tryptophan residue and lacks histidine, arginine and tyrosine residues. Rat liver nsL-TP was found to be identical to sterol carrier protein 2, stimulating the microsomal conversion of intermediates between lanosterol and cholesterol. Evidence was presented that nsL-TP binds cholesterol, suggesting that it acts as a carrier. On the other hand, failure to bind phospholipids disagrees with this proposed mode of action. A sensitive enzyme immunoassay was developed to determine levels of nsL-TP in rat tissues. By use of this assay, nsL-TP was found to be most prominently present in liver and intestinal mucosa (0.78 and 0.46 microgram nsL-TP per mg protein in 105 000 X g supernatant, respectively). Subfractionation studies showed that approx. 70% of nsL-TP was present in the membrane-free cytosol. However, application of an immunosorbent-purified antibody and protein A-linked gold particles to rat liver slices demonstrated a concentration of label over the peroxisomes. By way of immunoblotting it was shown that nsL-TP was absent from peroxisomes and that the immunoreactive material was a protein of mol. wt. 58 000. nsL-TP is capable of mediating net transfer of cholesterol to membranes, deficient in this lipid. Under such conditions of net transfer, nsL-TP stimulated the microsomal esterification of cholesterol and its conversion to pregnenolone by adrenal mitochondria. Levels of nsL-TP in Reuber H35 hepatoma cells was six per cent of that found in rat hepatocytes. This very low level of nsL-TP had no effect on de novo cholesterol biosynthesis and intracellular cholesterol esterification. These results raise doubts as to whether nsL-TP has a function in in situ cholesterol metabolism.     

Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes.

Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.     

Primary sequence and structural analysis of sterol carrier protein 2 from rat liver: homology with immunoglobulins

Sterol carrier protein 2 (SCP2) is involved in the later steps of cholesterol biosynthesis and in the intracellular transport of cholesterol. In the present investigation, the amino acid sequence of SCP2 from rat liver has been determined. It is a single polypeptide chain with 122 amino acid residues. Secondary structure prediction indicates an amphipathic alpha-helix region for residues 21-34 and antiparallel beta-sheet structure for residues 35-95. A major finding is the significant homology which exists over approximately 80 residues between SCP2 and the variable domains of the heavy chain of immunoglobulin G.     

New aspects of sterol carrier protein 2 (Nonspecific lipid-transfer protein) in fusion proteins and in peroxisomes

Sterol carrier protein 2 (SCP2) is a 13-kDa peroxisomal protein, identical to nonspecific lipidtransfer protein, and stimulates various steps of cholesterol metabolism in vitro. Although the name is reminiscent of acyl carrier protein, which is involved in fatty acid synthesis, SCP2 does not bind to lipids specifically or stoichiometrically. This protein is expressed either as a small precursor or as a large fusion (termed SCPx) that carries at its C-terminal the complete sequence of SCP2. SCPx exhibits 3-oxoacyl-CoA thiolase activity, as well as sterol-carrier and lipid-transfer activities. The N- and C-terminal parts of SCPx are similar to the nematode protein P-44 and the yeast protein PXP-18, respectively. P-44, which has no SCP2 sequence, thiolytically cleaved the side chain of bile acid intermediate at a rate comparable to that of SCPx. This, together with the properties of other fusions with SCP2-like sequence, suggests that the SCP2 part of SCPx does not play a direct role in thiolase reaction. PXP-18, located predominantly inside peroxisomes, is similar to SCP2 in primary structure and lipid-transfer activity, and protects peroxisomal acyl-CoA oxidase from thermal denaturation. PXP-18 dimerized at a high temperature, formed an equimolar complex with the oxidase subunit, and released the active enzyme from the complex when the temperature went down. This article attempts to gain insight into the role of SPC2, and to present a model in which PXP-18, a member of the SCP2 family, functions as a molecular chaperone in peroxisomes.     

Isolation and characterization of peroxisomes from the liver of normal untreated rats

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482-513] for the isolation of peroxisomes from rat liver involves the use of Triton WR-1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay-rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well-preserved peroxisomes from normal untreated animals. The livers were homogenized in buffered 0.25 M sucrose and a slightly modified 'light mitochondrial fraction' was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12-1.26 g/cm3) and subjected to an integrated force of 1.252 X 10(6) X (g X min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide-insensitive lipid beta-oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3'-diaminobenzidine-stained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.     

Characterization of a cDNA encoding rat sterol carrier protein2

Sterol carrier protein2 (SCP2) is a 13.2-kD protein that is thought to be involved in the intracellular transport of cholesterol. Using synthetic oligonucleotides based on the protein sequence of SCP2, a clone (SP43) was isolated from a rat liver cDNA library. The DNA sequence revealed that the cDNA could encode a polypeptide of 273 amino acids (28.9 kD) or 143 amino acids (15.3 kD) in which the carboxy-terminal 123 amino acids are identical to the SCP2 protein. RNA blot hybridization revealed that a variety of rat tissues contain a homologous RNA of a size similar to SP43 (approximately 1.5 kb). Levels of SCP2 mRNA increased in parallel with cytochrome P450scc mRNA in the immature gonadotropin-primed rat ovary. The isolation of a cDNA clone encoding SCP2 will facilitate studies on its role in cholesterol metabolism.     

Isolation of peroxisomes from rat liver using sucrose and Percoll gradients

Peroxisomes were isolated from the livers of both control and clofibrate-treated rats. Two procedures, one with a sucrose gradient, and a second with Percoll gradients, were utilized. The Percoll procedure allowed contamination of the isolated peroxisome fraction on protein basis, by lysosomes (8%), by mitochondria (5%) and by microsomes (2%). The peroxisome fraction isolated by the sucrose gradient showed no significant contamination with mitochondria, but the fraction contained 13% microsomes. In addition to established peroxisomal enzymes, the isolated peroxisomes also contained cytochrome b5, NADH-cytochrome c reductase and NADPH-isocitrate dehydrogenase. The peroxisomal membranes were also separated from the content, and they were found to have a relatively high phospholipid/protein ratio (0.55).     

Properties of peroxisomal 3-ketoacyl-coA thiolase from rat liver

Peroxisomal 3-ketoacyl-CoA thiolase has a molecular weight of 89,000 and consists of 2 polypeptide chains of identical size. The enzyme has no interchain disulfide bonds and is reversibly dissociated to an inactive monomer in the cold. Mitochondrial 3-ketoacyl-CoA thiolase and acetoacetyl-CoA specific thiolase have molecular weights of 154,000 and 149,000, respectively. They each consist of 4 polypeptide chains of identical size. Peroxisomal thiolase and mitochondrial 3-ketoacyl-CoA thiolase operate by a ping-pong mechanism. The catalytic properties, including substrate specificity, of the peroxisomal enzyme were compared to those of mitochondrial 3-ketoacyl-CoA thiolase.     

Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes

Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using affinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver cells carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.     

Nonspecific lipid transfer protein (sterol carrier protein-2) defective in patients with deficient peroxisomes

The biosynthesis and intracellular localization of nonspecific lipid transfer protein (nsLTP) in control human subjects and in patients with peroxisome-deficient disorders were investigated. The molecular mass of human nsLTP was indistinguishable from that of rat nsLTP (13 kDa) by immunoblot analysis. Intracellular localization was identical with that of catalase, a marker enzyme of peroxisomal matrix, by a double immunofluorescence study. The nsLTP was deficient in liver tissues or fibroblasts from patients with peroxisome-deficient disorders such as Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Pulse-chase experiments showed that nsLTP was synthesized as a large precursor in both the control and Zellweger fibroblasts. However, the processing to the 13 kDa mature protein was disturbed and the degradation was rapid in Zellweger fibroblasts. After somatic cell fusion using Zellweger fibroblasts from different genetic groups, the processing was normalized. These results suggest that the biosynthesis and localization of human nsLTP are similar to those of rat nsLTP and that the defect of nsLTP in peroxisome-deficient disorders is a phenomenon secondary to an abnormal transport mechanism of peroxisomal proteins. The defect of nsLTP may play an important role in metabolic disturbances in bile acid synthesis and steroidogenesis in peroxisome-deficient disorders.     

The primary structure of the nonspecific lipid transfer protein (sterol carrier protein 2) from bovine liver

The primary structure of the nonspecific lipid transfer protein (sterol carrier protein 2) from bovine liver has been determined. The protein consists of a single polypeptide chain of 121 amino acid residues with serine as the amino-terminal and alanine as the carboxy-terminal residue. The protein contains one single cysteine and tryptophan residue and lacks tyrosine, histidine and arginine.     

The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membrane-free cytosol as compared to 19% in the adrenal gland. In the latter tissue, the transfer protein is mainly found in the lysosomal/peroxisomal and the microsomal fraction at a level of 1093 and 582 ng per mg total protein, respectively (i.e., 17% and 35% of the total), and to a lesser extent in the mitochondrial fraction (11% of the total). Of all the membrane fractions isolated, the microsomal fraction from the liver and the mitochondrial fraction from the adrenal gland have the lowest levels of the transfer protein (i.e., 168 ng and 126 ng per mg total protein, respectively). These low levels correlate poorly with the active role proposed for this transfer protein in the conversion of cholesterol into bile acids and steroid hormones in these fractions. Using immunoblotting, it was demonstrated that in addition to the transfer protein (14 kDa) a cross-reactive 58 kD protein was present in the supernatant and the membrane fractions of both tissues. Cytochemical visualization in adrenal tissue with specific antibodies against the nonspecific lipid transfer protein showed that immunoreactive protein(s) were present mainly in the peroxisome-like structures.     

Localization of nonspecific lipid transfer protein (nsLTP = sterol carrier protein 2) and acyl-CoA oxidase in peroxisomes of pigment epithelial cells of rat retina.

We investigated the localization of nonspecific lipid transfer protein (nsLTP) in rat retina, especially in the pigment epithelial (RPE) cells, by the avidin-biotin-peroxidase complex method on cryosections for light microscopy and by the cryoimmunogold method for electron microscopy. Light microscopic observation revealed that the RPE, inner segment layer, nerve fiber layer, and Müller cells contain nsLTP. In the RPE cells gold particles were exclusively concentrated in the small peroxisomes (microperoxisomes; 0.1-0.3 micron in diameter), which were identified by double staining using anti-nsLTP and anti-catalase antibodies. In the peroxisomes gold particles were distributed homogeneously in the matrices and no preferential binding to the limiting membrane was observed. Acyl-CoA oxidase was also localized in the matrices of the peroxisomes. We suggest that the peroxisomes in RPE cells play important roles in the metabolism of lipids of the outer segment disk membranes, especially in the beta-oxidation of polyunsaturated long-chain and very long-chain fatty acids, such as docosahexaenoic acid which is composed of approximately one third of fatty acids in the disk membranes.     

Phospholipids, sterol carrier protein2 and adrenal steroidogenesis

Rat adrenocortical cells and preparations of plasma membrane and mitochondria have been employed to assess the effects of phospholipids and of sterol carrier protein2 (SCP2) on specific aspects of adrenal steroidogenesis. With intact cells, liposomal dispersions of cardiolipin caused significant stimulation of corticosterone output, while preparations of phosphatidylcholine, phosphatidylinositol, or the 4'-phosphate and the 4',5'-diphosphate derivatives of phosphatidylinositol were without effect. With the adrenal plasma membrane preparation, none of the added phospholipids affected either sodium fluoride or ACTH-responsive adenylate cyclase activity. With intact mitochondria, only cardiolipin, among the various phospholipids, tested, caused a concentration-dependent stimulation of pregnenolone production. However, even at the highest concentration of cardiolipin tested (500 microM), the stimulatory effect was only half that observed with 0.7 microM SCP2, and the two effectors were not synergistic. SCP2 caused a redistribution of cholesterol from mitochondrial outer to inner membranes, while cardiolipin, which is an activator of cytochrome P-450scc, had no effect on distribution of mitochondrial membrane cholesterol.