Jan Westerman

Transfer of cholesterol and oxysterol derivatives by the nonspecific lipid transfer protein (sterol carrier protein 2): a study on its mode of action.

The nonspecific lipid transfer protein (nsLTP) facilitates the transfer of both phospholipids and cholesterol between membrane interfaces. In this study, we have investigated the transport of 14C-labelled cholesterol, 7-ketocholesterol, 7 alpha-hydroxycholesterol and 25-hydroxycholesterol from a mixed lipid monolayer at the air/water interface to acceptor vesicles in the subphase. In the absence of nsLTP the transport of cholesterol was virtually nil, whereas the spontaneous transport of the oxysterol derivatives increased in the order 7-ketosterol less than 7 alpha-hydroxycholesterol less than 25-hydroxycholesterol. In the presence of nsLTP, the transport of both cholesterol and the oxysterol derivatives was greatly enhanced; the highest rate of transport was observed for 25-hydroxycholesterol. In the absence of vesicles, binding of cholesterol and of 25-hydroxycholesterol from the monolayer to nsLTP was negligible. Similarly, nsLTP did not bind cholesterol from radiolabeled bovine heart mitochondria under conditions where it stimulated the transfer of cholesterol to vesicles. In agreement with this failure to bind, nsLTP was unable to carry cholesterol between two separate monolayers. From the monolayer experiments it became apparent that nsLTP is highly surface-active. Measurement of the transport of cholesterol and of oxysterol derivatives by the monolayer-vesicles assay and of a series of pyrene-labeled phosphatidylcholine species by the fluorescent transfer assay showed a high correlation between the spontaneous and the nsLTP-mediated lipid transport. This supports the notion that nsLTP lowers the energy barrier for the lipid monomer-membrane interface equilibration process. In view of the above observations, we propose that nsLTP may facilitate the transfer of lipids by being part of a transient collisional complex between donor and acceptor membrane.

The complete primary structure of phospholipase A2 from human pancreas.

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.

Structure of apo‐phosphatidylinositol transfer protein α provides insight into membrane association

Phosphatidylinositol transfer protein α (PITPα) is a ubiquitous and highly conserved protein in multicellular eukaryotes that catalyzes the exchange of phospholipids between membranes in vitro and participates in cellular phospholipid metabolism, signal transduction and vesicular trafficking in vivo. Here we report the three‐dimensional crystal structure of a phospholipid‐free mouse PITPα at 2.0 Å resolution. The structure reveals an open conformation characterized by a channel running through the protein. The channel is created by opening the phospholipid‐binding cavity on one side by displacement of the C‐terminal region and a hydrophobic lipid exchange loop, and on the other side by flattening of the central β‐sheet. The relaxed conformation is stabilized at the proposed membrane association site by hydrophobic interactions with a crystallographically related molecule, creating an intimate dimer. The observed open conformer is consistent with a membrane‐bound state of PITP and suggests a mechanism for membrane anchoring and the presentation of phosphatidylinositol to kinases and phospholipases after its extraction from the membrane. Coordinates have been deposited in the Protein Data Bank (accession No. 1KCM).

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.

High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2)

Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to beta-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the beta-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible.

Complete exchange of phospholipids between microsomes and plasma lipoproteins mediated by liver phospholipid-exchange proteins

It is known that cholesterol and phospholipids are in dynamic equilibrium and spontaneously exchange both in vitro and in vivo between individual classes of plasma lipoproteins [l-4] . Spontaneous transfer of phospholipids has also been shown between lipoproteins and erythrocytes [5],mitochondria [6] ,liver microsomes [7] , liver slices [8] and cells in culture [9]. In these examples of phospholipid exchange it is presumed that transfer occurs upon formation of a collision complex. Evidence for a protein-facilitated transfer of lipoprotein phospholipids in serum is controversial [7,10]. On the other hand, eukaryotic cells contain proteins designated as phospholipid exchange proteins (PLEP) that do facilitate the transfer of phospholipids from one membrane to another [ 1 I-131 . A purified PLEP from beef liver shows a highly specific affinity for phosphatidylcholine (PC-PLEP [ 141). Two proteins have been isolated from bovine cerebral cortex which stimulate both the transfer of phosphatidylinositol (PI) and PC (PI-PLEP [IS] ). Recently, exchange proteins have been isolated from rat liver [ 161 and hepatoma [ 171 which lack specificity in the transfer of phospholipid. The purpose of the present study was to determine whether and if so, to what extent, purified PCand PI-exchange proteins catalyze the transfer of phospholipids between rat liver microsomes and the individual classes of lipoproteins. It will be shown that the protein-mediated transfer of labeled PC and PI from microsomes to very low density lipoproteins (VLDL), LDL and HDL was 5-lo-fold higher than

Human erythrocyte membranes are fluid down to −5°C

Abstract This first observation of the deuterium nuclear magnetic resonance ( 2 H-NMR) spectrum of phospholipid molecules incorporated into intact human erythrocyte ghosts shows that the liquid crystalline phase is stable down to a temperature of −5°C. The quality of the 3 H-NMR spectra indicate that it is now possible to carry out clinical studies of erythrocyte membranes using the techniques employed in this study.

Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli

The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction. The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E. coli strain. It was estimated that 5% of the total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography. Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2. These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2. The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver

The phospholipases A2, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate, isobutanol or dioxane to the native protein, under conditions where delipidation did not occur, greatly enhanced the hydrolytic action of the phospholipases. From these results it is concluded that phosphatidylcholine may be buried in the protein molecule.

The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding.

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.