Lipid-interacting properties of the N-terminal domain of human apolipoprotein C-III

L. Lins1, C. Flore2, L. Chapelle2, P.J. Talmud3, A. Thomas4 and R. Brasseur2,5

1 INSERM U447, IBL, 59021 Lille Cedex, France, 2 Centre de Biophysique Moléculaire Numérique, FSAGX, 5030 Gembloux, Belgium, 3 Division of Cardiovascular Genetics, Department of Medicine, Royal and Free University College Medical School, London WC1E 6JJ, UK and 4 INSERM U410, Faculté X.Bichat,75870 Paris Cedex 18, France


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The lipid-interacting properties of the N-terminal domain of human apolipoprotein C-III (apo C-III) were investigated. By molecular modeling, we predicted that the 6–20 fragment of apo C-III is obliquely orientated at the lipid/water interface owing to an asymmetric distribution of the hydrophobic residues when helical. This is characteristic of `tilted peptides' originally discovered in viral fusion proteins and later in various proteins including some involved in lipoprotein metabolism. Since most tilted peptides were shown to induce liposome fusion in vitro, the fusogenic capacity of the 6–20 fragment of apo C-III was tested on unilamellar liposomes and compared with the well characterized SIV fusion peptide. Mutants were designed by molecular modeling to assess the role of the hydrophobicity gradient in the fusion. FTIR spectroscopy confirmed the predominantly helical conformation of the peptides in TFE solution and also in lipid–peptide complexes. Lipid-mixing experiments showed that the apo C-III (6–20) peptide is able to increase the fluorescence of a lipophilic fluorescent probe. The vesicle fusion was confirmed by core-mixing and leakage assays. The hydrophobicity gradient plays a key role in the fusion process because the mutant with no hydrophobic asymmetry but the same mean hydrophobicity as the wild type does not induce significant lipid fusion. The apo C-III (6–20) fragment is, however, less fusogenic than the SIV peptide, in agreement with their respective mean hydrophobicity. Since lipid fusion should not be the physiological function of the N-terminal domain of apo CIII, we suggest that its peculiar distribution of hydrophobic residues is important for the lipid-binding properties of apo C-III and should be involved in apolipoprotein and lipid exchanges crucial for triglyceride metabolism.

Keywords: amphipathicity/lipid fusion/lipoprotein/molecular modeling/tilted peptide


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Apolipoprotein C-III (apo C-III) is a 79-residue glycoprotein. It is synthesized in the intestine and liver as part of the very low density lipoprotein (VLDL) and the high density lipoprotein (HDL) particles. Owing to its positive correlation with plasma triglyceride (Tg) levels, apo C-III is suggested to play a role in Tg metabolism (Shoulders et al., 1991Go; Bainton et al., 1992Go; Castelli, 1992Go) and is therefore of interest regarding atherosclerosis. However, unlike other apolipoproteins such as apo A-I, apo E or C-II for which many naturally occuring mutations are known (Assmann et al., 1992Go; Talmud, 1992Go), the structure–function relationships of apo C-III remains a subject of debate. One possibility is that apo C-III inhibits lipoprotein lipase (LPL) activity (Smith and Pownall, 1984Go), as shown by in vitro experiments (Lambert et al., 1996Go). Another suggestion, based on transgenic mice experiments (Aalto-Setala et al., 1992Go) and on more recent in vitro results (Breyer et al., 1999Go), is that elevated levels of apo C-III displace other apolipoproteins at the lipoprotein surface, modifying their clearance from plasma.

Like other apolipoproteins, apo C-III is made of amphipathic helices that are responsible for the lipid binding of the protein to the lipoprotein surface (Segrest et al., 1974Go; Li et al., 1988Go; Brasseur et al., 1992Go).

In a recent paper, we suggested the existence of an asymmetric distribution of hydrophobic residues in the N-terminal helix (residues 6–20) of apo C-III (Liu et al., 2000Go). This peculiar distribution is characteristic of so-called `tilted peptides' (Brasseur, 1991Go). Tilted peptides are not only amphipathic, but also their net hydrophobicity increases from one end of the helix to the other. Because of this asymmetry, we predicted by molecular modeling that such peptides should have an equilibrium tilted position at a hydrophobic/hydrophilic interface (Brasseur et al., 1997Go; Brasseur, 2000Go). This position should perturb the parallelism of lipid acyl chains when the interface is water/lipids. Recent neutron diffraction experiments have confirmed the existence of oblique-orientated peptides in bilayers (Bradshaw et al., 2000Go).

Tilted peptides were found in various proteins including viral fusion proteins (e.g. SIV GP32) (Horth et al., 1991Go; Martin et al., 1991Go, 1994Go), in signal sequences of proteins such as apoB 100 signal peptide (Talmud et al., 1996Go), in neurotoxic proteins (Pillot et al., 1996Go, 1997Go) and also in proteins implicated in lipoprotein metabolism such as LPL (lipoprotein lipase), HLP (hepatic lipase) (Brasseur et al., 1997Go), CETP (cholesteryl ester transport protein) (Brasseur et al., 1997Go), LCAT (lecithin cholesterol acyltransferase) (Perez-Mendez et al., 1998Go) or apo A-II (Lambert et al., 1998Go). Most of them were shown to induce liposome fusion in vitro when taken as isolated fragments (Martin et al., 1991Go, 1994Go; Pillot et al., 1996Go, 1997Go; Lambert et al., 1998Go; Perez-Mendez et al., 1998Go). Furthermore, when the peptides are mutated to modify the hydrophobicity distribution, their fusogenic properties are significantly decreased. When the same mutations are made from the whole protein, its activity is modified (Horth et al., 1991Go; Talmud et al., 1996Go). In apo C-III, site-directed mutations in the N-terminal domain clearly modify the binding of the protein to the lipids (Liu et al., 2000Go).

In this paper, we focus on the N-terminal tilted peptide of apo C-III (peptide 6–20) and on its liposome fusion activity. The potential role of the 6–20 peptide in the apo C-III function is discussed.


    Materials and methods
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 Materials and methods
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Materials

Egg phosphatidylcholine (PC), cholesterol (CHOL) and sphingomyelin (SM) were purchased from Sigma (St. Louis, MO, USA), egg phosphatidylethanolamine (PE) from Lipid Products (Redhill, Surrey, UK), octadecyl rhodamine chloride (R18) from Molecular Probes (Eugene, OR) and peptides (95% pure) from Syntem (Nîmes, France).

Molecular modeling of the peptide

Three-dimensional structures of the peptides were calculated as described previously (Brasseur, 1991Go, 1992; Pillot et al., 1996Go). The methods take account of the contribution of the lipid/water interface by the concomitant variation of the dielectric constant and the energy of transfer of atoms from a hydrophobic to a hydrophilic environment.

The peptides were then oriented at the hydrophobic/hydrophilic interface taking into account the hydrophobic and hydrophilic centres, calculated as described elsewhere (Brasseur, 1990Go).

Molecular hydrophobicity potential (MHP) calculations

MHP is a three-dimensional plot of the hydrophobicity potential of a molecule in order to visualize its amphipathy. The hydrophobicity of a molecule is calculated using its partition coefficient between water and octanol.

We postulate that the hydrophobicity induced by an atom i and measured at a point M in space decreases exponentially with the distance between this point M and the surface of an atom i according to the following equation (Brasseur, 1991Go):

where N is all atoms of the molecule, Etri is the transfer energy of atom i, ri is the radius of atom i and di is the distance between atom i and the point M. Etri is the energy required to transfer an atom i from a hydrophobic to a hydrophilic medium. Atomic Etri were calculated from the molecular transfer energies compiled by Tanford (Tanford, 1973Go) assuming that molecular Etr are the sum of their atomic Etr. Atomic Etr values were derived for seven different atom types (Brasseur, 1991Go).

The hydrophobic and hydrophilic isopotential surfaces were calculated by a cross-sectional computational method. A 1 Å mesh-grid plane was set to sweep across the molecule in steps of 1 Å. At each step, the sum of the hydrophobicity and hydrophilicity values at all grid nodes was calculated. The hydrophobic and hydrophilic MHP surfaces were then drawn by joining the isopotential values.

All calculations are performed on Pentium Pro processors, using PC-TAMMO+ and PC-PROT+ software. Graphs were drawn using WinMGM (Ab Initio Technology, Obernai, France).

Liposome preparation

Large unilamellar vesicles (LUV) were prepared by the extrusion technique of Hope et al. (Hope et al., 1985Go) using an extruder (Lipex Biomembranes, Vancouver, Canada). In brief, dry lipid films which are mixtures by weight of 26.6% phosphatidylcholine (PC), 26.6% sphingomyelin (SM), 26.6% phosphatidylethanolamine (PE) and 20.2% cholesterol were hydrated for 1 h at 37°C. A PC–PE (3:2 w/w ratio) mixture was also used. The resulting suspension was submitted to five successive cycles of freezing and thawing and thereafter extruded 10 times through two stacked polycarbonate filters (pore size 0.08 µm), under a nitrogen pressure of 20 bar.

The concentrations of the liposome suspensions were determined by phosphorus analysis (Mrsny et al., 1986Go).

Lipid-mixing experiments

Mixing of liposome membranes was followed by measuring the fluorescence increase of R18, a lipid-soluble probe, occurring after the fusion of labeled and unlabeled liposomes. Labeled liposomes were obtained by incorporating R18 in the dry lipid film at a concentration of 6.3% of the total lipid weight. Labeled and unlabeled liposomes were mixed at a weight ratio of 1:4 and a final concentration of 50 µM in 10 mM Tris, 150 mM NaCl, 0.01% EDTA, 1 mM NaN3 (pH 8). Fluorescence was recorded at room temperature ({lambda}exc 560 nm, {lambda}em 590 nm) on a Perkin-Elmer LS-50B fluorimeter.

Leakage of liposome vesicle contents

The ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid)–DPX [p-xylylenebis(pyridinium) bromide] assay of Ellens et al. (Ellens et al., 1985Go) was used to monitor vesicle leakage. The assay is based on the quenching of ANTS by DPX. ANTS and DPX are both encapsulated in the aqueous phase of the same liposomes. Leakage of vesicles was followed by measuring the dequenching of ANTS released into the medium. Fluorescence was recorded at room temperature ({lambda}exc 360 nm, {lambda}em 520 nm) on a Perkin-Elmer LS-50B fluorimeter.

Liposomes (LUV) were prepared as described above in 12.5 mM ANTS (45 mM NaCl), 45 mM DPX (20 mM NaCl), 10 mM Tris–HCl at pH 7.4. Vesicles containing encapsulated ANTS and DPX were eluted on a Sephadex G-75 column with 10 mM Tris–HCl, 150 mM NaCl (pH 7.4), to remove unencapsulated material.

Core-mixing experiments

The mixing of liposome contents was monitored using the core-mixing assay of Kendall and McDonald (Kendall and MacDonald, 1982Go). Liposomes (LUV) were prepared as described above in 10 mM Tris–HCl buffer, 150 mM NaCl, 1 mM NaN3 (pH 8.0) and containing calcein at 0.8 mM and CoCl2 at 1.0 mM or EDTA at 20 mM. Untrapped solutes were removed by a single elution on a Sephadex G-75 column with 10 mM Tris–HCl, 150 mM NaCl, 1 mM NaN3 buffer (pH 8.0). In a standard experiment, calcein, Co2+- and EDTA-containing vesicles were mixed at 1:1 molar ratio in a 10 mM Tris–HCl buffer (pH 8.0) (150 mM NaCl, 1 mM NaN3 ). When peptides were added, the calcein fluorescence was monitored at room temperature ({lambda}exc 490 nm, {lambda}em 520 nm) as a function of time on a Perkin-Elmer LS-50B fluorimeter. Co2+ (0.4 mM chelated with citrate at 1:1 mol/mol) was present in the medium to avoid fluorescence due to leakage of vesicle contents. The maximum fluorescence was determined in presence of 0.5% Triton X-100 (10 mM EDTA).

For the leakage, core-mixing and lipid-mixing experiments, assays were repeated at least three times.

Fourier transform infrared (FTIR) spectroscopic measurements

Attenuated total reflection (ATR) IR spectroscopy was used to determine the secondary structure of apo C-III peptides in solution and when bound to the lipids.

Spectra were recorded at room temperature on a Bruker Equinox 55 instrument equipped with a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector at a resolution of 2 cm-1, by averaging 512 scans. Free peptide samples (10 µg of peptide) were recorded in solution with increasing amounts of TFE. For the lipid-bound peptides (see preparation below), the internal reflection element was a germanium ATR plate (50x20x2 mm, Aldrich) with an aperture of 45° yielding 25 internal reflections. Reference spectra of a germanium plate were automatically recorded after a purge of 15 min with dry air and ratioed against the recently run sample spectra. Peptide–lipid complexes (100 µg of phospholipid) were spread out on the plate and slowly dried under a stream of N2. The plate was sealed in a universal sample holder and rehydrated by flushing the holder with N2 saturated with D2O for 3 h at room temperature.

Sample preparation

Amounts of 15 µg of peptides were added to 100 µg of liposomes prepared as described above. Lipid–peptide incubations were performed for 1 h at room temperature in 10 mM Tris–HCl, 150 mM NaCl buffer (pH 7.5). After incubation, the lipid–peptide mixture was filtered through an anisotropic hydrophilic YM membrane (cutoff 10 kDa) of a Centrifree micropartition system (Amicon) to separate lipid-associated from free peptides. Phospholipid concentration was determined as mentioned above.

Secondary structure determination

Vibrational bands, especially the amide I band (1600– 1700 cm-1), are sensitive to the secondary structures of the proteins. The C=O vibration is representative of 80% of the amide I band. This band accounts for all the secondary structures which have different vibration values. The combination of resolution-enhancement methods with curve-fitting procedures allows one to assign quantitatively different secondary structures such as {alpha}-helix, ß-sheets and unordered structures. Each band was assigned according to the frequency of its maximum. The areas of all bands assigned to a given secondary structure are then summed and divided by the sum of all areas. This gives the relative ratio of each secondary structure. The bands are assigned as follows (Goormaghtigh et al., 1999Go): {alpha}-helix, 1662–1645 cm-1; ß-sheets, 1689–1682 and 1637–1613 cm-1; random, 1644.5–1637 cm-1; ß-turns, 1682–1644.5 cm-1. It should be noted that the proteins spread on the plate are deuterated to avoid an overlap of {alpha}-helix and random-coil structures, as described previously (Goormaghtigh et al., 1999Go).


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The structure of the most stable helical conformation of the apo C-III (6–20) peptide was obtained by molecular modeling. The modeled peptide was then oriented at the lipid/water interface. Figure 1AGo shows that the helix axis is tilted towards the interface forming an angle of about 40° with the interface plane. The MHP surfaces clearly indicate that the tilt is due to an asymmetric distribution of the hydrophobicity along the helix axis (Figure 1BGo).



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Fig. 1. Molecular modeling of the apo C-III peptides at the lipid/water interface (orange grid). The hydrophobic phase is above and the hydrophilic phase below the plane. The N and C ends of the peptides are indicated. (A) WT 6–20 peptide; (C) mutant 0; (E) DM mutant (see Table IGo). The MHP (molecular hydrophibicity potential) calculating the hydrophilic (green envelopes) and hydrophobic (orange envelopes) environment of the peptides are also shown. (B) MHP around the WT peptide; (D) around the 0 mutant; (F) around the DM mutant.

 
Since most tilted peptides discovered up to now induce liposome fusion in vitro, the 6–20 N-terminal peptide of apo C-III was tested for fusogenic properties. Mutants were designed by molecular modeling to assess the role of the hydrophobicity gradient in the fusion process, as previously carried out for the viral and neurotoxic tilted peptides (Martin et al., 1994Go; Pillot et al., 1996Go, 1997Go). Mutants 0 and DM result from residue permutation in order to preserve the mean hydrophobicity of the fragment (Table IGo). Molecular modeling indicates that the 0 and DM mutants are oriented parallel and tilted, respectively (Figure 1C and EGo). The DM mutant is obtained by residue permutations from the 0 mutant sequence to yield a new tilted peptide whose sequence differs from that of the WT (Table IGo). All hydrophobic residues are located on the same side of the interface in the 0 mutant (only residue Ala6 is located on the other side) (Figure 1CGo), while they are asymmetrically distributed in the DM mutant (Figure 1EGo) as shown by the MHP envelopes (Figure 1D and FGo; Table IGo).


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Table I. Peptides used in this study
 
The secondary structure of the peptides was evaluated by FTIR spectroscopy in different TFE solutions. In 50% TFE, the three peptides are approximately 30–40% {alpha}-helix and 20–25% ß-sheet (Table IIGo). Increasing TFE does not change the ratio but significantly increases the area of the amide I band (Table IIIGo), indicating better solubilization of the peptides.


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Table II. Percentage of secondary structure ({alpha}, ß, coil and turn) of apo C-III peptides in TFE–D2O (1:1) solution as determined by FTIR spectroscopy
 

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Table III. Influence of % TFE on the secondary structure of the WT apo C-III peptide
 
The induction of vesicular lipid mixing by the different peptides was tested with PC/PE/SM/Chol LUVs as a measure of fusogenic activities. R18-labeled and R18-free liposomes were mixed and the increase in fluorescence intensity due to the dequenching of the probe is indicative of lipid fusion. Incubation of labeled and unlabeled vesicles in buffer alone (i.e. no peptide or TFE) did not modify the fluorescence intensity (data not shown). In the calibration assay, the SIV peptide, known for its fusogenic activity and its oblique insertion in lipids (Martin et al., 1991Go, 1994Go), was set to increase the fluorescence intensity (Figure 2Go). The same conditions were then used for the apo C-III peptides. At a 1:20 peptide to lipid molar ratio, the WT and the DM mutants induced an increase in fluorescence which is significant compared with TFE alone (Figure 2Go). Under the same conditions, the 0 mutant was at same level as the TFE alone. This observation supports the fact that the 0 mutant is not fusogenic. It should be noted that the SIV fragment had a larger effect than the WT apo C-III fragment.



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Fig. 2. Time course of lipid mixing of PC/PE/SM/Chol LUVs induced by different peptides in a 1:20 (peptide:lipid) mol/mol ratio, corresponding to a 150 µM peptide concentration in 100% TFE. Peptides were added to a mixture of labeled LUVs with R18 and unlabeled liposomes (1:4 w/w ratio). The increase in the R18 relative fluorescence due to probe dilution is followed at room temperature. In a control experiment, the same volume of TFE alone is added to the liposome mixture. The final TFE percentage is 1.6%. (•) apo C-III 6–20 WT; ({circ}) apo C-III mutant 0; ({blacktriangledown}) apo C-III DM mutant; ({triangleup}) SIV peptide; ({blacksquare}) TFE (blank).

 
Increasing amounts of apo C-III (6–20) peptides were added to liposomes. The R18 fluorescence intensity recorded 15 min after mixing showed that the peptide causes lipid mixing in a concentration-dependent fashion (Figure 3Go). TFE affected the process; at a lipid–peptide molar ratio of 1:20, increasing the TFE concentration significantly increased the inter-vesicular lipid mixing induced by the WT peptide (Figure 4Go). The optimum effect was at maximum TFE concentration. It should be noted that 100% TFE in the initial peptide preparation corresponds to 1.6% TFE in the final solution.



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Fig. 3. Influence of the peptide concentration on the extent of lipid mixing of PC/PE/SM/Chol LUVs induced by the apo C-III (6–20) WT peptide (in 100% TFE). The relative fluorescence of the R18 probe is measured after 15 min incubation. 100% corresponds to the relative fluorescence obtained with the peptide at 300 µM.

 


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Fig. 4. Effect of the percentage of TFE present in the Apo C-III (6–20) WT peptide samples on the lipid mixing of PC/PE/SM/Chol LUVs. Peptides were dissolved in a 10 mM Tris–HCl (150 mM NaCl, 1 mM NaN3, 0.01% EDTA) buffer (pH 8.0) and 20, 50 and 80% TFE or 100% TFE. The peptide to lipid molar ratio is 1:20. The R18 fluorescence is measured after 15 min. 100% relative fluorescence corresponds to that obtained for the peptide in 100% TFE after 15 min. The fluorescence of the TFE alone (at the same dilution as the peptide preparation) after 15 min was subtracted from the fluorescence induced by the peptide at the same time.

 
This must be compared with the FTIR experiments showing an increasing amide I band area under the same conditions, suggesting a higher yield of peptide solubilization in TFE. It supports the notion that the more the peptide is dissolved, the better it interacts with lipids.

In the presence of lipids, the secondary structure of the WT peptide remained predominantly helical (Table IIGo). This is consistent with the results obtained in the membrane core-mimicking solvent TFE (Table IIIGo).

To assess the leakage of vesicles due to their interaction with the peptides and the subsequent membrane destabilization, ANTS and DPX were encapsulated in the same liposomes. The WT and the DM apo C-III peptides induced an increase in the ANTS fluorescence, whereas the effect was not significant for the 0 mutant (Figure 5Go). The leakage process is almost immediate, the maximum value being reached during the first minute. It is worth noting that the SIV peptide is more potent than the WT apo C-III peptide, as it was for the lipid-mixing properties.



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Fig. 5. Leakage of liposomal contents of PC/PE/SM/Chol LUVs induced by the different apo C-III peptides monitored with the ANTS/DPX assay at room temperature. Peptide (dissolved in 100% TFE) is added to the liposome suspension in a 1:20 mol/mol ratio as described under Materials and methods. The increase in the ANTS fluorescence is monitored at 520 nm. 100% leakage is established by lysing the vesicles with 0.5% Triton X-100 (10 mM EDTA). (•) apo C-III (6–20) WT; ({circ}) apo C-III mutant 0; ({blacktriangledown}) apo C-III DM mutant; ({triangleup}) SIV peptide; ({blacklozenge}) TFE (blank).

 
Core-mixing experiments were also carried out to demonstrate further the fusogenic properties of the apo C-III (6–20) fragment. When the peptide was added to a mixture of calcein and Co2+- and EDTA-containing PC/PE/SM/Chol liposomes, a significant increase in calcein fluorescence was observed during the first minute (Figure 6Go). Whereas the SIV peptide still induced further mixing as a function of time, the apo CIII peptide did not (Figure 6Go). Since Co2+ ions were present in the medium, the effects observed should be due to the mixing of the liposome aqueous phases.



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Fig. 6. Core-mixing of PC/PE/SM/Chol LUVs induced by the apo C-III WT and the SIV peptides (dissolved in 100% TFE). Peptides are added to a mixture of calcein and Co2+- and EDTA-containing vesicles mixed at a 1:1 molar ratio (10 mM Tris–HCl, 150 mM NaCl, 1 mM NaN3 buffer, pH 8.0). The calcein fluorescence is monitored at 520 nm at room temperature as a function of time. 100% leakage is established by lysing the vesicles with 0.5% Triton X-100 (10 mM EDTA). (•) apo C-III (6–20) WT; ({circ}) SIV peptide; ({blacklozenge}) TFE (blank).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have analyzed the properties of the hydrophobic portion of the N-terminal domain of human apo C-III. While the 1–40 N-terminal region of apo C-III, corresponding to exon3, has been suggested to modulate the lipoprotein lipase (LPL) activity (McConathy et al., 1992Go; Jong et al., 1999Go) and to have poor lipid-binding capacities (because of its overall content in hydrophilic residues) (Sparrow et al., 1977Go; Jong et al., 1999Go), no information is available concerning the role of the hydrophobic part of that domain, particularly its role in lipid binding. We recently suggested that the 6–20 domain could have an asymmetric distribution of hydrophobic residues like the so-called `tilted peptides' that are able to destabilize lipid interfaces (Liu et al., 2000Go; Lins et al., 2001Go). This fragment is, however, less hydrophobic than other well-characterized tilted peptides such as the SIV fusion peptide (a mean hydrophobicity of 0.23 compared with 0.93), which is known to have fusogenic properties.

By computer modeling, we have calculated that the native apo C-III fragment tilts at a hydrophobic/water interface when helical, the helix axis forming an angle of about 40° with the interface plane. Mutants, either losing their hydrophobicity gradient (0 mutant) or recovering that character by additional permutation of residues (DM mutant), were also tested in order to assess the influence of the hydrophobicity distribution on the fusogenic activity.

In the modeling studies, a helical conformation of the peptide was assumed. While FTIR spectroscopy confirmed a predominant helical conformation of the peptide in TFE or in lipids, this was not 100% helix. Nevertheless, we can assume that the helical conformation plays a role in the fusion process, as previously suggested by others (Durell et al., 1997Go; Martin et al., 1999Go). Indeed, calculations were performed imposing a ß-strand and an {alpha}-helical conformation to the WT and the 0 mutant that is shown here to be non-fusogenic. There is no hydrophobicity gradient in the ß-strand conformation and simulations of WT ß-strand peptide assembly with lipids do not indicate any perturbation of the lipid organization. In contrast, the tilt of the helix at the interface (WT and DM, tilted; 0 mutant, parallel) does correlate with variations of experimental results on liposome fusion.

We carried out lipid mixing, vesicle leakage and core mixing assays for the apo C-III (6–20) peptide and its mutants and compared their activities with that of the SIV fusion peptide. A PC/PE/SM/Chol composition was used for the experiments since it was shown to be a good membrane model for other tilted peptides, notably for the SIV peptide (Martin et al, 1991Go, 1994Go), which is the positive control in our assays. Results were reproduced with PC–PE liposomes (3:2 w/w) (data not shown).

The lipid-mixing results clearly indicate that the apo C-III (6–20) peptide is able to induce the fusion of unilamellar liposomes. Dilution of the lipophilic probe R18 in the membrane of unlabeled liposomes induced an increase in fluorescence through a dequenching process. When the unlabeled LUV population was omitted, no change in fluorescence was observed. In the same way, mixing of labeled and unlabeled liposomes in the absence of the peptide (and of TFE) had little effect on the R18 fluorescence, suggesting that there is no significant vesicle instability. This also suggests that the effects observed with the peptide are not due to a simple exchange of the fluorescent probe and/or a modification of the shape of vesicles. However, aggregation of the LUVs with diffusion of the probe in the absence of true lipid fusion could not be completely ruled out at this stage. For this reason leakage and core-mixing experiments were performed. The apo C-III (6–20) peptide can alter the permeability of liposomes as measured by the release of small solutes such as ANTS and DPX. This could be due to the formation of hexagonal phases or other local changes in the lipid organization induced by the interaction with the peptide. Finally, the core-mixing experiments confirmed that the apo C-III peptide induces a true fusion of liposome vesicles. All effects are observed in the same concentration range. It should be mentioned that the mixing and permeability processes induced by the peptides are very fast, most of the activity occuring within the first minute. This has already been observed for other peptides (Martin et al, 1991Go, 1994Go; Pillot et al, 1996Go).

In contrast, mutant 0, which has a homogeneous distribution of hydrophobic residues (i.e. no hydrophobicity gradient) but the same mean hydrophobicity as the WT, is significantly less fusogenic and lipid-destabilizing than the WT. These results stress the importance of the oblique insertion into the lipids that mediates the destabilizing and fusogenic properties of peptides. This is further confirmed by the results obtained with the DM mutant in which the hydrophobicity gradient is restored. This peptide is as fusogenic as the WT while having a different linear sequence. The results strongly support that the fusogenic properties of tilted peptides are related to the asymmetric distribution of hydrophobicity rather than to its mean hydrophobicity value or to a sequence motif.

The fusion processes described in this paper are different from those observed during the formation of discoidal HDL-like particles and are specifically due to the peculiar hydrophobic properties of the apo C-III (6–20) peptide. While disk formation induces leakage, it only arises at the transition temperature of the lipid used and/or in presence of a detergent (Jonas, 1986Go). This is different from the lipid fusion induced by tilted peptides. At a sufficient peptide to lipid ratio, fusion occurs spontaneously. Furthermore, the results obtained with the 0 mutant mimicking lipid-binding amphipathic peptides clearly show that only `asymmetric' peptides induce lipid fusion.

We also compared the apo C-III peptides with the SIV fusion peptide, which has been extensively characterized by molecular modeling and by in vitro and in vivo approaches (Brasseur, 1991Go; Horth et al., 1991Go; Martin et al., 1991Go, 1994Go; Bradshaw et al., 2000Go). The WT and DM apo C-III peptides induce fusion at the same concentration range as the SIV peptide does but with a lower efficiency (2–3-fold less) in all three experiments (leakage, lipid- and core-mixing assays). This is in agreement with the lower mean hydrophobicity of the apo C-III fragment compared with most fusogenic peptides (viral fusion peptides, tilted peptides from Aß protein, prion, etc.). In a previous work, tilted peptides were classified according to their mean hydrophobicity (Brasseur et al., 1997Go). Apo C-III tilted peptide belongs to the low-hydrophobicity class, as does the oblique peptide of apo A-II, another protein involved in lipid metabolism (Lambert et al., 1998Go). Recently, we used a computational method called IMPALA to characterize these classes further (Lins et al., 2001Go). IMPALA computes the interactions of molecules with an implicit lipid bilayer using simple restraint functions (Ducarme et al., 1998Go). The results suggested that the high-hydrophobicity peptides have access to the two layers of the membrane and skip from one to the other during the Monte Carlo minimization, while the low-hydrophobicity peptides remain within a single lipid layer. The proteins interacting with membranes such as viral fusion proteins possess tilted peptides of the high-hydrophobicity class. In that case, destabilization of lipids would be maximum when the peptide can act on the two layers. Since lipoproteins are a monolayer of phospholipids surrounding a triglyceride core, only one lipid layer must be destabilized when the lipoproteins are interacting with proteins involved in lipid metabolism. The results presented in this paper are therefore in agreement with the classification, since the apo C-III peptide is less hydrophobic and less fusogenic than the SIV peptide. The apo A-II tilted peptide that is suspected to be involved in protein exchange between lipoproteins was also less potent in the fusion than the SIV peptide (Lambert et al., 1998Go).

As for apo A-II, it is tempting to suggest that the apo C-III tilted peptide is involved in the function of the whole protein. While lipid fusion is used here as an experimental index to evidence the destabilizing capacities of the N-terminal domain of apo C-III, fusion is unlikely to be the physiological role of this domain in vivo. Our study actually suggests that the imperfect amphipathy of the N-terminal domain of apo C-III should have a peculiar behavior when bound to lipids. While classical amphipathic helix is the motif involved in lipid-binding and protein–lipid complex stabilization for most apolipoproteins, the hydrophobic asymmetry should induce lipid binding but also lipid destabilization.

In a recent paper, Liu et al. introduced the mutations (L9T/T20L) corresponding to the 0 mutant in the whole apo C-III (Liu et al., 2000Go). This L9T/T20L mutant has a higher lipid binding efficiency and forms tighter and more stable complexes with lipids than the native apo C-III. Thus, the modification of the asymmetry of hydrophobic residues distribution in the N-terminal helix of apo C-III (in order to restore classical amphipathicity) enhances the stability of the lipid-bound apo C-III.

We can thus hypothesize that the tilted peptide of apo C-III is responsible for its loose binding to the lipoprotein particle. Through its lipid-destabilizing capacities, this domain should also be involved in the lipid exchange in vivo.

It is worth noting that the oblique insertion of the N-terminal domain (from residues 1 to 20) should also exist at the lipoprotein surface. Indeed, when we calculate the orientation of the 1–20 apo C-III domain using the IMPALA method, the 6–20 domain is inserted with a tilt in the lipid monolayer and the first five residues are located between the beginning of the acyl chains (for D5) and the surface (for E2) (data not shown).

In conclusion, the results presented here suggest that the presence of destabilizing fragments within some apolipoproteins (e.g. apo C-III and apo A-II) should facilitate the highly dynamic processes of protein and lipid exchanges crucial for lipid metabolism.


    Notes
 
5 To whom correspondence should be addressed. E-mail : brasseur.r{at}fsagx.ac.be Back


    Acknowledgments
 
R.B. is Research Director at the National Funds for Scientific Research of Belgium. P.J.T. is funded by the British Heart Foundation. This work was supported by the Interuniversity Poles of Attraction Program – Belgian State, Prime Minister's Office – Federal Office for Scientific, Technical and Cultural Affairs, contract No. P.4/03, the Belgian Loterie Nationale and the National Fund for Scientific Research of Belgium (FNRS).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received September 10, 2001; revised February 26, 2002; accepted March 8, 2002.