Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.     

Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. Two forms of PLTP exist in human plasma, one catalytically active (high activity form, HA-PLTP) and the other inactive (low activity form, LA-PLTP) (Oka, T., Kujiraoka, T., Ito, M., Egashira, T., Takahashi, S., Nanjee, N. M., Miller, N. E., Metso, J., Olkkonen, V. M., Ehnholm, C., Jauhiainen, M., and Hattori, H. (2000) J. Lipid Res. 41, 1651-1657). The two forms are associated with macromolecular complexes of different size. The apparent size of LA-PLTP is 520 kDa and that of HA-PLTP is 160 kDa. Of the circulating PLTP mass only a minor portion is in the HA-PLTP form in normolipidemic subjects. In the present study we have isolated and partially characterized the LA and HA forms of PLTP. Both LA- and HA-PLTP bind to heparin-Sepharose and can be separated by elution with 0-0.5 m NaCl gradient, with HA-PLTP displaying higher affinity for the matrix. LA-PLTP was further purified using hydrophobic butyl-Sepharose and anti-PLTP immunoaffinity chromatography steps. HA-PLTP was subjected to a second heparin-Sepharose step and hydroxylapatite chromatography. Analysis of the two forms of PLTP by SDS-PAGE, Western blotting, immunoprecipitation, and gel filtration demonstrates that LA-PLTP is complexed with apoA-I whereas HA-PLTP is not. Instead, HA-PLTP copurified with apoE. Based on these findings we suggest a model in which nascent PLTP enters the circulation as a high specific activity form not associated with apoA-I. During or after the transfer of lipolytic surface remnants to HDL, PLTP is transferred to apoA-I-containing HDL particles and thereby becomes part of the low activity complex.     

Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apolipoprotein A-I

Plasma phospholipid transfer protein (PLTP) is a factor that plays an important role in HDL metabolism. In this study we present data suggesting that PLTP has an inherent protease activity. After incubation of HDL3 in the presence of purified plasma PLTP, the d < 1.25 g/ml particles (fusion particles) contained intact 28.2 kDa apoA-I while the d > 1.25 g/ml fraction (apoA-I-PL complexes) contained, in addition to intact apoA-I, a cleaved 23 kDa form of apoA-I. Purified apoA-I was also cleaved by PLTP and produced a similar 23 kDa apoA-I fragment. The cleavage of apoA-I increased as a function of incubation time and the amount of PLTP added. The process displayed typically an 8-10 h lag or induction period, after which the cleavage proceeded in a time-dependent manner. This lag-phase was necessary for the development of the cleavage activity during incubation at 37 degrees C. The specific apoA-I cleavage activity of different PLTP preparations varied between 0.4-0.8 microg apoA-I degraded/h per 1000 nmol per h of PLTP activity. The 23 kDa apoA-I fragment reacted with monoclonal antibodies specific for the N-terminal part of apoA-I, indicating that the apoA-I cleavage occurred in the C-terminal portion. The apoA-I cleavage products were further characterized by mass spectrometry. The 23 kDa fragment yielded a mass of 22.924 kDa, demonstrating that the cleavage occurs in the C-terminal portion of apoA-I between amino acid residues 196 (alanine) and 197 (threonine). The intact apoA-I and the 23 kDa fragment revealed identical N-terminal amino acid sequences. The cleavage of apoA-I could be inhibited with APMSF and chymostatin, suggesting that it is due to a serine esterase-type of proteolytic activity. Recombinant PLTP produced in CHO cells or using the baculovirus-insect cell system caused an apoA-I cleavage pattern identical to that obtained with plasma PLTP. The present results raise the question of whether PLTP-mediated proteolytic cleavage of apoA-I might affect plasma HDL metabolism by generating a novel kinetic compartment of apoA-I with an increased turnover rate.     

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.     

Active plasma phospholipid transfer protein is associated with apoA-I- but not apoE-containing lipoproteins

Plasma phospholipid transfer protein (PLTP) is a multifaceted protein with diverse biological functions. It has been shown to exist in both active and inactive forms. To determine the nature of lipoproteins associated with active PLTP, plasma samples were adsorbed with anti-A-I, anti-A-II, or anti-E immunoadsorbent, and PLTP activity was measured in the resulting plasma devoid of apolipoprotein A-I (apoA-I), apoA-II, or apoE. Anti-A-I and anti-A-II immunoadsorbents removed 98 +/- 1% (n = 8) and 38 +/- 25% (n = 7) of plasma PLTP activity, respectively. In contrast, only 1 +/- 5% of plasma PLTP activity was removed by anti-E immunoadsorbent (n = 7). Dextran sulfate (DS) cellulose did not bind apoA-I, but it removed 83 +/- 5% (n = 4) of the PLTP activity in plasma. In size-exclusion chromatography, PLTP activity removed by anti-A-I or anti-A-II immunoadsorbent was associated primarily with particles of a size corresponding to HDL, whereas a substantial portion of the PLTP activity dissociated from DS cellulose was found in particles larger or smaller than HDL. These data show the following: 1) active plasma PLTP is associated primarily with apoA-I- but not apoE-containing lipoproteins; 2) active PLTP is present in HDL particles with and without apoA-II, and its distribution between these two HDL subpopulations varies widely among individuals; and 3) DS cellulose can remove active PLTP from apoA-I-containing lipoproteins, and this process creates new active PLTP-containing particles in vitro.     

Plasma phospholipid transfer activity is essential for increased atherogenesis in PLTP transgenic mice: a mutation-inactivation study

Plasma phospholipid transfer protein (PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene (mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration.     

ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP.In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.     

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in preβ-HDL generation

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce preβ-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.     

Inducible expression of phospholipid transfer protein (PLTP) in transgenic mice: acute effects of PLTP on lipoprotein metabolism

One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protein (PLTP), a plasma protein that is associated with HDL. In transgenic mice overexpressing human PLTP we found that elevated plasma PLTP levels dose-dependently increased the susceptibility to diet-induced atherosclerosis. This could be mainly due to the fact that most functions of PLTP are potentially atherogenic, such as decreasing plasma HDL levels. To further elucidate the role of PLTP in lipoprotein metabolism and atherosclerosis we generated a novel transgenic mouse model that allows conditional expression of human PLTP. In this mouse model a human PLTP encoding sequence is controlled by a Tet-On system. Upon induction of PLTP expression, our mouse model showed a strongly increased PLTP activity (from 3.0 ± 0.6 to 11.4 ± 2.8 AU, p < 0.001). The increase in PLTP activity resulted in an acute decrease in plasma cholesterol of 33% and a comparable decrease in phospholipids. The decrease in total plasma cholesterol and phospholipids was caused by a 35% decrease in HDL-cholesterol level and a 41% decrease in HDL-phospholipid level. These results demonstrate the feasibility of our mouse model to induce an acute elevation of PLTP activity, which is easily reversible. As a direct consequence of an increase in PLTP activity, HDL-cholesterol and HDL-phospholipid levels strongly decrease. Using this mouse model, it will be possible to study the effects of acute elevation of PLTP activity on lipoprotein metabolism and pre-existing atherosclerosis.     

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.     

Differences in reactivity of antibodies to active versus inactive PLTP significantly impacts PLTP measurement

Due to conflicting reports concerning the relationship between phospholipid transfer protein (PLTP) activity and mass in plasma, the protein concentration and activity of PLTP were assessed in fractions isolated by fast protein liquid chromatography from the plasma of healthy normolipidemic individuals. Using both polyclonal and monoclonal antibodies, PLTP was identified by Western blot analysis after both SDS and non-denaturing gradient gel electrophoresis, and quantitated by dot blot. PLTP activity was determined using a labeled vesicle/HDL assay. PLTP mass corresponded substantially with the activity distribution using the polyclonal antibody on dot blot with some inactive PLTP being present. However, the monoclonal antibody preferentially reacted with inactive PLTP, primarily associated with LDL and large HDL, overestimating inactive PLTP. Western blot analysis of non-denaturing gradient gels, using the polyclonal antibody, indicated that active PLTP was associated with numerous discrete HDL subpopulations (7.6-12.0 nm) with the major portion being 9-12 nm. Inactive PLTP was associated with particles of 12 to >17 nm. The monoclonal antibody demonstrated a different pattern of reactivity on gradient gels, showing strong reactivity with the inactive PLTP in particles of 12 to >17 nm, but less reactivity with particles of 7.6-12 nm. The differences in reactivities of antibodies for active versus inactive PLTP can account for some of the discrepancies reported in the literature regarding the relationship between PLTP mass and activity.     

Synthesis, modification, and flotation properties of rat hepatocyte apolipoproteins

We have studied apolipoprotein synthesis, intracellular modification and secretion by primary adult rat hepatocyte cultures using continuous pulse or pulse chase labeling with [35S]methionine, immunoprecipitation and two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis. The flotation properties of the newly secreted apolipoproteins were studied by discontinuous density gradient ultracentrifugation and one- and two-dimensional polyacrylamide gel electrophoresis. These studies showed that rat hepatocyte apoE is modified intracellularly to produce minor isoproteins that differ in size and charge. One of these minor isoproteins represents a monosialated apoE form (apoE3s1). Similarly, apoCIII is modified intracellularly to produce a disialated apoCIII form (apoCIIIs2), whereas newly synthesized apoA-I and apoA-IV are not glycosylated and overlap on two-dimensional gels with the proapoA-I and the plasma apoA-IV form, respectively. Both unmodified and modified apolipoproteins are secreted into the medium. Separation of secreted apolipoproteins by density gradient ultracentrifugation has shown that 50% of apoE, 80% of apoA-I, and more than 90% of apoA-IV and apoCIII are secreted in a lipid-poor form, whereas apoB-100 and apoB-48 are 100% associated with lipids. ApoB-100 floats in the VLDL and IDL regions, whereas apoB-48 is found in all lipoprotein fractions. ApoE and small amounts of apoA-I, apoA-IV and apoCIII float in the HDL region. Small amounts of apoE and apoCIII are also found in the VLDL and IDL regions, and apoE in the LDL region. Ultracentrifugation of nascent lipoproteins in the presence of rat serum promoted flotation of apoA-I and apoA-IV in the HDL fraction and resulted in increased flotation and distribution of apoE and apoCs in VLDL, IDL and LDL regions. These observations are consistent with the hypothesis that intracellular assembly of lipoproteins involves apoB-48 and apoB-100 forms, whereas a large portion of apoA-I, apoCIII and apoA-IV can be secreted in a lipid-poor form, which associates extracellularly with preexisting lipoproteins.     

Genetic variation of PLTP modulates lipoprotein profiles in hypoalphalipoproteinemia

Phospholipid transfer protein (PLTP) participates in key processes in lipoprotein metabolism, including interparticle phospholipid transfer, remodeling of HDL, cholesterol and phospholipid efflux from peripheral tissues, and the production of hepatic VLDL. The impact of PLTP on reverse cholesterol transport suggests that the gene may harbor sequence anomalies that contribute to disorders of HDL metabolism. The human PLTP gene was screened for sequence anomalies by DNA melting analysis in 276 subjects with hypoalphalipoproteinemia (HA) and 364 controls. The association with plasma lipid parameters was evaluated. We discovered 18 sequence variations, including four missense mutations and a novel polymorphism (c.-34G > C). In healthy controls, the c.-34G > C minor allele was associated with higher high density lipoprotein-cholesterol (HDL-C) and was depleted in subjects with HA. Linear regression models predict that possession of the rare allele decreases plasma triglyceride (TG) and TG/HDL-C and increases HDL-C independent of TG. Decreased PLTP activity was observed in one (p.R235W) of four (p.E72G, p.S119A, p.S124Y, and p.R235W) mutations in an in vitro activity assay. These findings indicate that PLTP gene variation is an important determinant of plasma lipoproteins and affects disorders of HDL metabolism.     

Distribution of human plasma PLTP mass and activity in hypo- and hyperalphalipoproteinemia

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism and reverse cholesterol transport. We have recently reported that plasma PLTP concentration correlates positively with plasma HDL cholesterol (HDL-C) but not with PLTP activity in healthy subjects. We have also shown that PLTP exists as active and inactive forms in healthy human plasma. In the present study, we measured plasma PLTP concentration and PLTP activity, and analyzed the distribution of PLTP in normolipidemic subjects (controls), cholesteryl ester transfer protein (CETP) deficiency, and hypo-alphalipoproteinemia (hypo-ALP). Plasma PLTP concentration was significantly lower (0.7 +/- 0.4 mg/l, mean +/- SD, n = 9, P < 0.001) in the hypo-ALP subjects, and significantly higher (19.5 +/- 4.3 mg/l, n = 17, P < 0.001) in CETP deficiency than in the controls (12.4 +/- 2.3 mg/l, n = 63). In contrast, we observed no significant differences in plasma PLTP activity between controls, hypo-ALP subjects, and CETP deficiency (6.2 +/- 1.3, 6.1 +/- 1.8, and 6.8 +/- 1.2 micro mol/ml/h, respectively). There was a positive correlation between PLTP concentration and plasma HDL-C (r = 0.81, n = 89, P < 0.001). By size exclusion chromatography analysis, we found that the larger PLTP containing particles without PLTP activity (inactive form of PLTP) were almost absent in the plasma of hypo-ALP subjects, and accumulated in the plasma of CETP deficiency compared with those of controls. These results indicate that the differences in plasma PLTP concentrations between hypo-ALP subjects, CETP deficiency, and controls are mainly due to the differences in the amount of the inactive form of PLTP.     

PLTP deficiency improves the anti-inflammatory properties of HDL and reduces the ability of LDL to induce monocyte chemotactic activity

We reported that phospholipid transfer protein (PLTP) deficiency decreased atherosclerosis in mouse models. Because the decreased atherosclerosis was accompanied by a significant decrease in plasma HDL levels, we examined the properties of PLTP knockout (PLTP0) HDL and tested its ability to prevent LDL-induced monocyte chemotactic activity in human artery wall cell cocultures. We isolated HDL and LDL from LDL receptor knockout/PLTP knockout (LDLr0/PLTP0) mice and from apolipoprotein B transgenic (apoBTg)/PLTP0 mice as well as their controls. PLTP0 HDL was relatively rich in protein and depleted in phosphatidylcholine. Turnover studies revealed a 3.5- to 4.0-fold increase in the turnover of protein and cholesteryl ester in HDL from PLTP0 mice compared with control mice. The ability of HDL from LDLr0/PLTP0 and apoBTg/PLTP0 mice to prevent the induction of monocyte chemotactic activity in human artery wall cell cocultures exposed to human LDL was dramatically better than that in controls. Moreover, LDL from PLTP0 mice was markedly resistant to oxidation and induced significantly less monocyte chemotactic activity compared with that in controls. In vitro, PLTP0 HDL removed significantly more oxidized phospholipids from LDL than did control HDL. We conclude that PLTP deficiency improves the anti-inflammatory properties of HDL in mice and reduces the ability of LDL to induce monocyte chemotactic activity.     

Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive

Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.     

Cholesteryl ester transfer protein and phospholipid transfer protein have nonoverlapping functions in vivo

Plasma phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are homologous molecules that mediate neutral lipid and phospholipid exchange between plasma lipoproteins. Biochemical experiments suggest that only CETP can transfer neutral lipids but that there could be overlap in the ability of PLTP and CETP to transfer or exchange phospholipids. Recently developed PLTP gene knock-out (PLTP0) mice have complete deficiency of plasma phospholipid transfer activity and markedly reduced high density lipoprotein (HDL) levels. To see whether CETP can compensate for PLTP deficiency in vivo, we bred the CETP transgene (CETPTg) into the PLTP0 background. Using an in vivo assay to measure the transfer of [(3)H]PC from VLDL into HDL or an in vitro assay that determined [(3)H]PC transfer from vesicles into HDL, we could detect no phospholipid transfer activity in either PLTP0 or CETPTg/PLTP0 mice. On a chow diet, HDL-PL, HDL-CE, and HDL-apolipoprotein AI in CETPTg/PLTP0 mice were significantly lower than in PLTP0 mice (45 +/- 7 versus 79 +/- 9 mg/dl; 9 +/- 2 versus 16 +/- 5 mg/dl; and 51 +/- 6 versus 100 +/- 9, arbitrary units, respectively). Similar results were obtained on a high fat, high cholesterol diet. These results indicate 1) that there is no redundancy in function of PLTP and CETP in vivo and 2) that the combination of the CETP transgene with PLTP deficiency results in an additive lowering of HDL levels, suggesting that the phenotype of a human PLTP deficiency state would include reduced HDL levels.     

Different phospholipid transfer protein complexes contribute to the variation in plasma PLTP specific activity

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids among lipoproteins. Over half of the PLTP in human plasma has been found to have little phospholipid transfer activity (inactive PLTP). We recently observed that plasma PLTP specific activity is inversely correlated with high-density lipoprotein (HDL) level and particle size in healthy adults. The purpose of this study was to evaluate the factors that contribute to the variation in plasma PLTP specific activity. Analysis of the specific activity of PLTP complexes in nine plasma samples from healthy adults revealed two clusters of inactive PLTP complexes with mean molecular weights (MW) of 342kDa and 146kDa. The large and small inactive PLTP complexes represented 52±8% (range 39-63%) and 8±8% (range 1-28%) of the plasma PLTP, respectively. Active PLTP complexes had a mean MW of 207kDa and constituted 40±6% (range 33-50%) of the plasma PLTP. The specific activity of active PLTP varied from 16 to 32μmol/μg/h. These data demonstrate for the first time the existence of small inactive plasma PLTP complexes. Variation in the amount of the two clusters of inactive PLTP complexes and the specific activity of the active PLTP contribute to the variation in plasma PLTP specific activity.     

Structure and phospholipid transfer activity of human PLTP: analysis by molecular modeling and site-directed mutagenesis.

The plasma phospholipid transfer protein (PLTP) is an important regulator of high density lipoprotein (HDL) metabolism. We have here, based on sequence alignments of the plasma LPS-binding/lipid transfer protein family and the X-ray structure of the bactericidal/permeability increasing protein (BPI), modeled the structure of PLTP. The model predicts a two-domain architecture with conserved lipid-binding pockets consisting of apolar residues in each domain. By site-directed mutagenesis of selected amino acid residues and transient expression of the protein variants in HeLa cells, the pockets are shown to be essential for PLTP-mediated phospholipid transfer. A solid phase ligand binding assay was used to determine the HDL-binding ability of the mutants. The results suggest that the observed decreases in phospholipid transfer activity of the N-terminal pocket mutants cannot be attributed to altered HDL-binding, but the C-terminal lipid-binding pocket may be involved in the association of PLTP with HDL. Further, the essential structural role of a disulfide bridge between cysteine residues 146 and 185 is demonstrated. The structural model and the mutants characterized here provide powerful tools for the detailed analysis of the mechanisms of PLTP function.     

Determination of human plasma phospholipid transfer protein mass and activity

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. PLTP is an 80-kDa glycoprotein that is expressed/secreted by a wide variety of tissues including lung, liver, adipose tissue, brain, and muscle. PLTP mediates a net transfer of phospholipids between vesicles and plasma HDLs. It also generates from small HDL particles large fused HDL particles with a concomitant formation of small lipid-poor apolipoprotein (apo) A-I-containing particles which are thought to act as the primary acceptors of cell-derived cholesterol from peripheral tissue macrophages. Another important function of PLTP is connected to lipolysis. Its role in the transfer of surface remnants from triglyceride-rich particles, very-low-density lipoproteins, and chylomicrons, to HDL is of importance for the maintenance of HDL levels. Recent observations from our laboratory have demonstrated that in circulation two forms of PLTP are present, one catalytically active (high-activity form, HA-PLTP) and the other a low-activity form (LA-PLTP). In view of the likely relevancy of PLTP in human health and disease, reliable and accurate methods for measuring plasma/serum PLTP activity and concentration are required. In this chapter, two radiometric PLTP activity assays are described: (i) exogenous, lipoprotein-independent phospholipid transfer assay and (ii) endogenous, lipoprotein-dependent phospholipid transfer assay. In addition, an ELISA method for quantitation of serum/plasma total PLTP mass as well as HA-PLTP and LA-PLTP mass is reported in detail.