Phosphatidylcholine Transfer Activity Research Articles

Structural basis of ligand recognition and transport by Sfh2, a yeast phosphatidylinositol transfer protein of the Sec14 superfamily.

Sec14-like phosphatidylinositol transfer proteins (PITPs) are involved in lipid metabolism and phosphatidylinositol 4-phosphate signaling by transporting phosphatidylinositol (PI) and a secondary ligand between the organellar membranes in eukaryotes. Yeast Sfh2 is a PITP that transfers PI and squalene without phosphatidylcholine transfer activity. To investigate the structural determinants for ligand specificity and transport in Sfh2, crystal structures of Sfh2 in complex with PI and squalene were determined at 1.5 and 2.4 Å resolution, respectively. The inositol head group of PI is recognized by highly conserved residues around the pocket entrance. The acyl chains of PI bind into a large hydrophobic cavity. Squalene is accommodated in the bottom of the cavity entirely by hydrophobic interactions. The binding of PI and squalene are mutually exclusive due to their overlapping binding sites, correlating with the role in lipid exchange. The binding mode of PI is well conserved in Sfh family proteins. However, squalene binding is unique to the Sfh2 homolog due to the specific hydrophobic residues forming a shape-complementary binding pocket. Recombinant apo Sfh2 forms a homodimer in vitro by the hydrophobic interaction of the gating α10-α11 helices in an open conformation. Ligand binding closes the lid and dissociates the dimer into monomers. This study reveals the structural determinants for the recognition of the conserved PI and a secondary ligand, squalene, and provides implications for the lipid-transfer function of Sfh2.

Lipid Binding Properties of Sec14‐like Homologues in the Yeast Saccharomyces cerevisiae

Lipids are hydrophobic molecules with several key functions in the cell. Due to the thermodynamic constraints placed upon lipids, they cannot freely diffuse across the cytosol. Their transport between two membrane compartments can be facilitated by lipid transfer proteins (LTPs). One of the most studied families of LTPs is S. cerevisiae’s Sec14p family. Proteins belonging to this group contain cellular retinaldehyde binding – triple functional (CRAL-TRIO) domain. In S. cerevisiae there are six members of this family, the founding member Sec14p and five of its homologues termed Sfh (Sec fourteen homologue) 1-5. The Sec14p has been extensively studied and it was determined that it possesses phosphatidylinositol (PI) and phosphatidylcholine (PC) binding and transfer activities (Bankaitis et al., 1990). Many of Sfh proteins have been implicated in various processes in cell, however, their exact roles remain to be discovered. Lipid binding and transfer by Sfh proteins were intensively studied (Li et al., 2000; Maeda et al., 2013; Tripathi et al., 2019; Holic et al., 2014; Pevalova et al., 2019) but some key questions remain open. Our objective is to determine lipids bound to S. cerevisiae’s Sec fourteen homologue proteins. For this we used HL-60 in situ lipid-binding assay, which employs co-incubation of permeabilized human HL-60 cells with recombinant protein of interest. Afterwards, the proteins are re-purified using affinity chromatography. The lipids extracted from re-isolated proteins are separated using thin layer chromatography and identified. Our results showed that Sfh1 binds PC and PI with approximately the same affinity. The remaining Sfh proteins, excluding Sfh5p, predominantly bind PI and neutral lipids (NLs). Sfh5 was found to bind only PI. Analysis of bound NLs identified squalene in Sfh2p as a likely binding partner and cholesterol in association with Sfh3p and Sfh4p. Moreover, it appears that both Sfh3p and Sfh4p can bind another NL in addition to cholesterol, which remains to be identified. Based on obtained results at least three out of five Sfh proteins are capable to bind NLs, predominantly sterols or precursors of the sterol biosynthetic pathway. It appears that binding of NLs is very specific, suggesting their crucial role in the function of Sfh proteins. Thus, the activity of some members of LTP family might be regulated by sterols or their precursors which in turn can connect and synchronize the sterol biosynthetic pathway with the phospholipid trafficking events. To identify the role of sterol binding by Sfh proteins in the physiology of the yeast cell, we will attempt to prepare mutants in sterol binding.

237: Phosphatidylcholine distribution between lipoprotein fractions in patients with CAD

Recent data have shown that phospholipids (mainly phosphatidylcholine) transfer activity is associated to metabolic syndrom and coronary artery disease (CAD). This transfer activity could modulate the phosphatidylcholine (PC) distribution between lipoprotein subclasses. In this study we separated lipoprotein fractions and we analyzed PC distribution. Ninety two subjects were recruited (47 CAD patients and 45 age- and sex-matched controls). Antropometric measurements, lipid profile and PC analysis were performed. Despite from an abnormal lipid profile: elevated serum triglycerides and LDL-C, and low HDL-C (but total cholesterol level was not significantly different between controls and CAD patients), serum PC centrations were slightly higher in CAD patients than in controls (respectively 2.53±0.84 and 2.36±0.43 mmol/L, ns). The level of PC was significanly higher in patients, compared to controls, concerning the (VLDL+LDL) fraction ( p <0.01). In concomitance, HDL-PC was descreased in CAD patients in comparison with controls ( p <0.05). Furthermore, serum PC level was colsely correlated to waist/hip ratio only in CAD patients ( p =0.0013). However, PC concentrations were less closely associated to BMI in the same group (p<0.035). Serum phospholipid status is modulated in patients with CAD. This involves a significant modulation of the PC distribution between lipoprotein subclasses and probably reflects an abnormality in the phospholipid transfer activity (PLTP activity) representing a useful marker of cardiovascular risk.

Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening

Phosphatidylcholine transfer protein (PC–TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC–TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC–TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC–TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC–TP activity, with IC 50 values that ranged from 4.1 to 95.0 μM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC–TP. Because mice with homozygous disruption of the PC–TP gene ( Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.

Phosphatidylcholine transfer activity of yeast Sec14p is not essential for its function in vivo

Yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, Sec14p, is essential for protein transport from the Golgi apparatus and for the cell viability. It is instrumental in maintaining the lipid composition of the Golgi membranes to be compatible with vesicle biogenesis and the secretory process by coordination of PC and PI metabolism. To address the question to which extent PC transfer ability of Sec14p is required for its essential in vivo function we generated a Sec14p mutant unable to transfer PC between membranes in the in vitro assay. Yeast cells with this modified Sec14p D115G as a sole Sec14p were viable with improved secretory activity compared to sec14 deficient strain. Thus, in vitro PC transfer ability of Sec14p is not required for its essential function(s) in living cells, however, yeast cells having PC transfer deficient Sec14p D115G as a sole Sec14p display regulatory abnormalities, including increased phospholipase D mediated PC turnover.

Genetic Evidence for Phospholipid-Mediated Regulation of the Rab GDP-Dissociation Inhibitor in Fission Yeast

We have previously identified mutant alleles of genes encoding two Rab proteins, Ypt3 and Ryh1, through a genetic screen using the immunosuppressant drug FK506 in fission yeast. In the same screen, we isolated gdi1-i11, a mutant allele of the essential gdi1+ gene encoding Rab GDP-dissociation inhibitor. In gdi1-i11, a conserved Gly267 was substituted by Asp. The Gdi1G267D protein failed to extract Rabs from membrane and Rabs were depleted from the cytosolic fraction in the gdi1-i11 mutant cells. Consistently, the Gdi1G267D protein was found mostly in the membrane fraction, whereas wild-type Gdi1 was found in both the cytosolic and the membrane fraction. Notably, overexpression of spo20+, encoding a phosphatidylcholine/phosphatidylinositol transfer protein, rescued gdi1-i11 mutation, but not ypt3-i5 or ryh1-i6. The gdi1-i11 and spo20-KC104 mutations are synthetically lethal, and the wild-type Gdi1 failed to extract Rabs from the membrane in the spo20-KC104 mutant. The phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppression of the gdi1-i11 mutation, suggesting that the phosphatidylcholine-transfer activity is important for the suppression. Furthermore, knockout of the pct1+ gene encoding a choline phosphate cytidyltransferase rescued the gdi1-i11 mutation. Together, our findings suggest that Spo20 modulates Gdi1 function via regulation of phospholipid metabolism of the membranes.

Plasma phospholipid transfer protein fused with green fluorescent protein is secreted by HepG2 cells and displays phosphatidylcholine transfer activity

Phospholipid transfer protein (PLTP) is a serum glycoprotein with a central role in high-density lipoprotein metabolism. We created a fusion protein in which enhanced green fluorescent protein (EGFP) was fused to the carboxyl-terminus of PLTP. Stably transfected HepG2 cells, which overexpress this fusion protein, were generated. PLTP-EGFP was translocated into the ER and fluoresced within the biosynthetic pathway, showing a marked concentration in the Golgi complex. The transfected cells secreted into the growth medium phospholipid transfer activity 7-fold higher than that of the mock-transfected controls. The medium of the PLTP-EGFP - expressing cells displayed EGFP fluorescence, demonstrating that both the PLTP and the EGFP moieties had attained a biologically active conformation. However, the specific activity of PLTP-EGFP in the medium was markedly reduced as compared with that of endogenous PLTP. This suggests that the EGFP attached to the carboxyl-terminal tail of PLTP interferes with the interaction of PLTP with its substrates or with the lipid transfer process itself. Fluorescently tagged PLTP is a useful tool for elucidating the intracellular functions of PLTP and the interaction of exogenously added PLTP with cells, and will provide a means of monitoring the distribution of exogenously added PLTP between serum lipoprotein subspecies.

Role of phospholipid transfer protein in rabbit lung development.

A 36-kDa phospholipid transfer protein (PLT-P(R)), which preferentially transfers phosphatidyl choline (PC) compared to phosphatidyl inositol (PI), was purified 827-fold from rabbit lung homogenate. Incorporation of cholesterol in unilamellar vesicles reduced the PC transfer activity of PLTP(R). Dipalmitoyl phosphatidyl choline uptake by alveolar type II cells was increased in the presence of the protein, and further enhanced in the presence of surfactant liposomes. However, a decrease in uptake was noted with cholesterol in host membranes. Incorporation of PI into host membranes had a low stimulatory effect on the process. All these effects were more pronounced in adult type II cells compared to premature, term and 3-day-old pups.

Measurement of Human Plasma Phospholipid Transfer Protein by Sandwich ELISA

Plasma phospholipid transfer protein (PLTP) plays a central role in the remodeling of HDLs. Reliable and accurate methods for assaying PLTP concentration are required. A sandwich ELISA for PLTP has been developed, using two monoclonal antibodies against recombinant human PLTP (rhPLTP) expressed in Chinese hamster ovary cells. The ELISA allows for the quantification of PLTP in the range 0.625-15.0 ng/assay (1.2-30.0 mg/L). Intra- and interassay CVs were <3.0% and <4.2% respectively. The assay was used to quantify plasma PLTP concentrations in 132 Japanese subjects (75 males and 57 females). PLTP concentrations were 12.0 +/- 3. 0 mg/L (mean +/- SD; range, 4.9-20.5 mg/L). No sex difference was observed. Plasma PLTP concentration was positively correlated with HDL-cholesterol (r = 0.72; P: <0.001), apolipoprotein (apo) A-I (r = 0.62; P: <0.001) and HDL(2)-cholesterol (r = 0.72; P: <0.001), and was negatively correlated with triacylglycerol (r = -0.45; P: <0. 001). There was no correlation with plasma apo A-II. These results agree with other evidence that plasma PLTP is associated with large apo A-I-containing lipoproteins. There was no correlation (r = -0. 01) between plasma PLTP and plasma phosphatidylcholine transfer activity (range, 3.5-10.5 micromol. mL(-1). h(-1)), suggesting that PLTP may exist in active and inactive forms. This new ELISA will be of value for further studies of PLTP in health and disease.

The Protein Kinase C-dependent Phosphorylation of Serine 166 Is Controlled by the Phospholipid Species Bound to the Phosphatidylinositol Transfer Protein α

The charge isomers of bovine brain PI-TPalpha (i.e. PI-TPalphaI containing a phosphatidylinositol (PI) molecule and PI-TPalphaII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V(max) values for PI-TPalphaI and II were 2.0 and 6.0 nmol/min, respectively; the K(m) values for both charge isomers were about equal, i.e. 0.7 micrometer. Phosphorylation of charge isomers of recombinant mouse PI-TPalpha confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo (32)P-labeled PI-TPalphas showed that serine was the major site of phosphorylation. Degradation of (32)P-labeled PI-TPalpha by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one (32)P-labeled peptide (amino acids 104-190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPalpha. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPalpha to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPalpha expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPalpha(S166A) and Myc-tagged PI-TPalpha(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPalpha is linked to the degradation of PI.

Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

Secretion of Phospholipid Transfer Protein by Human Hepatoma Cell Line, Hep G2, Is Enhanced by Sodium Butyrate

Hep G2 cells were used to study the synthesis and secretion of phospholipid transfer protein (PLTP). Upon incubation of the cells at confluence with serum-free Dulbecco’s modified Eagle’s medium (DMEM), phosphatidylcholine (PC) transfer activity was found to accumulate in the culture media. The PC transfer activity in the media was effectively inhibited by rabbit anti-human PLTP immunoglobulin (Ig)G, thus indicating that the PC transfer activity was due to secreted PLTP. The molecular weight of Hep G2 PLTP was ∼78 kDa by Western blot analysis, in agreement with the molecular weight obtained for purified human plasma PLTP. The PLTP secreted by Hep G2 also possessed an HDL conversion activity similar to that of human plasma PLTP. The addition of butyrate to the cell culture media resulted in a marked increase in the secretion of PLTP. After 24 h incubation with 4 mmol/L sodium butyrate, a more than twofold increase (P < 0.01) of PC transfer activity in the cell-conditioned media was obtained. The dose-dependent increase in the PC transfer activity in the media upon butyrate treatment was well correlated (r = 0.80, P < 0.01) with that of PLTP mass as determined by immuno-slot blot analysis of cell-conditioned media. The increased secretion of PLTP by Hep G2 treated with sodium butyrate was accompanied by a greater increase in the level of PLTP mRNA in the cells as determined by ribonuclease protection assay. In the presence of 4 mmol/L sodium butyrate, a fourfold increase (P < 0.01) in mRNA level was obtained at 24 h. No stabilizing effect of butyrate on PLTP mRNA was apparent upon treatment of the cultured cells with the RNA synthesis inhibitor, actinomycin D. Thus, the up-regulatory effect of butyrate on PLTP gene expression seemed to have occurred at the transcriptional level.

Submicellar bile salts stimulate phosphatidylcholine transfer activity of sterol carrier protein 2

Abstract To explore a potential role for sterol carrier protein 2 (SCP2, also known as non-specific lipid transfer protein) in hepatocellular phospholipid trafficking, we examined the influence of submicellar bile salt concentrations on phosphatidylcholine (PC) transfer activity of SCP2. We measured rate constants for first-order transfer of sn-1 palmitoyl, sn-2 parinaroyl PC, a naturally fluorescent self-quenching phospholipid between model membranes. Purified bovine liver SCP2 promoted transfer of PC from donor to acceptor small unilamellar vesicles. Taurine- and glycine-conjugated bile salts (anionic steroid detergent-like molecules), at concentrations well below their critical micellar concentrations, stimulated PC transfer activity of SCP2 80- to 140-fold. Rate constants increased in proportion to bile salt concentration, temperature, and bile salt–membrane binding affinity. Sodium taurofusidate, a conjugated fungal bile salt analog, also activated PC transfer whereas no effect was observed with the anionic and nonionic straight chain detergents sodium dodecyl sulfate and octylglucoside, respectively. Thermodynamic and kinetic analyses of PC transfer support a mechanism in which bile salts stimulate SCP2 activity by partitioning into donor vesicles and enhancing membrane association of SCP2. These results imply that under physiological conditions, SCP2 may contribute to hepatocellular selection and transport of biliary PCs.—Leonard, A. N., and D. E. Cohen. Submicellar bile salts stimulate phosphatidylcholine transfer activity of sterol carrier protein 2. J. Lipid Res. 1998. 39: 1981–1988.

Baculovirus-mediated expression of recombinant rat phosphatidylcholine transfer protein

Phosphatidylcholine transfer protein catalyzes intermembrane transfer of phosphatidylcholines exclusive of all other phospholipid classes. Although postulated to participate in phosphatidylcholine biosynthesis and biliary trafficking in liver, the molecular basis underlying the substrate specificity of phosphatidylcholine transfer protein remains to be elucidated. Having demonstrated the inability of Escherichia coli to express recombinant phosphatidylcholine transfer protein, we infected Spodoptera frugiperda (Sf9) cells with recombinant baculovirus. When assayed in vitro, cytosol of recombinant but not control infected cells demonstrated high levels of intermembrane phosphatidylcholine transfer activity and no transfer activity for phosphatidylethanolamine. A two-step purification protocol in which 10 mg of cytosolic protein was subjected to anion exchange chromatography followed by hydroxylapatite chromatography yielded 0.1 mg active protein which was >92% pure. The identity of purified protein was confirmed by matrix-assisted laser desorption–ionization mass spectrometry and by amino acid sequencing. Based on the recovery of 30% of PC transfer activity after purification, we estimate that recombinant rat phosphatidylcholine transfer protein accounted for ∼3–6% of cytosolic protein mass of infected cells. ▪ These results demonstrate the utility of baculovirus for expressing recombinant phosphatidylcholine transfer protein and should facilitate studies designed to elucidate the structural biology and physiological functions of this uniquely specific phospholipid transfer protein.—Feng, L., and D. E. Cohen. Baculovirus-mediated expression of recombinant rat phosphatidylcholine transfer protein.

The First 5 Amino Acids of the Carboxyl Terminus of Phosphatidylinositol Transfer Protein (PITP) α Play a Critical Role in Inositol Lipid Signaling: TRANSFER ACTIVITY OF PITP IS ESSENTIAL BUT NOT SUFFICIENT FOR RESTORATION OF PHOSPHOLIPASE C SIGNALING

Phosphatidylinositol transfer protein (PITP) is essential for phospholipase C signaling and for constitutive and regulated vesicular traffic. PITP has a single lipid-binding site that can reversibly bind phosphatidylinositol (PI) and phosphatidylcholine (PC) and transfer these lipids between membrane compartments in vitro. The role of the carboxyl terminus was examined by comparing wild-type PITPalpha with PITPalpha in which 5, 10, and 20 amino acids were deleted from the C terminus. Delta5- and Delta10-PITP had reduced PI and PC transfer activities compared with wild-type PITP, with the effect on PI transfer being more marked than that on PC transfer. Delta20-PITP was inactive at all concentrations tested. All three truncated mutants were unable to restore G-protein-mediated phospholipase Cbeta stimulation in HL-60 cells. Delta5- and Delta10-PITP, but not Delta20-PITP, inhibited the signaling function of wild-type protein without any effect on lipid transfer in vitro. We conclude that (a) the carboxyl terminus of PITP plays a critical role in phospholipase C signaling; (b) the transfer activity is not the only determining factor that dictates the restorative function of PITP in inositol lipid signaling; and (c) the dominant inhibitory effects of Delta5- and Delta10-PITP on wild-type PITP in phospholipase C signaling suggest the existence of a receptor for PITP.

Intrinsic sterol- and phosphatidylcholine transfer activities of 17β-hydroxysteroid dehydrogenase type IV

Previous studies have shown that the 80 kDa 17β-hydroxysteroid dehydrogenase (17β-HSD) type IV comprises distinct domains, including an N-terminal region related to the short chain alcohol dehydrogenase multigene family and a C-terminal part related to the lipid transfer protein sterol carrier protein 2 (SCP2). In this study, we have investigated whether the SCP2-related part of the 80 kDa protein leads to an intrinsic sterol and phospholipid transfer activity, as shown earlier for the 60 kDa SCP2-related peroxisomal 3-ketoacyl CoA thiolase with intrinsic sterol and phospholipid transfer activity called sterol carrier protein x (SCPx). Our results indicate that a fraction rich in the 80 kDa form of 17β-HSD type IV exhibits high transfer activities for 7-dehydrocholesterol and phosphatidylcholine. In addition, a purified recombinant peptide derived from the SCP2-related domain of the 17β-HSD type IV has about 30% of the transfer activities for 7-dehydrocholesterol and phosphatidylcholine seen with purified recombinant human SCP2. We conclude that the 80 kDa type IV 17β-HSD represents a potentially multifunctional protein with intrinsic in vitro sterol and phospholipid transfer activity in addition to its enzymatic activity.

Mutant rat phosphatidylinositol/phosphatidylcholine transfer proteins specifically defective in phosphatidylinositol transfer: implications for the regulation of phospholipid transfer activity.

The mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs) catalyze exchange of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. We find that Ser-25, Thr-59, Pro-78, and Glu-248 make up a set of rat (r) PI-TP residues, substitution of which effected a dramatic reduction in the relative specific activity for PI transfer activity without significant effect on PC transfer activity. Thr-59 was of particular interest as it is a conserved residue in a highly conserved consensus protein kinase C phosphorylation motif in metazoan PI-TPs. Replacement of Thr-59 with Ser, Gln, Val, Ile, Asn, Asp, or Glu effectively abolished PI transfer capability but was essentially silent with respect to PC transfer activity. These findings identify rPI-TP residues that likely cooperate to form a PI head-group binding/recognition site or that lie adjacent to such a site. Finally, the selective sensitivity of the PI transfer activity of rPI-TP to alteration of Thr-59 suggests a mechanism for in vivo regulation of rPI-TP activity.

Filamentous Fungi with High Cytosolic Phospholipid Transfer Activity in the Presence of Exogenous Phospholipid

The phospholipid transfer activity of cell extracts from 15 filamentous fungus strains grown on a medium containing phospholipids as the carbon source was measured by a fluorescence assay. This assay was based on the transfer of pyrene-labeled phosphatidylcholines forming the donor vesicles to acceptor vesicles composed of egg phosphatidylcholines. The highest phosphatidylcholine transfer activity was obtained with cell extracts from Aspergillus oryzae. The presence of exogenous phospholipids in the culture medium of A. oryzae was shown to increase markedly the activity of phospholipid transfer as well as the pool of exocellular proteins during the primary phase of growth. Modifications in the biochemical marker activities of cellular organelles were observed: succinate dehydrogenase, a mitochondrial marker; inosine diphosphatase, a Golgi system marker; and cytochrome c oxidoreductase, an endoplasmic reticulum marker, were increased 7.3-, 2-, and 22-fold, respectively, when A. oryzae was grown in the presence of phospholipids.

Structure-activity studies of human sterol carrier protein 2.

Recombinant human sterol carrier protein 2 (SCP2) variants were generated by site-directed mutagenesis and expression in Escherichia coli. The ability of the variants to stimulate microsomal conversion of 7-dehydrocholesterol to cholesterol (sterol carrier activity) and to transfer cholesterol and phosphatidylcholine from donor small unilamellar vesicles to acceptor membranes (cholesterol and phosphatidylcholine transfer activities) was compared with wild-type recombinant SCP2. Our results indicate that all measured activities of recombinant human pre-SCP2 (including the 20-amino acid leader sequence) and mature SCP2 were similar. Expressed glutathione S-transferase fusion proteins (GST-SCP2 and GST-pre-SCP2) possessed considerable activity, suggesting that steric obstruction at the amino terminus causes only minor inactivation. The effect of progressive removal of peptides from the carboxyl terminus showed that amino acids between Lys100 and Asn104 are essential for SCP2 activity. This conclusion was substantiated by the observation that replacing Asn104 with Asp or Ile caused considerable inactivation, whereas replacing Met105 with Leu had almost no effect. Since N-ethylmaleimide is known to inhibit SCP2 activity, substitutions were also introduced in the vicinity of Cys71. Whereas Val71 and Ser71 variants possessed wild-type activity, replacing Asp70 with Asn almost completely abolished SCP2 activity. Further, the importance of residues located close to the amino terminus was indicated by complete inactivation of a 10-amino-terminal amino acid deletion mutant and by replacing Leu20 with Glu. Circular dichroism results showed that Leu20 and Asp70 may serve to stabilize the overall fold, whereas residue 104 appears to play a role in the specific lipid binding and/or transfer activity of SCP2.

High density lipoprotein conversion mediated by human plasma phospholipid transfer protein.

Phospholipid transfer protein (PLTP) was purified from lipoprotein-free human plasma, obtained upon treatment of plasma with dextran sulfate and Ca2+, by employing a series of column chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified PLTP showed a single main band, corresponding to the molecular mass of 78 kDa. However, isoelectric focusing of the purified preparation gave multiple bands with pI ranging from 4.3 to 5.1, indicative of microheterogeneity. Purified PLTP was shown to possess not only phospholipid transfer activity, but also high density lipoprotein (HDL) conversion activity (Tu, A.-Y., Nishida, H. I., and Nishida, T. (1990), FASEB J. 4, A2148; Jauhiainen, M., Metso, J., Pahlman, R., Blomqvist, S., van Tol, A., and Ehnholm, C. (1993) J. Biol. Chem. 268, 4032-4036). Isolated HDL3 was enlarged to the size of HDL2b upon incubation with purified PLTP for 6 h at 37 degrees C at the PLTP/HDL3 molar ratio of approximately 1:45. Both the HDL conversion and the phosphatidylcholine transfer activities of purified PLTP were effectively inhibited by rabbit anti-PLTP immunoglobulin G. The primary importance of PLTP in the HDL enlargement that occurs in human plasma upon incubation at 37 degrees C was shown by the strong inhibitory effect of the anti-PLTP immunoglobulin G. The process of PLTP-mediated HDL enlargement was accompanied by the release of apoproteins, primarily apoA-I. HDL3 enlargement mediated by PLTP was effectively inhibited by the addition of free fatty acids.