To assess the functional properties of apolipoprotein (apo) AII and to investigate the mechanism leading to the displacement of apo AI from native and reconstituted high-density lipoproteins (HDL and r-HDL) by apo AII, wild-type and variant apo AII peptides were synthesized. The wild-type peptides, residues 53Ϫ70 and 58Ϫ70, correspond to the C-terminal helix of apo AII and are predicted to insert at a tilted angle into a lipid bilayer. We demonstrate that both the apo AII-(53Ϫ70) peptide, and to a lesser extent the apo AII-(58Ϫ70) peptide are able to induce fusion of unilamellar lipid vesicles together with membrane leakage, and to displace apo AI from HDL and r-HDL. Two variants of the apo AII-(53Ϫ70)-wild-type (WT) peptide, designed either to be parallel to the water/lipid interface [apo AII-(53Ϫ70)-0°] or to retain an oblique orientation [apo AII-(53Ϫ70)-30°], were synthesized in order to test the influence of the obliquity on their fusogenic properties and ability to displace apo AI from HDL. The parallel variant did not bind lipids, due to its self-association properties. However, the apo AII-(53Ϫ70)-30°v ariant was fusogenic and promoted the displacement of apo AI from HDL. Moreover, the extent of fusion of the apo AII-(53Ϫ70)-WT, apo AII-(58Ϫ70)-WT and apo AII-(53Ϫ70)-30°peptides was related to the A-helical content of the lipid-bound peptides measured by infrared spectroscopy. Infrared measurements using polarized light also confirmed the oblique orientation of the helical component of the three peptides. In native and r-HDL, the tilted insertion of the C-terminal helix of apo AII resulting in a partial destabilization of the HDL external lipid layer might contribute to the displacement of apo AI by apo AII.Keywords : apolipoprotein AII ; apolipoprotein AI; membrane fusion; lipid-binding; synthetic peptide.The physiological role of apolipoprotein (apo) AII, in the HDL seem less efficient than apo AI-only particles in promoting regulation of plasma high-density lipoprotein (HDL) metabolism the uptake and esterification of cell-derived cholesterol [10], includes the ability to displace apo AI from nascent HDL [1Ϫ contributing to the reverse-cholesterol transport mechanism. The 3], and thereby the negative control on the activity of lecithin amount of cholesterol efflux induced by these particles is how-:cholesterol acyltranferase (LCAT) in these particles [4Ϫ6]. Be-ever highly variable and depends upon the type of cells [11,12]. sides its lipid-binding properties and its ability to displace apo Apo AII further seems less protective against the development AI, apo AII was reported to either activate [7], or inhibit [8,9] of atherosclerosis than apo AI, as trangenic mice overexpressing hepatic lipase, depending upon the assay conditions. Apo AI:AII either human or murine apo AII develop more lesions than human apo AI transgenic or control mice [13Ϫ16], although oppo- Abbreviations. apo, apolipoprotein; HDL, high-density lipoproteins;The C-terminal region of apo AII (i.e. residues 47Ϫ77) was nal domains of ap...

Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments of LCAT with lipases suggest that residues 50-74 form an interfacial recognition site and this hypothesis was tested by site-directed mutagenesis. The (delta56-68) deletion mutant had no activity on any substrate. Substitution of W61 with F, Y, L or G suggested that an aromatic residue is required for full enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid substrate. The decreased activity of the single R52A and K53A mutants on HDL and LDL and the severer effect of the double mutation suggested that these conserved residues contribute to the folding of the LCAT lid. The membrane-destabilizing properties of the LCAT 56-68 helical segment were demonstrated using the corresponding synthetic peptide. An M65N-N66M substitution decreased both the fusogenic properties of the peptide and the activity of the mutant enzyme on all substrates. These results suggest that the putative interfacial recognition domain of LCAT plays an important role in regulating the interaction of the enzyme with its organized lipoprotein substrates.

A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167Ϫ171]. Due to an asymmetric distribution of hydrophobic residues along the axis of the Ahelix, such peptides adopt an oblique orientation which can destabilise membranes or lipid cores. Variants of these oblique peptides, designed to have an homogeneous distribution of hydrophobic and hydrophilic residues along the helical axis, are classified as regular amphipathic peptides. These peptides are expected to lie parallel to the polar/apolar interface with their hydrophobic residues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was identified at residues 56Ϫ68 in the sequence of the lecithin-cholesterol acyltransferase (LCAT), enzyme. This peptide is predicted to penetrate a lipid bilayer at an angle of 40°through its more hydrophobic Cterminal end and thereby induce the destabilisation of a membrane or a lipid core. The LCAT-(56Ϫ68) wild-type peptide was synthesised together with the LCAT-(56Ϫ68, 0°) variant, in which the hydrophobicity gradient was abolished through residue permutations. In two other variants, designed to keep their oblique orientation, the W61 residue was shifted either towards the more hydrophilic N-terminal at residue 57, or to position 68 at the hydrophobic C-terminal end of the peptide. Peptide-induced vesicle fusion was demonstrated by fluorescence measurements using pyrene-labeled vesicles and by monitoring of vesicle size by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emission of the peptides. Fluorescence polarisation measurements, using diphenyl hexatriene, were carried out to follow changes in the lipid fluidity. The LCAT-(56Ϫ68) wild-type peptide and the two oblique variants, induced fusion of unilamellar dimyristoylglycerophosphocholine vesicles. Tryptophan fluorescence emission measurements showed a 12Ϫ14 nm blue shift upon addition of the wild-type peptide and of the W61→68 variant to lipids, whereas the fluorescence of the W61→57 variant did not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as predicted by the theoretical calculations. In contrast, the 0°variant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid transition temperature was decreased after addition of the wild-type, the W61→68 and W61→57 fusogenic peptides, whereas the opposite effect was observed with the 0°variant. The behaviour of the wild-type and variant LCAT-(56Ϫ68) peptides stresses the contribution of the hydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter consequently influences the structur...

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of action of fusogenic peptides, this hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion. To assess the role of the hydrophobicity gradient upon the fusogenic activity, two of these fusogenic peptides and several variants were synthesized. The LCAT-(57-70) peptide, which is part of the sequence of the lipolytic enzyme lecithin cholesterol acyltransferase, forms stable beta-sheets in lipids, while the apolipoprotein A-II (53-70) peptide remains predominantly helical in membranes. The variant peptides were designed through amino acid permutations, to be either parallel, perpendicular, or to retain an oblique orientation relative to the lipid-water interface. Peptide-induced vesicle fusion was monitored by lipid-mixing experiments, using fluorescent probes, the extent of peptide-lipid association, the conformation of lipid-associated peptides and their orientation in lipids, were studied by Fourier Transformed Infrared Spectroscopy. A comparison of the properties of the wild-type and variant peptides shows that the hydrophobicity gradient, which determines the orientation of helical peptides in lipids and their fusogenic activity, further influences the secondary structure and lipid binding capacity of these peptides.

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