The interfacial properties of ApoA-I and an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at the triolein/water interface

Apolipoprotein A-I (apoA-I) is the major protein in high density lipoprotein (HDL). During lipid metabolism, apoA-I moves among HDL and triacylglycerol-rich lipoproteins. The main structure and the major lipid binding motif of apoA-I is the amphipathic alpha-helix. To understand how apoA-I behaves at hydrophobic lipoprotein interfaces, the interfacial properties of apoA-I and an amphipathic alpha-helical consensus sequence peptide (CSP) were studied at the triolein/water (TO/W) interface. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-residue tandem repeat sequences that form amphipathic alpha-helices modeling the central part of apoA-I. ApoA-I or CSP added into the aqueous phase surrounding a triolein drop lowered the interfacial tension (gamma) of TO/W in a concentration- and time-dependent fashion. The gamma(TO/W) was lowered approximately 16 millinewtons (mN)/m by apoA-I at 1.4 x 10(-6) m and approximately 15 mN/m by CSP at 2.6 x 10(-6) m. At equilibrium gamma, both apoA-I and CSP desorbed from the interface when compressed and readsorbed when expanded. The maximum surface pressure CSP could withstand without being ejected (PiMAX) was 16 mN/m. The PiMAX) of apoA-I was only 14.8 mN/m, but re-adsorption kinetics suggested that only part of the apoA-I desorbed at Pi between 14.8 and 19 mN/m. However, above approximately 19 mN/m (PiOFF) the entire apoA-I molecule desorbed into the water. ApoA-I was more flexible at the TO/W interface than CSP and showed more elasticity at oscillation periods 4-128 s even at high compression, whereas CSP was elastic only at faster periods (4 and 8 s) and moderate compression. Flexibility and surface pressure-mediated desorption and re-adsorption of apoA-I probably provides lipoprotein stability during metabolic-remodeling reactions in plasma.     

Apolipoprotein C-I binds more strongly to phospholipid/triolein/water than triolein/water interfaces: a possible model for inhibiting cholesterol ester transfer protein activity and triacylglycerol-rich lipoprotein uptake

Apolipoprotein C-I (apoC-I) is an important constituent of high-density lipoprotein (HDL) and is involved in the accumulation of cholesterol ester in nascent HDL via inhibition of cholesterol ester transfer protein and potential activation of lecithin:cholesterol acyltransferase (LCAT). As the smallest exchangeable apolipoprotein (57 residues), apoC-I transfers between lipoproteins via a lipid-binding motif of two amphipathic α-helices (AαHs), spanning residues 7-29 and 38-52. To understand apoC-I's behavior at hydrophobic lipoprotein surfaces, oil drop tensiometry was used to compare the binding to triolein/water (TO/W) and palmitoyloleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. When apoC-I binds to either interface, the surface tension (γ) decreases by ~16-18 mN/m. ApoC-I can be exchanged at both interfaces, desorbing upon compression and readsorbing on expansion. The maximal surface pressures at which apoC-I begins to desorb (Π(max)) were 16.8 and 20.7 mN/m at TO/W and POPC/TO/W interfaces, respectively. This suggests that apoC-I interacts with POPC to increase its affinity for the interface. ApoC-I is more elastic on POPC/TO/W than TO/W interfaces, marked by higher values of the elasticity modulus (ε) on oscillations. At POPC/TO/W interfaces containing an increasing POPC:TO ratio, the pressure at which apoC-I begins to be ejected increases as the phospholipid surface concentration increases. The observed increase in apoC-I interface affinity due to higher degrees of apoC-I-POPC interactions may explain how apoC-I can displace larger apolipoproteins, such as apoE, from lipoproteins. These interactions allow apoC-I to remain bound to the interface at higher Π values, offering insight into apoC-I's rearrangement on triacylglycerol-rich lipoproteins as they undergo Π changes during lipoprotein maturation by plasma factors such as lipoprotein lipase.     

Surface study of apoB1694�C1880, a sequence that can anchor apoB to lipoproteins and make it nonexchangeable

Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein. During lipoprotein assembly, it recruits phospholipids and triacylglycerols (TAG) into TAG-rich lipoprotein particles. It remains bound to secreted lipoproteins during lipid metabolism in plasma. The β1 region (residues 827–1880) of apoB has a high amphipathic β strand (AβS) content and is proposed to be one region anchoring apoB to lipoproteins. The AβS-rich region between apoB37 and apoB41 (residues 1694–1880) was cloned, expressed, and purified. The interfacial properties were studied at the triolein/water (TO/W) and air/water (A/W) interfaces. ApoB[37–41] is surface-active and adsorbs to the TO/W interface. After adsorption the unbound apoB[37–41] was removed from the aqueous phase. Adsorbed apoB[37–41] did not desorb and could not be forced off by increasing the surface pressure up to 23 mN/m. ApoB[37–41] adsorbed on the TO/W interface was completely elastic when compressed and expanded by ±13% of its area. On an A/W interface, the apoB[37–41] monolayer became solid when compressed to 4 mN/m pressure indicating extended β-sheet formation. It could be reversibly compressed and expanded between low pressure and its collapse pressure (35 mN/m). Our studies confirm that the AβS structure of apoB[37–41] is a lipid-binding motif that can irreversibly anchor apoB to lipoproteins.     

Surface behavior of apolipoprotein A-I and its deletion mutants at model lipoprotein interfaces

Apolipoprotein A-I (apoA-I) has a great conformational flexibility to exist in lipid-free, lipid-poor, and lipid-bound states during lipid metabolism. To address the lipid binding and the dynamic desorption behavior of apoA-I at lipoprotein surfaces, apoA-I, Δ(185-243)apoA-I, and Δ(1-59)(185-243)apoA-I were studied at triolein/water and phosphatidylcholine/triolein/water interfaces with special attention to surface pressure. All three proteins are surface active to both interfaces lowering the interfacial tension and thus increasing the surface pressure to modify the interfaces. Δ(185-243)apoA-I adsorbs much more slowly and lowers the interfacial tension less than full-length apoA-I, confirming that the C-terminal domain (residues 185-243) initiates the lipid binding. Δ(1-59)(185-243)apoA-I binds more rapidly and lowers the interfacial tension more than Δ(185-243)apoA-I, suggesting that destabilizing the N-terminal α-helical bundle (residues 1-185) restores lipid binding. The three proteins desorb from both interfaces at different surface pressures revealing that different domains of apoA-I possess different lipid affinity. Δ(1-59)(185-243)apoA-I desorbs at lower pressures compared with apoA-I and Δ(185-243)apoA-I indicating that it is missing a strong lipid association motif. We propose that during lipoprotein remodeling, surface pressure mediates the adsorption and partial or full desorption of apoA-I allowing it to exchange among different lipoproteins and adopt various conformations to facilitate its multiple functions.     

C-Terminus of Apolipoprotein A-I Removes Phospholipids from a Triolein/Phospholipids/Water Interface, but the N-Terminus Does Not: A Possible Mechanism for Nascent HDL Assembly

Apolipoprotein A-I (ApoA-I) is the principle protein component of HDL, also known as “good cholesterol,” which is an inverse marker for cardiovascular disease. The N-terminal 44 amino acids of ApoA-I (N44) are predicted to be responsible for stabilization of soluble ApoA-I, whereas the C-terminal 46 amino acids (C46) are predicted to initiate lipid binding and oligomerization. In this work, we apply what we believe to be a novel application of drop tensiometry to study the adsorption and desorption of N44 and C46 at a triolein/POPC/water (TO/POPC/W) interface. The amount of peptide that adsorbed to the surface was dependent on the surface concentration of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and pressure (Π) before adsorption. At a TO/POPC/W interface, the exclusion pressure (Π_EX) of C46 was 25.8 mN/m, and was 19.3 mN/m for N44. Once adsorbed, both peptides formed a homogeneous surface with POPC but were progressively ejected from the surface by compression. During a compression, C46 removed POPC from the surface whereas N44 did not. Repeated compressions caused C46 to deplete entirely the surface of phospholipid. If full-length ApoA-I could also remove phospholipid, this could provide a mechanism for the transfer of surface components of chylomicrons and very low density lipoprotein to high density lipoprotein with the assistance of phospholipid transfer protein.     

Conformation and lipid binding of the N-terminal (1-44) domain of human apolipoprotein A-I

Because of its role in reverse cholesterol transport, human apolipoprotein A-I is the most widely studied exchangeable apolipoprotein. Residues 1-43 of human apoA-I, encoded by exon 3 of the gene, are highly conserved and less well understood than residues 44-243, encoded by exon 4. In contrast to residues 44-243, residues 1-43 do not contain the 22 amino acid tandem repeats thought to form lipid binding amphipathic helices. To understand the structural and functional roles of the N-terminal region, we studied a synthetic peptide representing the first 44 residues of human apoA-I ([1-44]apoA-I). Far-ultraviolet circular dichroism spectra showed that [1-44]apoA-I is unfolded in aqueous solution. However, in the presence of n-octyl beta-d-glucopyranoside, a nonionic lipid mimicking detergent, above its critical micelle concentration ( approximately 0.7% at 25 degrees C), sodium dodecyl sulfate, an ionic detergent, above its CMC ( approximately 0.2%), trimethylamine N-oxide, a folding inducing organic osmolyte, or trifluoroethanol, an alpha-helix inducer, alpha-helical structure was formed in [1-44]apoA-I up to approximately 45%. Characterization by density gradient ultracentrifugation and visualization by negative staining electron microscopy demonstrated that [1-44]apoA-I interacts with dimyristoylphosphatidylcholine (DMPC) over a wide range of lipid:peptide ratios from 1:1 to 12:1 (w/w). At 1:1 DMPC:[1-44]apoA-I (w/w) ratio, discoidal complexes with composition approximately 4:1 (w/w) and approximately 100 A diameter were formed in equilibrium with free peptide. At higher ratios, discoidal complexes were shown to exist together with a heterogeneous population of lipid vesicles with peptide bound also in equilibrium with free peptide. When bound to DMPC, [1-44]apoA-I has approximately 60% helical structure, independent of whether it forms discoidal or vesicular complexes. This helical content is consistent with that of the predicted G helix (residues 8-33). Our data provide the first strong and direct evidence that the N-terminal region of apoA-I binds lipid and can form discoidal structures and a heterogeneous population of vesicles. In doing so, approximately 60% of this region folds into alpha-helix from random coil. The composition of the 100 A discoidal complex is approximately 5 [1-44]apoA-I and approximately 150 DMPC molecules per disk. The helix length of 5 [1-44]apoA-I molecules in lipid-bound form is just long enough to wrap around the DMPC bilayer disk once.     

Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films

The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer.     

Interfacial properties of amphipathic beta strand consensus peptides of apolipoprotein B at oil/water interfaces

The region between residues 968 and 1882 of apolipoprotein B (apoB-21 to apoB-41) is rich in amphipathic beta strands (AbetaSs) and promotes the assembly of primordial triacylglyceride (TAG)-rich lipoproteins. To understand the importance of AbetaS in recruiting TAG, the interfacial properties of two AbetaS consensus peptides, P12 and P27, were studied at dodecane/water (DD/W) and triolein/water (TO/W) interfaces. P12 (acetyl-LSLSLNADLRLK-amide) and P27 (acetyl-LSLSLNADLRLKNGNLSLSLNADLRLK-amide), when added into the aqueous phase surrounding a suspended oil drop (dodecane or triolein), decreased the interfacial tension (gamma) in a concentration-dependent manner. At the DD/W interface, 1 x 10(-5) M P12 decreased gamma to approximately 20 mN/m and 6.6 x 10(-6) M P27 decreased gamma to approximately 13 mN/m. At the TO/W interface, 1.5 x 10(-5) M P12 decreased gamma to approximately 14 mN/m and 9.0 x 10(-6) M P27 decreased gamma to approximately 12 mN/m. The surface area of both peptides was between 11.2 and 15.1 angstroms2 per residue, consistent with beta sheets lying flat on DD/W and TO/W interfaces. P12 and P27 are almost purely elastic on DD/W, TO/W, and air/water interfaces. When P12 and P27 were compressed beyond the equilibrium gamma to as low as 4 mN/m, they could not be readily desorbed from either interface. These properties probably help in assembling nascent TAG-rich lipoproteins, and AbetaS may anchor apoB to beta lipoproteins.     

Conformation and lipid binding of a C-terminal (198-243) peptide of human apolipoprotein A-I

Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into approximately 60% alpha-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46-residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10 and 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far-ultraviolet circular dichroism spectra show that [198-243]apoA-I is also unfolded in aqueous solution. However, self-association induces approximately 50% alpha-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde, and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid-mimicking detergents, above their CMC, approximately 60% alpha-helix was induced in the peptide. In contrast, SDS, an anionic lipid-mimicking detergent, induced helical folding in the peptide at a concentration of approximately 0.003% (approximately 100 microM), approximately 70-fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solubilize dimyristoylphosphatidylcholine (DMPC) liposomes and fold into approximately 60% alpha-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy demonstrated that the peptide binds to DMPC with a high affinity to form at least two sizes of relatively homogeneous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide weight ratio, diameter, and density) of both complexes are similar to those of plasma A-I/DMPC complexes formed under similar conditions: small discoidal complexes (approximately 3:1 weight ratio, approximately 110 A, and approximately 1.10 g/cm3) formed at an initial 1:1 weight ratio and larger discoidal complexes (approximately 4.6:1 weight ratio, approximately 165 A, and approximately 1.085 g/cm3) formed at initial 4:1 weight ratio. The cross-linking data for the peptide on the complexes of two sizes is consistent with the calculated peptide numbers per particle. Compared to the approximately 100 A disk-like complex formed by the N-terminal peptide in which helical structure was insufficient to cover the disk edge by a single belt, the compositions of these two types of complexes formed by the C-terminal peptide are more consistent with a "double belt" model, similar to that proposed for full-length apoA-I. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this C-terminal peptide model can mimic the interaction with the phospholipid of plasma apoA-I to form two sizes of homogeneous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.     

Effects of the core lipid on the energetics of binding of ApoA-I to model lipoprotein particles of different sizes

Interaction of apolipoproteins (apo) with lipid surfaces plays crucial roles in lipoprotein metabolism and cholesterol homeostasis. To elucidate the thermodynamics of binding of apoA-I to lipid, we used lipid emulsions composed of triolein (TO) and egg phosphatidylcholine (PC) as lipoprotein models. Determination of the level of binding of wild-type (WT) apoA-I and some deletion mutants to large (120 nm diameter; LEM) and small (35 nm diameter; SEM) emulsions indicated that N-terminal (residues 44-65) and C-terminal (residues 190-243 and 223-243) deletions have large effects on lipid interaction, whereas deletion of the central region (residues 123-166) has little effect. Substitution of amino acids at either L230 or L230, L233, and Y236 with proline residues also decreases the level of binding, indicating that an alpha-helix conformation in this C-terminal region is required for efficient lipid binding. Calorimetry showed that binding of WT apoA-I to SEM generates endothermic heat (DeltaH approximately 30 kcal/mol) in contrast to the exothermic heat (ca. -85 kcal/mol) generated upon binding to LEM and egg PC small unilamellar vesicles (SUV). This exothermic heat arises from an approximately 25% increase in alpha-helix content, and it drives the binding of apoA-I to LEM and SUV. There is a similar increase in alpha-helix content of apoA-I upon binding to either SEM or SUV, but the binding of apoA-I to SEM is an entropy-driven process. These results suggest that the presence of a core triglyceride modifies the highly curved SEM surface packing and thereby the thermodynamics of apoA-I binding in a manner that compensates for the exothermic heat generated by alpha-helix formation.     

Electron paramagnetic resonance spectroscopy of site-directed spin labels reveals the structural heterogeneity in the N-terminal domain of apoA-I in solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.     

The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.     

Structure and interfacial properties of chicken apolipoprotein A-IV

To gain insight into the evolution and function of apolipoprotein A-IV (apoA-IV) we compared structural and interfacial properties of chicken apoA-IV, human apoA-IV, and a recombinant human apoA-IV truncation mutant lacking the carboxyl terminus. Circular dichroism thermal denaturation studies revealed that the thermodynamic stability of the alpha-helical structure in chicken apoA-IV (DeltaH = 71.0 kcal/mol) was greater than that of human apoA-IV (63.6 kcal/mol), but similar to that of human apoA-I (73.1 kcal/mol). Fluorescence chemical denaturation studies revealed a multiphasic red shift with a 65% increase in relative quantum yield that preceded loss of alpha-helical structure, a phenomenon previously noted for human apoA-IV. The elastic modulus of chicken apoA-IV at the air/water interface was 13.7 mN/m, versus 21.7 mN/m for human apoA-IV and 7.6 mN/m for apoA-I. The interfacial exclusion pressure of chicken apoA-IV for phospholipid monolayers was 31.1 mN/m, versus 33.0 mN/m for human A-I and 28.5 mN/m for apoA-IV.We conclude that the secondary structural features of chicken apoA-IV more closely resemble those of human apoA-I, which may reflect the evolution of apoA-IV by intraexonic duplication of the apoA-I gene. However, the interfacial properties of chicken apoA-IV are intermediate between those of human apoA-I and apoA-IV, which suggests that chicken apoA-IV may represent an ancestral prototype of mammalian apoA-IV, which subsequently underwent further structural change as an evolutionary response to the requisites of mammalian lipoprotein metabolism.     

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.     

Activation of lipoprotein lipase by N-alpha-palmitoyl (56-79) fragment of apolipoprotein C-II

The effect of apolipoprotein C-II (apoC-II) and a synthetic fragment of apoC-II corresponding to residues 56-79 on the lipoprotein lipase (LpL) catalyzed hydrolysis of trioleoylglycerol in a monolayer of egg phosphatidylcholine and of dipalmitoylphosphatidylcholine vesicles was examined. Synthetic peptide 56-79, which does not associate with lipid, did not activate LpL at surface pressures greater than 30 mN/m; apoC-II is active up to 34 mN/m. However, acylation of the NH2-terminus of peptide 56-79 with palmitoyl chloride gave nearly identical LpL activating properties as compared to apoC-II. We conclude that at high surface pressures the lipid-binding region of apoC-II (residues 44-55) plays an essential role in LpL activation.     

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.     

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.     

Antibodies to the carboxyl terminus of human apolipoprotein A-I. The putative cellular binding domain of high density lipoprotein 3 and carboxyl-terminal structural homology between apolipoproteins A-I and A-II.

We have studied the binding of 125I-labeled high density lipoproteins (HDL3) to liver plasma membranes, which are thought to contain specific HDL receptor sites, using anti-peptide antibodies directed against two sites in the carboxyl-terminal region of human apoA-I. Two distinct antibody populations raised to peptides corresponding to amino acid residues 205-220 and 230-243, respectively, recognized regions of apoA-I that are exposed in the lipid environment of HDL3. However, anti-AI[230-243] IgG, but not anti-AI[205-220] IgG, recognized HDL2, suggesting that residues 205-220 of apoA-I are expressed differently in the two HDL populations. In addition, anti-AI[230-243] IgG showed strong cross-reactivity toward apoA-II. Epitope mapping studies showed that anti-AI[230-243] binds to an epitope located in the carboxyl-terminus of apoA-II, demonstrating significant structural homology between the carboxyl-terminal of apoA-II, demonstrating significant structural homology between the carboxyl-terminal regions of apoA-I and A-II, two candidate proteins for mediating the specific cellular interaction of HDL3. Fab fragments from anti-AI[205-220] and anti-AI[230-243] inhibited the binding of 125I-HDL3 to liver plasma membranes by approximately 80% and 60%, respectively. These findings are in agreement with our recent work using isolated CNBr fragments of apoA-I (Morrison, J., Fidge, N. H., and Tozuka, M. (1991) J. Biol. Chem. 266, 18780-18785), which suggest that the carboxyl-terminal region of apoA-I contains a binding domain which mediates the specific interaction of HDL3 with liver plasma membranes, possibly through the involvement of specific HDL receptors.     

Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1

To explore the functional interactions between apoA-I and ABCA1, we correlated the cross-linking properties of several apoA-I mutants with their ability to promote cholesterol efflux. In a competitive cross-linking assay, amino-terminal deletion and double amino- and carboxy-terminal deletion mutants of apoA-I competed effectively the cross-linking of WT (125)I-apoA-I to ABCA1, while the carboxy-terminal deletion mutant apoA-I[Delta(220-243)] competed poorly. Direct cross-linking of WT apoA-I, amino-terminal, and double deletion mutants of apoA-I to ABCA1 showed similar apparent K(d) values (49-74 nM), whereas the apparent K(d) values of the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] were increased 3-fold. Analysis of several internal deletions and point mutants of apoA-I showed that apoA-I[Delta(61-78)], apoA-I[Delta(89-99)], apoA-I[Delta(136-143)], apoA-I[Delta(144-165)], apoA-I[D102A/D103A], apoA-I[E125K/E128K/K133E/E139K], apoA-I[L141R], apoA-I[R160V/H162A], and WT apoA-I had similar ABCA1-mediated lipid efflux, and all competed efficiently the cross-linking of WT (125)I-apoA-I to ABCA1. WT apoA-I and ABCA1 could be cross-linked with a 3 A cross-linker. The WT apoA-I, amino, carboxy and double deletion mutants of apoA-I showed differences in the cross-linking to WT ABCA1 and the mutant ABCA1[W590S]. The findings are consistent with a direct association of different combinations of apoA-I helices with a complementary ABCA1 domain. Mutations that alter ABCA1/apoA-I association affect cholesterol efflux and inhibit biogenesis of HDL.     

A novel cell line (Caco-2) for the study of intestinal lipoprotein synthesis

Lipoprotein synthesis by the colonic adenocarcinoma cell line Caco-2 was investigated to assess the utility of this cell line as a model for the in vitro study of human intestinal lipid metabolism. Electron micrographic analysis of conditioned medium revealed that under basal conditions of culture post-confluent Caco-2 cells synthesize and secrete lipoprotein particles. Lipoproteins of density (d) less than 1.063 g/ml consist of a heterogeneous population of particles (diameter from 10 to 90 nm). This fraction consists of very low density lipoproteins (d less than 1.006 g/ml) and low density lipoproteins (d = 1.019-1.063 g/ml). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled Caco-2 lipoproteins revealed that very low density lipoproteins contain apolipoprotein E (apoE) and C apolipoproteins, while low density lipoproteins contained apoB-100, apoE, apoA-I, and C apolipoproteins. The 1.063-1.21 g/ml density fraction contained two morphological entities, discoidal (diameter 15.6 +/- 3.9 nm) and round high density lipoprotein particles (diameter 10.2 +/- 2.3 nm). The high density lipoproteins contained apoA-I, apoB-100, apoB-48, apoE, and the C apolipoproteins. Using isoelectric focusing polyacrylamide gel electrophoresis newly secreted apoA-I was identified as pro-apoA-I. ApoE and apoC-III released by Caco-2 cells were highly sialylated. mRNA species for apoA-I, apoC-III, and apoE, but not apoA-IV were identified by Northern blot analysis. ApoA-I, apoB, and apoE were visualized in Caco-2 cells by immunolocalization analysis. This intestinal cell line may be useful for in vitro studies of nutritional and hormonal regulation of lipoprotein synthesis.