NMR structure of the sterol carrier protein-2: implications for the biological role.

The determination of the NMR structure of the sterol carrier protein-2 (SCP2), analysis of backbone (15)N spin relaxation parameters and NMR studies of nitroxide spin-labeled substrate binding are presented as a new basis for investigations of the mode of action of SCP2. The SCP2 fold is formed by a five-stranded beta-sheet and four alpha-helices. Fatty acid binding to a hydrophobic surface area formed by amino acid residues of the first and third helices, and the beta-sheet, which are all located in the polypeptide segment 8-102, was identified with the use of the spin-labeled substrate 16-doxylstearic acid. In the free protein, the lipid-binding site is covered by the C-terminal segment 105-123, suggesting that this polypeptide segment, which carries the peroxisomal targeting signal (PTS1), might be involved in the regulation of ligand binding.

Crystallization and initial X-ray analysis of rabbit mature sterol carrier protein 2.

Sterol carrier protein 2 (SCP2) is a basic intracellular protein which facilitates the in vitro intermembrane transfer of cholesterol, phospholipids and glycolipids. SCP2 was expressed in Escherichia coli, purified to apparent electrophoretic homogeneity and crystallized. Single crystals were obtained by hanging-drop vapour diffusion using ammonium sulfate as precipitant. These crystals belong to space group P4(1)2(1)2 or its enantiomorph, with unit-cell parameters a = b = 57.5, c = 86.5 A, and have one molecule in the crystallographic asymmetric unit. Intensity data to 1.8 A resolution were collected from native SCP2 crystals using synchrotron radiation, were processed and scaled with an R(linear) = 4.9%.

In pre-sterol carrier protein 2 (SCP2) in solution the leader peptide 1-20 is flexibly disordered, and residues 21-143 adopt the same globular fold as in mature SCP2.

The preform of the rabbit sterol carrier protein 2 (pre-rSCP2) was cloned, the uniformly 15N-labelled protein expressed in Escherichia coli and studied by three-dimensional 15N-resolved nuclear magnetic resonance spectroscopy. In spite of its low solubility in aqueous solution of only approximately 0.3 mM, sequential 15N and 1H backbone resonance assignments were obtained for 105 out of the 143 residues. From comparison of the sequential and medium-range nuclear Overhauser effects (NOEs) in the two proteins, all regular secondary structures previously determined in mature human SCP2 (hSCP2) [Szyperski et al. (1993) FEBS Lett. 335: 18-26] were also identified in pre-rSCP2. Near-identity of the backbone 15N and 1H chemical shifts and 1:1 correspondence of 24 long-range NOEs to backbone amide groups in the two proteins show that the residues 21-143 adopt the same globular fold in pre-rSCP2 and mature hSCP2. The N-terminal 20-residue leader peptide of pre-rSCP2 is flexibly disordered in solution and does not observably affect the conformation of the polypeptide segment 21-143.

Identification of a receptor mediating absorption of dietary cholesterol in the intestine.

Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily.

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd approximately 0.37 microM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLDbeta, from Arabidopsis.

A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and nanomolar concentrations of calcium, has been identified in Arabidopsis. This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLDbeta for this PIP2-dependent PLD and PLDalpha for the previously characterized PIP2-independent PLD that requires millimolar Ca2+ for optimal activity. The PLDbeta cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLDbeta cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLDbeta, but not PLDalpha, activity. The activity of the expressed PLDbeta is dependent on PIP2 and submicromolar amounts of Ca2+, inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLDbeta is evolutionarily divergent from PLDalpha and that its N terminus contains a regulatory Ca2+-dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.

Structural heterogeneity of phospholipase D in 10 dicots.

The occurrence of multiple forms of phospholipase D (EC 3.1.4.4) was investigated in different tissues of castor bean (Ricinus communis) and in other plant species. Phospholipase D variants were resolved by nondenaturing and isoelectric focusing polyacrylamide gel electrophoresis and detected by immunoblotting using anti-phospholipase D antibodies and by enzyme activity assay. Three phospholipase D variants were produced differentially in the roots, endosperm, cotyledons, and hypocotyl of 5-day postgermination seedlings of castor bean. Furthermore, different patterns of phospholipase D variants were found in the different regions of hypocotyl (elongated and hook). Multiple phospholipase D forms were found in florets of cauliflower and broccoli, leaves of cabbage, celery, tomato, and potato, and alfalfa sprouts, suggesting that structural heterogeneity of phospholipase D occurs widely in plants.

Multiple Forms of Phospholipase D following Germination and during Leaf Development of Castor Bean.

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were identified and partially characterized in endosperm of germinated seeds and leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel electrophoresis, isoelectric focusing, and size-exclusion chromatography. PLD was detected with both a PLD activity assay and immunoblots with PLD-specific antibodies. There were three major forms of PLD, designated types 1, 2, and 3, based on their mobility during nondenaturing polyacrylamide gel electrophoresis. Molecular masses of the PLD variants were estimated at 330, 230, and 270 kD for the types 1, 2, and 3, respectively. Isoelectric points of the native type 1, 2, and 3 PLDs were approximately 6.2, 4.9, and 4.8. Under the in vitro assay conditions used, the three forms of PLD exhibited the same substrate specificity, hydrolyzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but not phosphatidylserine (PS) and phosphatidylinositol (PI). The three forms of PLD differed in their substrate preferences, and the order of activities was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG = PC. The Km values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM, respectively. These PLDs were expressed differentially following seed germination and during leaf development. Type 1 was found in the early stages of seedling growth and in young leaves, type 2 was present in all the tissues and growth stages examined, and type 3 was expressed in senescent tissues. The PLDs shifted from largely cytosolic to predominantly membrane-associated forms during leaf development. The present studies demonstrate the structural heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of multiple molecular forms of PLD in cellular metabolism is discussed.

Purification and immunological analysis of phospholipase D from castor bean endosperm.

Phospholipase D (EC 3.1.4.4) has been implicated in diverse cellular processes, but its physiological role is not well established in plants. In order to develop immunological and molecular biology approaches to address the problem, we report here the immunological analysis and N-terminal amino acid sequence of a cytosolic phospholipase D from castor bean (Ricinus communis L.). The enzyme was purified to apparent homogeneity from germinating castor bean endosperm. The specific activity of the purified enzyme was enhanced by approximately 670-fold with an overall yield of 4%. Its molecular mass was estimated at 92 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this enzyme was KLVENIEETVGFGKG. Polyclonal antibodies were raised against the purified enzyme. The antibodies inhibited the activity of transphosphatidylation more than that of hydrolysis of phospholipase D. The differential effect on the two activities of this enzyme implies that different active sites on this enzyme may be involved in the two reactions. Immunoblot analyses showed that the amounts of phospholipase D protein relative to the total endosperm proteins increased during the first 5 days of germination. The antibodies cross-reacted to proteins from several tested plant species, and those proteins had molecular masses similar to that of castor bean phospholipase D. These results indicate that the expression of phospholipase D in castor bean changes according to growth stages and that phospholipase D enzymes of different plant species are structurally related.