The N-terminal globular domain and the first class A amphipathic helix of apolipoprotein A-I are important for lecithin:cholesterol acyltransferase activation and the maturation of high density lipoprotein in vivo.

To investigate the role of the N terminus of apolipoprotein A-I (apoA-I) in the maturation of high density lipoproteins (HDL), two N-terminal mutants with deletions of residues 1-43 and 1-65 (referred to as Delta 1-43 and Delta 1-65 apoA-I) were studied. In vitro, these deletions had little effect on cellular cholesterol efflux from macrophages but LCAT activation was reduced by 50 and 70% for the Delta 1-43 and Delta 1-65 apoA-I mutants, respectively, relative to wild-type (Wt) apoA-I. To further define the role of the N terminus of apoA-I in HDL maturation, we constructed recombinant adenoviruses containing Wt apoA-I and two similar mutants with deletions of residues 7-43 and 7-65 (referred to as Delta 7-43 and Delta 7-65 apoA-I, respectively). Residues 1-6 were not removed in these mutants to allow proper cleavage of the pro-sequence in vivo. Following injection of these adenoviruses into apoA-I-deficient mice, plasma concentrations of both Delta 7-43 and Delta 7-65 apoA-I were reduced 4-fold relative to Wt apoA-I. The N-terminal deletion mutants, in particular Delta 7-65 apoA-I, were associated with greater proportions of pre beta-HDL and accumulated fewer HDL cholesteryl esters relative to Wt apoA-I. Wt and Delta 7-43 apoA-I formed predominantly alpha-migrating and spherical HDL, whereas Delta 7-65 apoA-I formed only pre beta-HDL of discoidal morphology. This demonstrates that deletion of the first class A amphipathic alpha-helix has a profound additive effect in vivo over the deletion of the globular domain alone (amino acids 1-43) indicating its important role in the production of mature alpha-migrating HDL. In summary, the combined in vitro and in vivo studies demonstrate a role for the N terminus of apoA-I in lecithin:cholesterol acyltransferase activation and the requirement of the first class A amphipathic alpha-helix for the maturation of HDL in vivo.

Dynamic and stable pools of apoE differ functionally at the HepG2 cell surface.

Synthesis of apolipoprotein (apo)E in hepatocytes leads to both secretion and retention in cell surface pools. Inclusion of Brefeldin A to HepG2 cells prompted a rapid decrease of cell surface apoE to about 37% of control values after a 3-h incubation. The t(1/2) for this dynamic pool was estimated to be 15 min. In contrast, a stable fraction of apoE (t(1/2) > 20 h) was found in association with the extracellular matrix (ECM). Increased content of apoE on the ECM correlated with decreased binding of VLDL. Decreased apoE on the cell surface correlated with increased binding of VLDL to cells. Collectively, this suggests that glycosaminoglycan-bound apoE can occlude binding sites for apoE-containing lipoproteins on glycosaminoglycans. In solid-phase assays, heparin, suramin, and chondroitin sulfates A and B efficiently inhibited the binding of apoE to heparan sulfate proteoglycans, but were unable to displace apoE from this glycosaminoglycan. Finally, decreasing cell surface apoE with suramin subsequently decreased the apoE content on secreted apoB-containing lipoproteins without affecting the overall secretion of apoE or apoB to the extracellular medium. In summary, cell surface apoE comprises both dynamic fractions, which can be donated to newly secreted lipoproteins, and stable fractions, which may act to minimize the unproductive binding of lipoproteins to the ECM.

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Proteolytic degradation and impaired secretion of an apolipoprotein A-I mutant associated with dominantly inherited hypoalphalipoproteinemia.

We have devised a combined in vivo, ex vivo, and in vitro approach to elucidate the mechanism(s) responsible for the hypoalphalipoproteinemia in heterozygous carriers of a naturally occurring apolipoprotein A-I (apoA-I) variant (Leu(159) to Arg) known as apoA-I Finland (apoA-I(FIN)). Adenovirus-mediated expression of apoA-I(FIN) decreased apoA-I and high density lipoprotein cholesterol concentrations in both wild-type C57BL/6J mice and in apoA-I-deficient mice expressing native human apoA-I (hapoA-I). Interestingly, apoA-I(FIN) was degraded in the plasma, and the extent of proteolysis correlated with the most significant reductions in murine apoA-I concentrations. ApoA-I(FIN) had impaired activation of lecithin:cholesterol acyltransferase in vitro compared with hapoA-I, but in a mixed lipoprotein preparation consisting of both hapoA-I and apoA-I(FIN) there was only a moderate reduction in the activation of this enzyme. Importantly, secretion of apoA-I was also decreased from primary apoA-I-deficient hepatocytes when hapoA-I was co-expressed with apoA-I(FIN) following infection with recombinant adenoviruses, a condition that mimics secretion in heterozygotes. Thus, this is the first demonstration of an apoA-I point mutation that decreases LCAT activation, impairs hepatocyte secretion of apoA-I, and makes apoA-I susceptible to proteolysis leading to dominantly inherited hypoalphalipoproteinemia.

Influence of caveolin-1 on cellular cholesterol efflux mediated by high-density lipoproteins.

Caveolin-1 is a principal structural component of caveolae membranes. These membrane microdomains participate in the regulation of signaling, transcytosis, and cholesterol homeostasis at the plasma membrane. In the present study, we determined the effect of caveolin-1 expression on cellular cholesterol efflux mediated by high-density lipoprotein (HDL). We evaluated this effect in parental NIH/3T3 cells as well as in two transformed NIH/3T3 cell lines in which caveolin-1 protein levels are dramatically downregulated. Compared with parental NIH/3T3 cells, these two transformed cell lines effluxed cholesterol more rapidly to HDL. In addition, NIH/3T3 cells harboring caveolin-1 antisense also effluxed cholesterol more rapidly to HDL. However, this effect was not due to changes in total cellular cholesterol content. We further showed that chronic HDL exposure reduced caveolin-1 protein expression in NIH/3T3 cells. HDL exposure also inhibited caveolin-1 promoter activity, suggesting a direct negative effect of HDL on caveolin-1 gene transcription. Moreover, we showed that HDL-induced downregulation of caveolin-1 prevents the uptake of oxidized low-density lipoprotein in human endothelial cells. These data suggest a novel proatherogenic role for caveolin-1, i.e., regarding the uptake and/or transcytosis of modified lipoproteins.

Secondary structure of human apolipoprotein A-I(1-186) in lipid-mimetic solution.

The solution structure of an apoA-I deletion mutant, apoA-I(1-186) was determined by the chemical shift index (CSI) method and the torsion angle likelihood obtained from shift and sequence similarity (TALOS) method, using heteronuclear multidimensional NMR spectra of [u-(13)C, u-(15)N, u-50% (2)H]apoA-I(1-186) in the presence of sodium dodecyl sulfate (SDS). The backbone resonances were assigned from a combination of triple-resonance data (HNCO, HNCA, HN(CO)CA, HN(CA)CO and HN(COCA)HA), and intraresidue and sequential NOEs (three-dimensional (3D) and four-dimensional (4D) 13C- and 15N-edited NOESY). Analysis of the NOEs, H(alpha), C(alpha) and C' chemical shifts shows that apoA-I(1-186) in lipid-mimetic solution is composed of alpha-helices (which include the residues 8-32, 45-64, 67-77, 83-87, 90-97, 100-140, 146-162, and 166-181), interrupted by short irregular segments. There is one relatively long, irregular and mostly flexible region (residues 33-44), that separates the N-terminal domain (residues 1-32) from the main body of protein. In addition, we report, for the first time, the structure of the N-terminal domain of apoA-I in a lipid-mimetic environment. Its structure (alpha-helix 8-32 and flexible linker 33-44) would suggest that this domain is structurally, and possibly functionally, separated from the other part of the molecule.

Apolipoprotein A-I: structure-function relationships.

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.

Distinct central amphipathic alpha-helices in apolipoprotein A-I contribute to the in vivo maturation of high density lipoprotein by either activating lecithin-cholesterol acyltransferase or binding lipids.

Recombinant adenoviruses with cDNAs for human apolipoprotein A-I (wild type (wt) apoA-I) and three mutants, referred to as Delta4-5A-I, Delta5-6A-I, and Delta6-7A-I, that have deletions removing regions coding for amino acids 100-143, 122-165, and 144-186, respectively, were created to study structure/function relationships of apoA-I in vivo. All mutants were expressed at lower concentrations than wt apoA-I in plasma of fasting apoA-I-deficient mice. The Delta5-6A-I mutant was found primarily in the lipid-poor high density lipoprotein (HDL) pool and at lower concentrations than Delta4-5A-I and Delta6-7A-I that formed more buoyant HDL(2/3) particles. At an elevated adenovirus dose and earlier blood sampling from fed mice, both Delta5-6A-I and Delta6-7A-I increased HDL-free cholesterol and phospholipid but not cholesteryl ester. In contrast, wt apoA-I and Delta4-5A-I produced significant increases in HDL cholesteryl ester. Further analysis showed that Delta6-7A-I and native apoA-I could bind similar amounts of phospholipid and cholesterol that were reduced slightly for Delta5-6A-I and greatly for Delta4-5A-I. We conclude from these findings that amino acids (aa) 100-143, specifically helix 4 (aa 100-121), contributes to the maturation of HDL through a role in lipid binding and that the downstream sequence (aa 144-186) centered around helix 6 (aa 144-165) is responsible for the activation of lecithin-cholesterol acyltransferase.

Deletion of the C-terminal domain of apolipoprotein A-I impairs cell surface binding and lipid efflux in macrophage.

The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.

Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phospholipid efflux.

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins.

Effect of apolipoprotein A-I lipidation on the formation and function of pre-beta and alpha-migrating LpA-I particles.

A unique class of lipid-poor high-density lipoprotein, pre-beta1 HDL, has been identified and shown to have distinct functional characteristics associated with intravascular cholesterol transport. In this study we have characterized the structure/function properties of poorly lipidated HDL particles and the factors that mediate their conversion into multimolecular lipoprotein particles. Studies were undertaken with homogeneous recombinant HDL particles (LpA-I) containing apolipoprotein (apo) A-I and various amounts of palmitoyloleoylphosphatidylcholine (PC) and cholesterol. Complexation of apoA-I with small amounts of PC and cholesterol results in the formation of discrete lipoprotein structures that have a hydrated diameter of about 6 nm but contain only one molecule of apoA-I (Lp1A-I). While the molecular charge and alpha-helix content of apoA-I are unaffected by lipidation, the thermodynamic stability of the protein is reduced significantly (from 2.4 to 0.9 kcal/mol of apoA-I). Evaluation of apoA-I conformation by competitive radioimmunoassay with monoclonal antibodies shows that addition of small amounts of PC and cholesterol to apoA-I significantly increases the immunoreactivity of a number of domains over the entire molecule. Increasing the ratio of PC:apoA-I to 10:1 in the Lp1A-I complex is associated with increases in the alpha-helix content and stability of apoA-I. However, incorporation of 10-15 mol of PC destabilizes the Lp1A-I complex and promotes the formation of more thermodynamically stable (1.8 kcal/mol of apoA-I) bimolecular structures (Lp2A-I) that are approximately 8 nm in diameter. The formation of an Lp2A-I particle is associated with an increased immunoreactivity of most of the epitopes studied, with the exception of one central domain (residues 98-121), which becomes significantly less exposed. This structural change parallels a significant increase in the net negative charge on the complex. Characterization of the ability of these lipoproteins to act as substrates for lecithin:cholesterol acyltransferase (LCAT) shows that unstable Lp1A-I complexes stimulate a higher rate of cholesterol esterification by LCAT than the small but more stable Lp2A-I particles (Vmax values are 5.8 and 0.3 nmol of free cholesterol esterified/h, respectively). The ability of LCAT to interact with lipid-poor apoA-I suggests that LCAT does not need to bind to the lipid interface on an HDL particle but that LCAT may directly interact with apoA-I. The data suggests that lipid-poor HDL particles may be metabolically reactive particles because they are thermodynamically unstable.

ApoE of the HepG2 cell surface includes a major pool associated with chondroitin sulfate proteoglycans.

We have investigated the association of apolipoprotein E (apoE) with the HepG2 cell surface (i.e. plasma membrane and extracellular matrix) using domain specific monoclonal antibodies against apoE. Growth in beta-D-xyloside decreased the incorporation of 35S into glycosaminoglycans by 31% and cell surface apoE by 45% with a concomitant increase in apoE secretion (4.3-fold), underlining the importance of glycosaminoglycan association of apoE. Heparinase (3-10 U/mL) or heparin (1 mg/mL) decreased apoE by 25 and 30.5%, respectively, which suggests that some apoE is associated with cell surface heparan sulfate proteoglycans. Chondroitinase ABC (1.5 U/mL) reduced cell surface apoE by 40%, indicating that a major pool of apoE is associated with chondroitin sulfate proteoglycans. Further enzymatic and displacement analysis suggested that cell surface apoE associates specifically with GAGs containing chondroitin-4-sulfates. 3H1, a monoclonal antibody that recognizes an epitope within the lipid-binding C-terminal domain of apoE, decreased binding of apoE to chondroitin sulfate proteoglycans in solid-phase assays by 77% and to heparan sulfate proteoglycans by 46%, suggesting that this region is of increased importance for binding to chondroitin sulfate proteoglycans. Previous studies with 3H1 demonstrated that apoE of the extracellular matrix is lipid-poor (Burgess, J. W., Gould, D. R., and Marcel, Y. L. (1998) J. Biol. Chem. 273, 5645-5654), but we show here that apoE on the remaining cell surface is lipid-associated. In summary, lipidated apoE associates with the HepG2 plasma membrane through interactions with chondroitin-4-sulfate containing GAGs and, to a lesser extent, HSPG.

Importance of central alpha-helices of human apolipoprotein A-I in the maturation of high-density lipoproteins.

We have studied the role of amphipathic alpha-helices in the ability of apoA-I to promote cholesterol efflux from human skin fibroblasts and activate lecithin:cholesterol acyltransferase (LCAT). Three apoA-I mutants were designed, each by deletion of a pair of predicted adjacent central alpha-helices [Delta(100-143), Delta(122-165), Delta(144-186)], and expressed in Escherichia coli. This strategy was used to minimize disruption of the predicted secondary structure of the resulting protein. These three central deletion mutants have been previously shown to be expressed as stable folded proteins but to exhibit altered phospholipid-binding properties. When recombined with phospholipids to form homogeneous LpA-I containing equivalent amounts of POPC and tested for their ability to promote diffusional cholesterol efflux from normal [3H]cholesterol-labeled fibroblasts, each mutant and the wild-type recombinant protein (Rec.-apoA-I) promoted cholesterol efflux with very similar rates at all the concentrations tested. These experiments showed that all LpA-I could acquire cellular cholesterol with similar affinity and binding capacity. However, when the cell-incubated LpA-I were incubated with purified LCAT, two mutants, Delta(122-165) and Delta(144-186), appeared incapable of activating the enzyme. To directly determine their ability to activate LCAT, each mutant and the control were recombined with equivalent amounts of cholesterol and phospholipid and incubated with the purified enzyme. The results show that whereas deletion of residues 100-143 has little effect on LCAT activation, deletion of residues 122-165 or 144-186 results in an inability of the mutants to promote cholesterol esterification. In conclusion, our results show that no specific sequence in the central domain of apoA-I is required for efficient diffusional cholesterol efflux from normal fibroblasts; however, residues 144-186 appear critical for optimum LCAT activation and cholesteryl ester accumulation. Since deletion of residues 144-186 also perturbs phospholipid association and prevents the formation of large LpA-I particles [Frank, P. G., Bergeron, J., Emmanuel, F., Lavigne, J. P., Sparks, D. L., Denèfle, P., Rassart, E., and Marcel, Y. L. (1997) Biochemistry 36, 1798-1806], the data show that this pair of alpha-helices plays an important role in the maturation of HDL. Sequence analysis of these apoA-I helices further identifies specific residues that appear essential to this activity.

The HepG2 extracellular matrix contains separate heparinase- and lipid-releasable pools of ApoE. Implications for hepatic lipoprotein metabolism.

We have examined the association of apoE with the extracellular matrix (ECM) of HepG2 cells. Comparison of ECM prepared by previously published methods demonstrated that cytochalasin B-prepared material yielded the highest endogenous apoE, representing 23.6% of that in cell monolayers. ECM prepared with EDTA or Triton X-100 exhibited decreased levels of apoE, 3 and 6%, respectively. ECM bound very low density lipoprotein poorly (5-6% of the monolayer capacity); however, these incubations dramatically increased the apoE content of the ECM. Heparinase or suramin decreased apoE of the ECM by 19.6 and 37.3%, respectively, suggesting association with heparin sulfate proteoglycans. EDTA or EGTA also displaced 35% of the apoE, suggesting a Ca2+-dependent association. Incubation with phosphatidylcholine vesicles (PCV) displaced 30% of the apoE, suggesting that lipid content affects association of apoE with the ECM. Data derived from sequential incubations with combinations of suramin, EGTA, and PCV were consistent with the presence of two distinct pools of apoE on the HepG2 ECM, one releasable with suramin and EGTA and the other releasable with lipids. Exogenously applied lipid-free apoE readily bound to the ECM; however, increasing the lipid content decreased its association. Lipid-free apoE could be equally displaced from the ECM with PCV or suramin. When lipid-free apoE adsorbed to microtiter wells was incubated with a triglyceride emulsion or palmitoyloleyl phosphatidylcholine micelles, the immunoreactivity of 3H1 (but not other antibodies), a monoclonal antibody against an epitope in the C-terminal domain of apoE, increased about 4-fold. In a similar manner, incubation of ECM with lipid dramatically increased the immunoreactivity of 3H1, indicating that apoE of the ECM exists in a lipid-poor form. Scatchard analysis demonstrated that the increased immunoreactivity was due to an increase in the number of antibody binding sites. In conclusion, the ECM contains two pools of lipid-poor apoE. One pool associates with the ECM through heparin sulfate proteoglycans- and Ca2+-dependent interactions. A second pool of apoE dissociates from the ECM upon lipidation. The lipid-sensitive pool of apoE may participate in secretion or efflux of lipids or in the capture of lipoproteins by providing the apoE needed for receptor-mediated uptake.

Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I.

The interaction of plasma phospholipid transfer protein (PLTP) with HDL has not been characterized in detail, although we have reported that the apoA-I/apoA-II molar ratio in the HDL particle influences PLTP-mediated HDL conversion, but not phospholipid transfer. The aim of this study was to examine whether PLTP binds apoA-I or apoA-II, and if this occurs, then determine the PLTP-binding domain of the apoA-I molecule. To study the PLTP/apolipoprotein interaction we used a solid phase ligand binding assay, the ELISA technique, and apoA-I and apoA-II affinity chromatography. PLTP bound to both apoA-I and apoA-II affinity columns, a finding subsequently utilized in the purification of PLTP. PLTP also bound to both apoA-I and apoA-II on ELISA plates in a concentration-dependent manner, and the binding could be displaced by preincubating the PLTP sample with purified apolipoproteins. To determine which portion of apoA-I is recognized by PLTP, we coated ELISA plates with either recombinant full-length apoA-I or three shortened apoA-I forms sequentially truncated from the C-terminus. To characterize the PLTP binding ability of the C-terminal region of apoA-I, we used both C-terminal CNBr-fragment and a synthetic C-terminal peptide of apoA-I. To further confirm the identity of the binding region, we probed the interaction with a polyclonal and several monoclonal anti-apoA-I antibodies. The antibodies that inhibited the interaction between PLTP and apoA-I were directed towards apoA-I epitopes localized between amino acids 27-141. The polyclonal antibody, R33, and the monoclonal antibody A-I-1 (epitope between amino acids 27-48) were most effective and reduced PLTP binding by 70%. These results show that PLTP binds to both apoA-I and apoA-II, and that the PLTP binding domain of apoA-I resides in the amino terminal region.

Evolution of mammalian apolipoprotein A-I and conservation of antigenicity: correlation with primary and secondary structure.

We have evaluated the immunoreactivity of 20 monoclonal antibodies (mAbs) directed against human apolipoprotein (apo)A-I with a panel of high density lipoproteins (HDL) from 13 mammalian species. The pattern of cross-reactivity showed that 20 mAbs had different specificity. While not all mAbs recognized apoA-I from all of the different species, the antigenicity of some sequences was well conserved. Thus, mAb A05 cross-reacted with all species except guinea pig and rat. In contrast, the mAb 4H1, which recognized residues 2-8, required a specific proline in position 3, as no immunoreactivity was found in the species missing this amino acid. Furthermore, the presence of a threonine residue in place of serine (in position 6) in the cynomolgus monkey was associated with a 20-fold loss of immunoreactivity in radioimmunometric assay with 4H1. As most of the epitopes were found in CNBr fragments 2 and 3, we sequenced these regions in four species (horse, goat, sheep, and cat) and analyzed the alignment of most known sequences to evaluate their consensus. Except for the rat and the chicken, considerable identity was observed. This permitted us to deduce the involvement of the residues in some antigenic epitopes. In the middle of apoA-I, a conservative mutation Asp103-->Glu was found sufficient to eliminate all reactivity of this epitope for A11 (residues 99-105 ... 12l6-132) in five species (rabbit, cow, goat, sheep, and rat). The residues essential to the expression of two other epitopes overlapping with A11 were also characterized. Edmundson-wheel representation of 18-residue repeated sequences of the different apoA-I species (for the eight amphipatic helices of residues 46-63, 68-85, 101-118, 123-140, 143-160, 167-184, 189-206, and 222-239) showed that secondary structure of apoA-I was more conserved than the antigenic epitopes. The N-terminal region, residues 1 to about 98, is rich in both strictly preserved sequences and epitope expression in most of the species surveyed. This evolutionary conservation of the N-terminal domain suggests an important yet unknown function.

Deletion of central alpha-helices in human apolipoprotein A-I: effect on phospholipid association.

In order to better understand the structure-function properties of apolipoprotein (apo) A-I, we have constructed and expressed three apoA-I mutants using a system previously described for the expression of human apolipoprotein A-I (Rec.-apoA-I). These mutants (corresponding to deletion of apoA-I residues 100-143, 122-165, 144-186) have been studied for their ability to form reconstituted apoA-I-containing lipoproteins (LpA-I) with POPC and DMPC, and for their structural and physical properties. Rec.- and native apoA-I can form homogeneous discoidal Lp2A-I over a wide range of POPC/apoA-I ratios [(20-130)/1] and exhibit sizes ranging from 9.5 to 10.5 nm. When recombined with varying POPC content [(20-130)/1, POPC/A-I)], the three mutants produce homogeneous discoidal Lp2A-I that contain a low POPC/A-I molar ratio [(20-40)/l for all mutants] and exhibit a nearly constant size [7.5-7.6 nm for delta (100-143) and 7.9-8.0 nm for the other two mutants]. Kinetics of association of these proteins with DMPC are similar for delta (100-143) and Rec.-apoA-I (t 1/2 of 4.0 and 4.4 min, respectively) but appear significantly reduced for delta (122-165) and delta (144-186) (t 1/2 of 7.5 and 6.9 min, respectively). While in the lipid-free form, all proteins have a similar thermodynamic stability with a very comparable free energy of unfolding (delta GD degree) for the alpha-helical structure, as determined by isothermal denaturation studies. delta-(100-143) has a significantly lower alpha-helical content (33%) as compared to the other proteins [40, 41, and 45% for Rec.-apoA-I. delta (122-165), and delta (144-186), respectively]. When associated to POPC, delta (122-165) and delta (144-186) have a higher alpha-helicity (63 and 63%) and an enhanced stability (2.5 and 2.3 kcal/mol, respectively) as compared to delta (100-143) (49% and 1.8 kcal/mol) and Rec.-apoA-I (52% and 1.9 kcal/mol). These results suggest that the amphipathic alpha-helices within residues 100-186 are directly involved in interactions with phospholipids. The helical region 100-121 appears to be more important to the stabilization of the lipid-apoprotein complex formed whereas helices within residues 122-186 appear to be critical to the initial rates of association of the apoprotein with DMPC. These data suggest that an important role of the central domain 100-186 may be to maintain the plasticity of apoA-I and its ability to form different classes of HDL particles. Therefore, it is likely that this region may also play an important role in the functional properties of this apoprotein.

Characterization of human apolipoprotein A-I expressed in Escherichia coli.

Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)6-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/1 of culture medium. We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques. The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine. His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles. Lipid-bound native apoA-I and His-apoA-I showed very similar alpha-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I). The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes. In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values. Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I. The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture. In conclusion, we show that His-apoA-I expressed in E. coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux.

Effect of the apolipoprotein A-I and surface lipid composition of reconstituted discoidal HDL on cholesterol efflux from cultured fibroblasts.

Five series of reconstituted discoidal HDL (LpA-I) particles have been prepared, and their constituents, apolipoprotein A-I (apoA-I), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), unesterified cholesterol (UC), phosphatidylinositol (PI), or sphingomyelin (SM), have been systematically varied to elucidate the relationship between HDL composition and cholesterol efflux from non-cholesterol-loaded human skin fibroblasts. The physical properties, such as hydrodynamic diameters, alpha-helix contents, and surface potentials, of these LpA-I have been measured and related to the ability of the LpA-I to accept cellular cholesterol. The results show that for LpA-I particles containing 2, 3, or 4 apoA-I per particle, Lp4A-I are the best acceptors of cellular cholesterol, followed by Lp3A-I and then Lp2A-I particles. Discoidal Lp2A-I with variations in POPC content, from 121 to 266 mol/particle; show no difference in their abilities to promote cholesterol efflux. Similarly, inclusion of 7 and 15 mol of free cholesterol to Lp2A-I also does not affect their ability to accept cellular cholesterol. However, increasing the content of either PI or SM, up to 20 mol/particle, is associated with significantly increased abilities of the LpA-I to promote cholesterol efflux. The efflux of cellular cholesterol to discoidal LpA-I particles is independent of specific changes in apoA-I conformation and charge, but appears to be positively related to major changes in the size of the lipoprotein particle. The study suggests that in contrast to interlipoprotein cholesterol transfers, the efflux of cholesterol from cultured fibroblasts is less sensitive to factors that affect the frequency of molecular collisions and more dependent on the ability of an HDL particle to absorb and retain cholesterol molecules. Since SM and PI appear to modulate this adsorption/desorption of cholesterol to HDL, variations in the concentration of these lipids within HDL would be expected to affect plasma cholesterol homeostasis.

Role of Lp A-I and Lp A-I/A-II in cholesteryl ester transfer protein-mediated neutral lipid transfer. Studies in normal subjects and in hypertriglyceridemic patients before and after fenofibrate therapy.

The two major subclasses of HDL contain apo A-I only (Lp A-I) or both apo A-I and apo A-II (Lp A-I/A-II). We have carried out experiments to quantify the participation of Lp A-I and Lp A-I/A-II in the neutral lipid transfer reaction in normal and hypertriglyceridemic subjects. Thirteen hypertriglyceridemic subjects were studied before and after fenofibrate therapy. Fenofibrate treatment resulted in decreases in total cholesterol, triglycerides (TG), and VLDL cholesterol of 19%, 48%, and 70%, respectively, and a 28% increase in HDL cholesterol, with no significant change in the proportion of Lp A-I and Lp A-I/A-II particles. The abundance of cholesteryl ester transfer protein (CETP) mRNA in peripheral adipose tissue decreased with treatment in four of five patients studied; however, no change occurred in plasma CETP mass. Using an isotopic transfer assay, we demonstrated that both Lp A-I and Lp A-I/A-II participated in the CE transfer reaction, with no change after fenofibrate therapy. This finding suggests that the marked increase in HDL cholesterol during fenofibrate therapy is due to normalization of plasma TG and hence decreased opportunity for mass transfer of lipid between HDL and TG-rich proteins in vivo. In this population of hypertriglyceridemic subjects, CETP was distributed in both the Lp A-I and Lp A-I/A-II subfractions of HDL, with preferential association with the smaller Lp A-I poor. In contrast, in nine normal subjects studied, negligible amounts of CETP were associated with Lp A-I/A-II. Nonetheless, the Lp A-I/A-II fraction of HDL contributed significantly to total CE mass transfer in normolipidemic plasma. Lp A-I/A-II is an efficient donor for CE transfer to TG-rich lipoproteins, and its low affinity for CETP may in fact facilitate neutral lipid transfer either by a shuttle mechanism or by formation of a ternary complex.