PLTP regulates STAT3 and NFκB in differentiated THP1 cells and human monocyte-derived macrophages.

Phospholipid transfer protein (PLTP) plays an important role in regulation of inflammation. Previously published studies have shown that PLTP binds, transfers and neutralizes bacterial lipopolysaccharides. In the current study we tested the hypothesis that PLTP can also regulate anti-inflammatory pathways in macrophages. Incubation of macrophage-like differentiated THP1 cells and human monocyte-derived macrophages with wild-type PLTP in the presence or absence of tumor necrosis factor alpha (TNFα) or interferon gamma (IFNγ) significantly increased nuclear levels of active signal transducer and activator of transcription 3, pSTAT3(Tyr705) (p<0.01). Similar results were obtained in the presence of a PLTP mutant without lipid transfer activity (PLTP(M159E)), suggesting that PLTP-mediated lipid transfer is not required for activation of the STAT3 pathway. Inhibition of ABCA1 by chemical inhibitor, glyburide, as well as ABCA1 RNA inhibition, reversed the observed PLTP-mediated activation of STAT3. In addition, PLTP reduced nuclear levels of active nuclear factor kappa-B (NFκB) p65 and secretion of pro-inflammatory cytokines in conditioned media of differentiated THP1 cells and human monocyte-derived macrophages. Our data suggest that PLTP has anti-inflammatory capabilities in macrophages.

Linkage and association of phospholipid transfer protein activity to LASS4.

Phospholipid transfer protein activity (PLTPa) is associated with insulin levels and has been implicated in atherosclerotic disease in both mice and humans. Variation at the PLTP structural locus on chromosome 20 explains some, but not all, heritable variation in PLTPa. In order to detect quantitative trait loci (QTLs) elsewhere in the genome that affect PLTPa, we performed both oligogenic and single QTL linkage analysis on four large families (n = 227 with phenotype, n = 330 with genotype, n = 462 total), ascertained for familial combined hyperlipidemia. We detected evidence of linkage between PLTPa and chromosome 19p (lod = 3.2) for a single family and chromosome 2q (lod = 2.8) for all families. Inclusion of additional marker and exome sequence data in the analysis refined the linkage signal on chromosome 19 and implicated coding variation in LASS4, a gene regulated by leptin that is involved in ceramide synthesis. Association between PLTPa and LASS4 variation was replicated in the other three families (P = 0.02), adjusting for pedigree structure. To our knowledge, this is the first example for which exome data was used in families to identify a complex QTL that is not the structural locus.

Association of plasma phospholipid transfer protein activity with IDL and buoyant LDL: impact of gender and adiposity.

Current data suggest that phospholipid transfer protein (PLTP) has multiple metabolic functions, however, its physiological significance in humans remains to be clarified. To provide further insight into the role of PLTP in lipoprotein metabolism, plasma PLTP activity was measured, and lipoproteins were analyzed in 134 non-diabetic individuals on a controlled diet. Insulin sensitivity index (Si) and body fat composition were also determined. Plasma PLTP activity was comparable between men (n=56) and women (n=78). However, in women but not in men, plasma PLTP activity was positively correlated with cholesterol, triglyceride, low density lipoprotein (LDL) cholesterol, and apolipoprotein (apo) B (r=0.38-0.45, P< or =0.001), and with body mass index (BMI), subcutaneous and intra-abdominal fat (SCF, IAF) (r=0.27-0.29, P<0.02). Among the different apo B-containing lipoproteins (LpB) in women, PLTP was most highly correlated with intermediate density lipoproteins (IDL) and buoyant LDL (r=0.45-0.46, P<0.001). The correlation with IDL was significant only in women with BMI < or =27.5 kg/m(2) (n=56). In men with BMI < or =27.5 kg/m(2) (n=35), PLTP activity was significantly correlated with buoyant LDL (r=0.40, P<0.02) and high density lipoprotein (HDL) (r=0.43, P<0.01). These data provide evidence for a role of PLTP in LpB metabolism, particularly IDL and buoyant LDL. They also suggest that gender and obesity-related factors can modulate the impact of PLTP on LpB.

Plasma phospholipid transfer protein activity in patients with low HDL and cardiovascular disease treated with simvastatin and niacin.

Plasma phospholipid transfer protein (PLTP) is an important modulator of high-density lipoprotein (HDL) metabolism, regulating its particle size, composition, and mass. In patients with low HDL and cardiovascular disease (CVD), plasma PLTP activity is positively correlated with the concentration of HDL particles containing apo A-I but not apo A-II (Lp(A-1)). We recently completed a study to determine the effect of simvastatin and niacin (S-N) therapy on disease progression/regression in these patients, and found that this therapy selectively increased Lp(A-I). To determine if PLTP was also increased with this drug therapy, we measured the PLTP activity in the plasma of 30 of these patients obtained at baseline and after 12 months of therapy, and compared the changes to a similar group of 31 patients who received placebo for the drugs. No significant increase in PLTP activity was observed in either group of patients. However, changes in apo A-I and A-II between these two time points were correlated with the corresponding change in PLTP activity. The correlation coefficients were r=0.57 (P=0.001) and r=0.43 (P=0.02) for apo A-I, and r=0.54 (P=0.002) and r=0.41 (P=0.02) for apo A-II in the placebo and S-N group, respectively. At baseline, PLTP activity correlated positively with the percent of plasma apo A-I associated with Lp(A-I) (r=0.38, P=0.04) and the amounts of apo A-I in these particles (r=0.43, P=0.02). These relationships persisted in patients who took placebo for 12 months (r=0.46, P=0.009 and r=0.37, P=0.04, respectively), but was attenuated in those treated with S-N. These data indicate that S-N-induced increase in Lp(A-I) was PLTP-independent. It also confirms our previous observation that an interrelationship exists between PLTP and apo-specific HDL particle subclasses in CVD patients with low HDL, and that this relationship is altered by drug intervention.

Lipopolysaccharide-binding protein and phospholipid transfer protein release lipopolysaccharides from gram-negative bacterial membranes.

Although animals mobilize their innate defenses against gram-negative bacteria when they sense the lipid A moiety of bacterial lipopolysaccharide (LPS), excessive responses to this conserved bacterial molecule can be harmful. Of the known ways for decreasing the stimulatory potency of LPS in blood, the binding and neutralization of LPS by plasma lipoproteins is most prominent. The mechanisms by which host lipoproteins take up the native LPS that is found in bacterial membranes are poorly understood, however, since almost all studies of host-LPS interactions have used purified LPS aggregates. Using native Salmonella enterica serovar Typhimurium outer membrane fragments (blebs) that contained (3)H-labeled lipopolysaccharide (LPS) and (35)S-labeled protein, we found that two human plasma proteins, LPS-binding protein (LBP) and phospholipid transfer protein (PLTP), can extract [(3)H]LPS from bacterial membranes and transfer it to human high-density lipoproteins (HDL). Soluble CD14 (sCD14) did not release LPS from blebs yet could facilitate LBP-mediated LPS transfer to HDL. LBP, but not PLTP, also promoted the activation of human monocytes by bleb-derived LPS. Whereas depleting or neutralizing LBP significantly reduced LPS transfer from blebs to lipoproteins in normal human serum, neutralizing serum PLTP had no demonstrable effect. Of the known lipid transfer proteins, LBP is thus most able to transfer LPS from bacterial membranes to the lipoproteins in normal human serum.

Plasma lipoproteins promote the release of bacterial lipopolysaccharide from the monocyte cell surface.

When bacterial lipopolysaccharide (LPS) enters the bloodstream, it is thought to have two general fates. If LPS binds to circulating leukocytes, it triggers innate host defense mechanisms and often elicits toxic reactions. If instead LPS binds to plasma lipoproteins, its bioactivity is largely neutralized. This study shows that lipoproteins can also take up LPS that has first bound to leukocytes. When monocytes were loaded with [(3)H]LPS and then incubated in plasma, they released over 70% of the cell-associated [(3)H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [(3)H]LPS remained tightly associated with the cells. The transfer reaction could be reproduced in the presence of pure native lipoproteins or reconstituted high density lipoprotein. Plasma immunodepletion experiments and experiments using recombinant LPS transfer proteins revealed that soluble CD14 significantly enhances LPS release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell surface can be released. Lipoprotein-mediated LPS release was accompanied by a reduction in several cellular responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo.

Phospholipid transfer protein enhances removal of cellular cholesterol and phospholipids by high-density lipoprotein apolipoproteins.

High-density lipoprotein (HDL) apolipoproteins remove excess cholesterol from cells by an active transport pathway that may protect against atherosclerosis. Here we show that treatment of cholesterol-loaded human skin fibroblasts with phospholipid transfer protein (PLTP) increased HDL binding to cells and enhanced cholesterol and phospholipid efflux by this pathway. PLTP did not stimulate lipid efflux in the presence of albumin, purified apolipoprotein A-I, and phospholipid vesicles, suggesting specificity for HDL particles. PLTP restored the lipid efflux activity of mildly trypsinized HDL, presumably by regenerating active apolipoproteins. PLTP-stimulated lipid efflux was absent in Tangier disease fibroblasts, induced by cholesterol loading, and inhibited by brefeldin A treatment, indicating selectivity for the apolipoprotein-mediated lipid removal pathway. The lipid efflux-stimulating effect of PLTP was not attributable to generation of prebeta HDL particles in solution but instead required cellular interactions. These interactions increased cholesterol efflux to minor HDL particles with electrophoretic mobility between alpha and prebeta. These findings suggest that PLTP promotes cell-surface binding and remodeling of HDL so as to improve its ability to remove cholesterol and phospholipids by the apolipoprotein-mediated pathway, a process that may play an important role in enhancing flux of excess cholesterol from tissues and retarding atherogenesis.

Introduction of the human PLTP transgene suppresses the atherogenic diet-induced increase in plasma phospholipid transfer activity in C57BL/6 mice.

The human plasma phospholipid transfer protein (PLTP) has been shown to facilitate the transfer of phospholipids between lipoproteins and convert high-density lipoproteins into larger and smaller particles in vitro. To explore the lipid transport function in vivo, transgenic C57BL/6 mice that express the human PLTP gene, driven by its natural promoter, were generated. Little difference in PLTP activity and lipoprotein lipids was observed between transgenic mice and non-transgenic control mice fed the chow diet. In response to an atherogenic high-fat, high-cholesterol, cholic acid containing diet, the PLTP activity increased significantly with time in control mice (62% in males and 34% in females after the high-fat diet for 18 weeks). In contrast, the PLTP activity did not change appreciably in the transgenic mice fed the atherogenic diet. Thus, the introduction of the human transgene suppressed the diet-induced increase in plasma PLTP activity, as evidenced by a decrease in PLTP mRNA in a variety of tissues. High-density lipoprotein levels decreased in mice fed the atherogenic diet, but there was a proportionally greater decrease in transgenic animals than in controls. After 18 weeks on the atherogenic diet, the transgenic animals had high-density lipoprotein-cholesterol and PLTP activity approximately one-half of that of control animals. Non-denaturing gradient gel electrophoresis of plasma indicated that the atherogenic diet decreased the high-density lipoprotein size distribution in control mice. However, high-density lipoprotein particle size distribution of the transgenic mice was shifted to smaller particles compared with control animals (P < 0.001). These findings suggest that PLTP activity can modulate the effects of an atherogenic diet on high-density lipoproteins.

Differential expression of tumor necrosis factor receptor subtypes on leukocytes in systemic inflammatory response syndrome.

OBJECTIVE: To determine the expression of tumor necrosis factor (TNF) receptor in patients with systemic inflammatory response syndrome (SIRS). DESIGN: Prospective study. SETTING: Intensive care unit and central laboratory. PATIENTS: Blood specimens from 18 healthy volunteers (controls) and 16 patients with SIRS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Using monoclonal antibodies, fluorescence labeling, and high sensitivity flow cytometry, we measured the expression of membrane TNF receptor subtypes TNF-R55 and TNF-R75 on peripheral blood leukocytes. Receptor expression is expressed as mean fluorescence intensity +/- SD (units: detection channel number). In controls, TNF-R55 was only weakly expressed (monocytes: 2.5+/-1.8; neutrophils: 0.7+/-0.8), whereas expression of TNF-R75 was higher (monocytes: 28.6+/-9.0; neutrophils: 4.8+/-1.0) and was also found on lymphocytes (on CD8+ lymphocytes: 5.7+/-1.8; CD16+: 5.5+/-1.2; CD4+: 9.7+/-3.7). In SIRS, we observed increased expression of TNF-R55 on monocytes (6.9+/-3.4, p<.001) and neutrophils (2.2+/-1.9, p<.01), as well as decreased expression of TNF-R75 on monocytes (17.3+/-13.2; p<.001). The extent of TNF-R55 up-regulation did not correlate with that of TNF-R75 down-regulation. TNF-R55 on monocytes and neutrophils strongly correlated with body temperature but not with survival, whereas monocyte TNF-R75 was considerably lower in nonsurvivors, albeit not significantly (12.3+/-7.1 vs. 23.9+/-16.7; p = .07). CONCLUSIONS: These data indicate that leukocyte TNF-R55 and TNF-R75 react differentially and probably serve different functions in SIRS, which prompts the investigation of receptor subtype-specific therapeutic approaches.

Relationship between plasma phospholipid transfer protein activity and HDL subclasses among patients with low HDL and cardiovascular disease.

Low levels of high density lipoproteins (HDL) are associated with an increased risk for premature cardiovascular disease. The plasma phospholipid transfer protein (PLTP) is believed to play a critical role in lipoprotein metabolism and reverse cholesterol transport by remodeling HDL and facilitating the transport of lipid to the liver. Plasma contains two major HDL subclasses, those containing both apolipoproteins (apo) A-I and A-II, Lp(A-I, A-II), and those containing apo A-I but not A-II, Lp(A-I). To examine the potential relationships between PLTP and lipoproteins, plasma PLTP activity, lipoprotein lipids, HDL subclasses and plasma apolipoproteins were measured in 52 patients with documented cardiovascular disease and low HDL levels. Among the patients, plasma PLTP activity was highly correlated with the percentage of plasma apo A-I in Lp(A-I) (r=0.514, p < 0.001) and with the apo A-I, phospholipid and cholesterol concentration of Lp(A-I) (r=0.499, 0.478, 0.457, respectively, p < 0.001). Plasma PLTP activity was also significantly correlated with plasma apo A-I (r=0.413, p=0.002), HDL cholesterol (r=0.308, p=0.026), and HDL, and HDL3 cholesterol (r=0.284 and 0.276, respectively, p < 0.05), but no significant correlation was observed with Lp(A-I, A-I), plasma cholesterol, triglycerides, or apo B, very low density lipoprotein cholesterol or low density lipoprotein cholesterol. These associations support the hypothesis that PLTP modulates plasma levels of Lp(A-I) particles without significantly affecting the levels of Lp(A-I, A-II) particles.

Relationship between phospholipid transfer protein activity and HDL level and size among inbred mouse strains.

Because of the paucity of data on phospholipid transfer protein (PLTP) activity and lipoprotein phospholipid in mouse strains, plasma PLTP activity (PLTA), plasma phospholipid and cholesterol, HDL phospholipid and cholesterol, and HDL size distribution were determined in 15 inbred mouse strains. The 15 inbred mouse strains differed in their relatedness to one another and consisted of six largely unrelated groups: Castaneus, Swiss, C57BL, AKR, DBA, and NZB. Lipid and PLTA analyses were performed on plasma pools from male and female mice that had fasted for 4 h prior to blood draw. Among the representative unrelated strains fed the chow diet, there was a highly significant relationship between PLTA and plasma phospholipid (r(s) = 0.727, P < 0.01), HDL phospholipid (r(s) = 0.762, P < 0.01), HDL cholesterol (r(s) = 0.699, P < 0.02), percentage of large HDL particles (r(s) = 0.699, P < 0.02), and HDL peak size (r(s) = 0.776, P < 0.01). Similar results were obtained among these strains fed a high fat, high cholesterol diet. PLTA increased in all strains fed the high fat diet (chix = 94%, range 6 to 221%). Strain SM having relatively low PLTA and HDL was crossed with strain NZB having high PLTA and HDL. The F1 progeny from this cross were backcrossed to strain SM and 41 male backcross progeny collected. Among these individual backcrossed animals, PLTA was highly correlated with plasma phospholipid (r(s) = 0.508, P = 0.001), HDL phospholipid (r(s) = 0.566, P < 0.001), HDL cholesterol (r(s) = 0.532, P < 0.001), and percentage of large HDL particles (r(s) = 0.446, P = 0.020). Therefore, we conclude that PLTP is a determinant of HDL level and size in mice.-Albers, J. J., W. Pitman, G. Wolfbauer, M. C. Cheung, H. Kennedy, A-Y. Tu, S. M. Marcovina, and B. Paigen. Relationship between phospholipid transfer protein activity and HDL level and size among inbred mouse strains.

Toward a new reference method for the leukocyte five-part differential.

A flow cytometric method performing a five-part leukocyte differential based on three-color staining with anti-CD45-fluorescein isothiocyanate (FITC), anti-CD-14-phycoerythrin (PE)/Cy5, and a cocktail of PE-labeled anti-CD2, anti-CD16, and anti-HLA-DR antibodies was evaluated. Results obtained by using three different sample preparation procedures and two different flow cytometers were compared with those of a 1,000-cell manual differential for evaluation of accuracy. We observed excellent correlations with the manual differential for all leukocyte subclasses and even higher correlations between the different flow cytometric methods. Flow cytometric basophil results were identical to the manual counts, regardless of which sample preparation technique or flow cytometer was used. Therefore, we propose our flow cytometric method as the first acceptable automated reference method for basophil counting. The flow cytometric results for the other leukocyte subclasses were apparently influenced by the sample preparation, which could not be explained by cell loss during washing steps. Moreover, a small influence of the flow cytometer was also observed. Assessing the influence of sample storage, we found only minimal changes within 24 h. In establishing reference values, high precision of flow cytometric results facilitated detection of a significantly higher monocyte count for males (relative count: 7.08 +/- 1.73% vs. 6.44 +/- 1.33%, P < 0.05; absolute count: 0.536 +/- 0.181 x 10(9)/liter vs. 0.456 +/- 139 x 10(9)/liter, P < 0.01). Our data indicate that monoclonal antibody-based flow cytometry is a highly suitable reference method for the five-part differential: It also shows, however, that studies will have to put more emphasis on methodological issues to define a method that shows a high interlaboratory reproducibility.

Molecular and macromolecular specificity of human plasma phospholipid transfer protein.

Phospholipid transfer protein (PLTP), also known as lipid transfer protein 2 (LTP-2), mediates a transfer of phospholipids between high-density lipoproteins (HDL). The molecular and macromolecular specificities of recombinant human PLTP were studied using a fluorometric assay based on the excimer fluorescence of pyrenyl lipids. To determine lipoprotein specificity of PLTP, donor very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and HDL were labeled with 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PPyDPC) and incubated with unlabeled acceptor VLDL, LDL, and HDL in every pairwise combination. The highest rate of PPyDPC transfer mediated by PLTP occurred between donor HDL and acceptor HDL. Reassembled HDL (rHDL) consisting of 1-palmitoyl-2-oleoylphosphatidylcholine, apolipoprotein A-I, and pyrene lipids (100:1:4) were used to demonstrate that PLTP transfers diacylglyceride > phosphatidic acid > sphingomyelin > phosphatidylcholine (PC) > phosphatidylglycerol > cerobroside > phosphatidylethanolamine. Thus, PLTP transfers a variety of lipids with two carbon chains and a polar head group. Unsaturation of one PC acyl chain greatly increased transfer rate, whereas increasing chain length and exchanging sn-1/sn-2 position had only small effects. The rate of PPyDPC transfer by PLTP decreases with increasing free cholesterol content in rHDL and with decreasing HDL size. In contrast to spontaneous transfer, PLTP mediates the accumulation of PC in small rHDL particles. PLTP may be important in vivo in the recycling of PC from mature HDL to nascent HDL, the latter of which are the initial acceptors of cholesterol from peripheral tissue for reverse cholesterol transport to the liver.

DNA sequences essential for transcription of human phospholipid transfer protein gene in HepG2 cells.

The present study was conducted to determine the essential DNA sequences required for the transcription of the human phospholipid transfer protein gene. Truncation studies revealed that DNA sequences between -230 and -159, particularly those at the upstream region, were responsible for the full promoter activity. This region was able to compete with AP-2 and GRE oligonucleotides for the binding to HepG2 cell nuclear extract as shown by gel mobility shift assay. Further analysis, using site-directed mutagenesis, indicated that DNA sequences identical to Sp1 and highly homologous to GRE and Ap-2 consensus sequences were essential for the transcription. These findings support the concept that several elements, spread over the entire functional promoter, synergistically drive the basal transcription.

Plasma phospholipid mass transfer rate: relationship to plasma phospholipid and cholesteryl ester transfer activities and lipid parameters.

Human plasma phospholipid transfer protein (PLTP) has been shown to facilitate the transfer of phospholipid from liposomes or isolated very low and low density lipoproteins to high density lipoproteins. Its activity in plasma and its physiological function are presently unknown. To elucidate the role of PLTP in lipoprotein metabolism and to delineate factors that may affect the rate of phospholipid transfer between lipoproteins, we determined the plasma phospholipid mass transfer rate (PLTR) in 16 healthy adult volunteers and assessed its relationship to plasma lipid levels, and to phospholipid transfer activity (PLTA) and cholesteryl ester transfer activity (CETA) measured by radioassays. The plasma PLTR in these subjects was 27.2 +/- 11.8 nmol/ml per h at 37 degrees C (mean +/- S.D.), and their PLTA and CETA were 13.0 +/- 1.7 mumol/ml per h and 72.8 +/- 15.7 nmol/ml per h, respectively. Plasma PLTR was correlated directly with total, non-HDL, and HDL triglyceride (rs = 0.76, P < 0.001), total and non-HDL phospholipid (rs > 0.53, P < 0.05), and inversely with HDL free cholesterol (rs = -0.54, P < 0.05), but not with plasma PLTA and CETA. When 85% to 96% of the PLTA in plasma was removed by polyclonal antibodies against recombinant human PLTP, phospholipid mass transfer from VLDL and LDL to HDL was reduced by 50% to 72%, but 80% to 100% of CETA could still be detected. These studies demonstrate that PLTP plays a major role in facilitating the transfer of phospholipid between lipoproteins, and suggest that triglyceride is a significant modulator of intravascular phospholipid transport. Furthermore, most of the PLTP and CETP in human plasma is associated with different particles. Plasma PLTA and CETA were also measured in mouse, rat, hamster, guinea pig, rabbit, dog, pig, and monkey. Compared to human, PLTA in rat and mouse was significantly higher and in rabbit and guinea pig was significantly lower while the remaining animal species had PLTA similar to humans. No correlation between PLTA and CETA was observed among animal species.

Neutralization and transfer of lipopolysaccharide by phospholipid transfer protein.

Phospholipid transfer protein (PLTP) and lipopolysaccharide-binding protein (LPB) are lipid transfer proteins found in human plasma. PLTP shares 24% sequence similarity with LBP. PLTP mediates the transfer and exchange of phospholipids between lipoprotein particles, whereas LBP transfers bacterial lipopolysaccharide (LPS) either to lipoprotein particles or to CD14, a soluble and cell-surface receptor for LPS. We asked whether PLTP could interact with LPS and mediate the transfer of LPS to lipoproteins or to CD14. PLTP was able to bind and neutralize LPS: incubation of LPS with purified recombinant PLTP (rPLTP) resulted in the inhibition of the ability of LPS to stimulate adhesive responses of neutrophils, and addition of rPLTP to blood inhibited cytokine production in response to LPS. Transfer of LPS by rPLTP was examined using fluorescence dequenching experiments and native gel electrophoresis. The results suggested that rPLTP was able to mediate the exchange of LPS between micelles and the transfer of LPS to reconstituted HDL particles, but it did not transfer LPS to CD14. Consonant with these findings, rPLTP did not mediate CD14-dependent adhesive responses of neutrophils to LPS. These results suggest that while PLTP and LBP both bind and transfer LPS, PLTP is unable to transfer LPS to CD14 and thus does not mediate responses of cells to LPS.

Molecular biology of phospholipid transfer protein.

Lipid transfer proteins play an essential role in the intravascular dynamics of lipids among lipoproteins and between lipoproteins and cell membranes. Phospholipid transfer protein has been known for over a decade but, unlike cholesteryl ester transfer protein, has been investigated relatively little with regard to its physiological importance. The recent determination of the phospholipid transfer protein complementary DNA sequence as well as the further characterization of its gene structure will direct future studies toward the understanding of its structure-function correlations, physiological regulation, and clinical assessment at the molecular level. As a member of the lipid-transfer lipopolysaccharide-binding protein gene family, phospholipid transfer protein will attract investigators to studying its possible involvement in lipopolysaccharide or endotoxin interactions in addition to its phospholipid transfer activity.

Functional characterization of the promoter region of the human phospholipid transfer protein gene.

We have identified the functional promoter region of the human phospholipid transfer protein gene. Primer extension analysis indicates multiple sites for transcription initiation. Sequence analysis reveals that the promoter consists of TATA box, high GC region, and several consensus sequences for the potential binding of transcription factors. To assay promoter activity, DNA fragments with various lengths of the 5'-flanking region were fused upstream to the luciferase gene and transfected into HepG2, COS-7, and CHO cells. A minimal promoter of 159 base pairs between -230 and -72 relative to the first transcriptional initiation site is responsible for the full activity. Two motifs, Sp1 and AP-2, are located within this area. It may suggest that the PLTP promoter activity relies primarily on the putative cis-elements in the functional region.

Functional expression of human and mouse plasma phospholipid transfer protein: effect of recombinant and plasma PLTP on HDL subspecies.

The molecular cloning of mouse plasma phospholipid transfer protein (PLTP) and the eukaryotic cell expression of complementary DNA for mouse and human PLTP are described. Mouse PLTP was found to share 83% amino acid sequence identity with human PLTP. PLTP was produced in baby hamster kidney cells. Conditioned medium from BHK cells expressing PLTP possessed both phospholipid transfer activity and high density lipoprotein (HDL) conversion activity. PLTP mRNA was detected in all 16 human tissues examined by Northern blot analysis with ovary, thymus, and placenta having the highest levels. PLTP mRNA was also examined in eight mouse tissues with the highest PLTP mRNA levels found in the lung, brain, and heart. The effect of purified human plasma-derived PLTP and human recombinant PLTP (rPLTP) on the two human plasma HDL subspecies Lp(A-I) and Lp(A-I/A-II) was evaluated. Plasma PLTP or rPLTP converted the two distinct size subspecies of Lp(A-I) into a larger species, an intermediate species, and a smaller species. Lp(A-I/A-II) particles containing multiple size subspecies were significantly altered by incubation with either plasma or rPLTP with the largest but less prominent subspecies becoming the predominant one, and the smallest subspecies increasing in concentration. Thus, PLTP promoted the conversion of both Lp(A-I) and Lp(A-I/A-II) to populations of larger and smaller particles. Also, both human PLTP and mouse rPLTP were able to convert human or mouse HDL into larger and smaller particles. These observations suggest that PLTP may play a key role in extracellular phospholipid transport and modulation of HDL particles.

Purification and molecular cloning of human apolipoprotein F.

In our effort to study proteins that are involved in high density lipoprotein metabolism, we have identified apolipoprotein F and isolated a full length cDNA clone. Apolipoprotein F, with an apparent molecular mass of 29 kilodaltons, was purified from human high density lipoproteins using a modified two dimensional electrophoresis procedure. The cDNA, with a size of 1735 base pairs, was cloned from a Hep G2 cDNA library. The cDNA encodes apolipoprotein F, which is composed of 162 amino acids, and predicts that apolipoprotein F is a proteolytic product of a larger protein. Northern blot analysis indicates that apolipoprotein F mRNA is detected only in liver for the tissues examined. The gene was mapped to human chromosome number 12 using a human/rodent somatic cell hybrid mapping panel.