Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Frank Peelman1, Berlinda Vanloo1, Oscar Perez-Mendez1, Anne Decout1, Jean-Luc Verschelde2, Christine Labeur1, Nicole Vinaimont1, Annick Verhee2, Nicolas Duverger3, Robert Brasseur4, Joël Vandekerckhove2, Jan Tavernier2 and Maryvonne Rosseneu1,5

1 Laboratory for Lipoprotein Chemistry and 2 Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research, Faculty of Medicine, Department of Biochemistry, University of Gent, B-9000 Gent, Belgium, 3 Rhône-Poulenc-Rorer-GENCELL, Cardiovascular Department,Vitry-sur Seine, France and 4 Centre de Biophysique Moléculaire Numérique, Faculté des Sciences Agronomiques de Gembloux, Gembloux, Belgium


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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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 ({Delta}56–68) 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.

Keywords: cholesterol/interfacial recognition domain/lecithin/lecithin cholesterol acyltransferase/lipoprotein/membrane destabilization


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lecithin cholesterol acyltransferase (LCAT) is a key enzyme in lipoprotein metabolism, as it accounts for the synthesis of most of the plasma cholesteryl esters (Glomset, 1968Go). This enzyme catalyses the transacylation of the sn-2 fatty acid of lecithin to the free 3ß-hydroxyl group of cholesterol whereby lysolecithin and cholesteryl ester are formed. LCAT is active on both low-density (LDL) and high-density (HDL) lipoproteins and several mutations in the LCAT gene lead to impaired enzymatic activity. Carriers of mutations causing familial LCAT deficiency (FLD) lose activity on both substrates, whereas a partial loss of activity on HDL only is characteristic of mutations causing fish-eye disease (FED) (Kuivenhoven et al., 1997Go). We have recently shown, using structural homology calculations based upon threading methods, that LCAT belongs to the lipase family (Peelman et al., 1998aGo). The LCAT enzyme has an {alpha}/ß hydrolase fold like the lipases and its catalytic triad includes D345 and H377 (Peelman et al., 1998aGo), together with S181 (Francone and Fielding, 1991Go). Using pancreatic lipase and C.antarctica lipase as templates, we built a 3D model for the central domain of LCAT, in which the architecture of the lipases is conserved. We further extended these homology calculations to the identification of a potential `lid' domain at residues 50–74 of LCAT, closed by a disulphide bridge, as in several other lipases (Winkler et al., 1990Go; Grochulski et al., 1993Go). This domain was also proposed by Adimoolam and Jonas (1997) as the interfacial recognition site of this enzyme. Deletion of this segment was found by both groups (Adimoolam and Jonas, 1997Go; Peelman et al., 1998aGo) to decrease the enzymatic activity significantly. The primary determinants of the differential LCAT activity on its lipoprotein substrates are not known. The major apolipoprotein component of HDL, apo AI, is required as a co-factor for the {alpha}-activity on HDL. The catalytic action of LCAT, which has both a phospholipase and an acyltransferase activity, requires the access of both a cholesterol and a lecithin molecule to the catalytic site. The enzymatic activity of LCAT therefore depends both on the surface properties and on the packing of the organized lipid core of the lipoprotein substrates. Lipase lids, in their open conformation, come into close contact with lipid substrates. These lids are moreover characterized by the presence of an amphipathic {alpha}-helix, in both their open and closed conformations. The hydrophobic side of this helix is oriented towards the core of the protein in the closed conformation of the lid, whereas it is exposed in the open conformation, thus creating a large hydrophobic surface contributing to the interaction with the lipid interface and to substrate binding (Derewenda et al., 1992Go; Grochulski et al., 1994Go; Egloff et al., 1995Go; Kyu Kim et al., 1997). In analogy with lipases, such a lid domain might be involved in the primary interaction between LCAT and its lipoprotein substrates. We proposed previously that the interfacial recognition domain of lipolytic enzymes, such as lipases and LCAT, should not only serve as a binding site for the hydrophobic substrate but further include a `tilted' peptide (Brasseur et al., 1997Go). Such a peptide destabilizes a micellar or bilayer lipid substrate and facilitates the diffusion of a monomeric phospholipid or triglyceride into the active site cavity of the enzyme.

In this work, we investigated more closely the structural and functional properties of the 50–74 segment of LCAT and especially aimed at identifying residues in this domain which are critical for the enzymatic function of LCAT. Brasseur and co-workers (Brasseur, 1991Go; Brasseur et al., 1997Go) hypothesized that the lid or the interfacial recognition domain of lipolytic enzymes should be able to destabilize the organized lipid substrate and that this destabilization might occur through the action of an oblique helical peptide. Oblique helical peptides are characterized by a hydrophobicity gradient along their longitudinal axis. The more hydrophobic end of the helix inserts in the membrane and the helix makes a tilted angle with respect to the lipid/water interface, thus perturbing the regular lipid organization. We identified an oblique peptide at residues 56–68, with sequence similarity to the amphipathic helical segment of the lipase lids. We investigated the lipid-destabilizing properties of the synthetic LCAT 56–68 peptide and of variants and measured the activity of the corresponding LCAT mutants on different substrates. A conserved tryptophan residue at position 61 in the helical segment of the LCAT lid is also present in the lid of other lipases (Van Tilbeurgh et al., 1993Go; Martinelle et al., 1996Go). The role of this Trp residue and that of the conserved basic residues R52 and K53 was investigated by site-directed mutagenesis and transient expression of the mutants. The activity of the various mutants was tested on a monomeric substrate, on HDL and on LDL, in order to assess the contribution of the mutated residues to the substrate recognition and binding. These results provide experimental evidence that the 50–74 loop of LCAT, and especially tryptophan 61, modulates the enzyme activity on an organized lipid substrate and that it contributes to the selective activity of LCAT on either HDL or LDL.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
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Materials

L-{alpha}-Phosphatidylcholine (PC) from egg yolk, L-{alpha}-phosphatidylethanolamine (PE), free cholesterol (FC) and 1-pyrenebutanoic acid were purchased from Sigma. [3H] Cholesterol was obtained from Amersham. 1-Palmitoyl-2-pyrene(14)-phosphatidylcholine (Pyr-PC), 1,2-bis(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine and 1-pyrenehexanoic acid were purchased from Molecular Probes (Junction City, OR). All reagents for peptide synthesis and sequencing were supplied by Applied Biosystems (Foster City, CA). 1,1,1,3,3,3-Hexafluoropropan-2-ol (HFP) was of the highest grade from Sigma. Dimethyl sulphoxide (DMSO) was purchased from Merck (Darmstadt, Germany).

Molecular modelling of the peptides by energy minimization

Molecular modelling of the peptides was carried out as described previously (Brasseur, 1991Go; Brasseur and Ruysschaert, 1986Go). The method used to predict the conformational structure of the peptides accounted for the contribution of the lipid/water interface, the concomitant variation of the dielectric constant and the transfer energy of atoms from a hydrophobic to a hydrophilic environment. The structure, mode of insertion and orientation of the peptides were studied in a dipalmitoylphosphatidylcholine monomolecular layer. In this model, the interaction energy (sum of the contributions from Van der Waals interactions, torsional potential energy, electrostatic interactions and transfer energy) between the peptide and dipalmitoylphosphatidylcholine in the monolayer was calculated and minimized until the lowest energy state of the entire aggregate was reached. All calculations were performed on an Olivetti CP486 computer, using PC-TAMMO+ (Theoretical Analysis of Molecular Membrane Organization) and PC-PROT+ (Protein Plus Analysis) software. Graphs were drawn with the PC-MGM+ (Molecular Graphics Manipulation) program.

Synthesis and purification of peptides

All peptides were synthesized by the standard Fmoc solid-phase method on an Applied Biosystems Model 413A peptide synthesizer, as described previously (Lambert et al., 1998Go). The purity and mass of the peptides were verified by electrospray ionization mass spectrometry using a Fisons/VG (Manchester, UK) platform mass spectrometer. Peptide concentration was determined by phenylalanine quantitation by reversed-phase high-performance liquid chromatography (HPLC) (Vercaemst et al., 1989Go).

Lipid-mixing experiments

Small unilamellar vesicles (SUV) were prepared from a mixture of PC–PE–FC (50:25:7.5, w/w), when necessary in the presence of 2.5 mol% of Pyr-PC (Pillot et al., 1996Go). Fusion of pyrene-labelled and unlabelled vesicles was measured using a fluorescence probe dilution assay (Pillot et al., 1996Go; Perez-Mendez et al., 1998Go). The pyrene excimer/monomer (E/M) ratio was calculated from the excimer and monomer fluorescence intensities measured at 475 and 398 nm, with an excitation wavelength of 346 nm, in an Aminco SPF 500 spectrofluorimeter. All fusion experiments were performed at a phospholipid concentration of 50 µM in 5 mM citrate buffer (pH 5) containing 150 mM NaCl, 3 mM Na2EDTA and 1 mM NaN3. The final peptide concentrations were in the range 0.2–2 µM.

Tryptophan fluorescence measurements

The environment of the single tryptophan residue of the lipid-free and lipid-associated LCAT 56–68 peptides was probed by measurement of the intrinsic fluorescence emission spectrum. Dimyristoylphosphatidylcholine (DMPC) vesicles were added to the peptides in order to minimize the light scattering caused by the aggregation of PC–PE–FC vesicles (Perez-Mendez et al., 1998Go). Emission spectra were recorded at wavelengths between 300 and 450 nm, with the excitation wavelength set at 290 nm. Measurements were performed on an Aminco SPF-500 spectrofluorimeter using a circulating water-bath for temperature control.

Site-directed mutagenesis

Mutagenesis was carried out in the pXL 3105 plasmid vector (Peelman et al., 1998aGo) using the Quick Change Site-Directed Mutagenesis method (Stratagene). Mutations were built in by PCR using PfU DNA polymerase. Additional restriction sites were introduced to facilitate screening. After DpnI digestion of the parental dam-methylated template, the synthesized mutated DNA was transformed into Escherichia coli XL1-Blue supercompetent cells. Candidate clones were screened by restriction analysis and mutants were sequenced on an ALF automated sequencer (Pharmacia Biotech). Transient expression of the LCAT cDNA in Cos-1 cells was carried out by lipofectamine (Gibco) transfection. After transfection, the cell culture media (Dulbecco's modified Eagle's medium; Life Technologies) were changed to Optimem after 16 h and harvested after 48 h.

LCAT activity assays and mass measurements

The activity of WT LCAT and mutants was measured on three different substrates: r-HDL, LDL and a momomeric substrate (Peelman et al., 1998aGo). Reconstituted HDL (rHDL) consisting of 1-palmitoyl-2-linoleoylphosphatidylcholine–cholesterol–apo AI complexes at a molar ratio of 100:10:1 were prepared by the cholate-dialysis method (Matz and Jonas, 1982Go). The percentage of cholesteryl esters formed during the enzymatic reaction was determined by HPLC (Vercaemst et al., 1989Go; Vanloo et al., 1992Go; Peelman et al., 1998aGo). LDL was purified from plasma by sequential ultracentrifugation at densities between 1.007 and 1.063 g/ml and dialysed against 10 mM Tris–HCl buffer (pH 8.0), 0.15 M NaCl, 3 mM EDTA, 1 mM NaN3. After heat inactivation at 56°C, LDL was radiolabelled with [3H]cholesterol (Stokke and Norum, 1971Go). The reaction was initiated by adding 350 µl of the cell culture medium to a mixture containing labelled LDL (20 µM free cholesterol), 0.15 M NaCl, 3 mM EDTA, 1 mM NaN3, 4 mM ß-mercaptoethanol and 4 mg/ml BSA in 10 mM Tris–HCl buffer, pH 8). The enzymatic reaction was stopped after 3 h by addition of 4 ml of hexane–propan-2-ol (3:2, v/v). Unesterified cholesterol and cholesteryl esters were separated by thin-layer chromatography on silica gel plates developed with hexane–diethyl ether–acetic acid (90:20:1, v/v) and quantitated by liquid scintillation counting. The LCAT phospholipase activity on a monomeric substrate was monitored by fluorescence measurements, using 1,2-bis(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine as substrate (Bonelli and Jonas, 1992Go). For quantitative measurement of the phospholipase activity, the amount of 1-pyrenebutanoic acid formed by LCAT hydrolysis of 1,2-bis(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine was determined by HPLC. The assay mixture contained 0.5–2 µM 1,2-bis(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine, 4 mM ß-mercaptoethanol, 4 mg/ml BSA, to which 350 µl of cell culture medium and 10 mM Tris–HCl buffer (pH 8.0), 0.15 M NaCl, 3 mM EDTA, 1 mM NaN3 were added to a final volume of 0.5 ml. The mixture was incubated at 37°C for 10 min and the reaction was stopped by addition of 4 ml of chloroform–methanol (2:1, v/v) containing 1-pyrenehexanoic acid as internal standard. 1-Pyrenebutanoic acid was quantified by isocratic HPLC (Waters 600E) on a reversed-phase ODS C18 column (Merck LiChroCART 2504), eluted with acetonitrile–water–trifluoroacetic acid (70:30:0.1, v/v). This compound was detected by absorbance measurement using a Waters 486 UV detector set at 342 nm, at a sensitivity of 10–9 mol. LCAT mass was assayed by solid-phase enzyme immunoassay using chicken antibodies specific to LCAT and purified recombinant human LCAT as a standard ( Peelman et al., 1998aGo).


    Results
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Localization of the interfacial recognition domain (IRD) in LCAT and alignment with lipases

Threading alignment of the LCAT sequence against a library of solved three-dimensional protein structures showed that LCAT has an {alpha}/ß hydrolase fold (Peelman et al., 1998aGo). The 123D threading alignment of the LCAT sequence with that of Candida rugosa lipase suggests that there is structural similarity between the lid domains at residues C60–C97 of C.rugosa lipase and residues C50–C74 of LCAT. Both segments are closed by a disulphide bridge (Yang et al., 1987Go; Grochulski et al., 1993Go). An amphipathic {alpha}-helix was identified between residues 74 and 84, in the closed lid of C.rugosa lipase (Figure 1AGo), which is shifted N-terminal towards residues 72–82, upon opening of the lid (Grochulski et al., 1994Go). The analysis of the lid of the crystallized human pancreatic lipase identified an {alpha}-helix at residues 248–253 in the closed lid. This helix is shifted to residues 250–259 in the open conformation of the lid (Egloff et al., 1995Go) (Figure 1AGo). Figure 1BGo illustrates the similarity between residues 56–68 in the proposed LCAT lid with the sequences of the amphipathic helices in the C.rugosa and the human pancreatic lipase lids described above. There is significant sequence conservation between the 72–82 segment of C.rugosa lipase and the 56–68 helix of LCAT, as residues L78, D79, L80 and M82 of the hydrophobic face of the amphipathic helix in C.rugosa lipase correspond to residues L62, D63, L64 and M66 in the amphipathic helix of LCAT. Although the sequence identity is lower between LCAT residues 56–68 and the helix of the pancreatic lipase lid, residues I60, W61 and F67 of LCAT are conserved in the pancreatic lipase sequence. A helical-wheel projection of the three helical sequences shows that the residues of the hydrophobic side of the pancreatic lipase helix are conserved in the 56–68 LCAT segment (Figure 1CGo). The proposed interfacial recognition domain at residues 56–68 in LCAT is characterized by a patch of aromatic residues including W61 and F67, which are aligned with W252 and F258 in the human pancreatic lipase lid.



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Fig. 1. Alignment of residues 56–68 in LCAT with residues 72–84 in C.rugosa lipase and residues 247–259 in human pancreatic lipase. (A) Secondary structure of the 72–84 segment in the lid of C.rugosa lipase in its open and closed conformation and of the 247–259 segment in the lid of human pancreatic lipase in its open and closed conformation. {alpha} = {alpha}-helical conformation; ß = ß-conformation (Grochulski et al., 1994Go; Egloff et al., 1995Go). (B) Alignment of residues 56–68 in LCAT with residues 72–84 in the lid of C.rugosa lipase and residues 247–259 in the lid of human pancreatic lipase. * = Identical residues; . = homologous residues. (C) Helical wheel projection of residues 56–68 in LCAT, residues 72–84 in C.rugosa lipase and residues 247–259 in human pancreatic lipase.

 
As residues C50 and C74 of LCAT form a disulphide bridge (Yang et al., 1987Go), the positively charged residues R52 and K53, N-terminal of the loop, come close to the C-terminal negatively charged residue D73, with which they might form a salt bridge. The positive charge at position 52 is evolutionarily conserved, as R52 is either conserved or replaced by K52 in C.elegans LCAT (Peelman et al., 1998bGo). The charge conservation at position 53 seems less stringent, as there is a K53Q substitution in C.elegans LCAT.

Computer modelling and characterization of the LCAT-(56–68) peptides.

Brasseur and co-workers (Brasseur, 1991Go; Brasseur et al., 1997Go) hypothesized that the lid or the interfacial recognition domain of lipolytic enzymes should include a helical asymmetric hydrophobic peptide, which inserts in membranes with a tilted angle, thus destabilizing the organized substrate. The orientation of the LCAT 56–68 peptide was verified using the programs developed by Brasseur (1991). According to these calculations, residues 56–68 of LCAT should qualify as an oblique helical membrane-destabilizing peptide. The orientation of this and of variant peptides at a lipid/water interface is illustrated in Figure 2Go, showing that the 56–68 WT LCAT is tilted at ~40° at the lipid/water interface.



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Fig. 2. Computer modelling of the mode of insertion of the LCAT peptides in a lipid matrix. For reasons of simplicity, lipids were not drawn. The horizontal line represents the interface between the hydrophobic (upper) and the hydrophilic (lower) phase. The peptides are (A) LCAT-(56–68) WT, (B) LCAT-(56–68, 0°), (C) LCAT-(56–68, W61->68) and (D), LCAT-(56–68, W61->57).

 
The sequences and properties of the synthetic LCAT-(56–68) peptides are summarized in Table IGo. Residue W61 was shifted either N-terminal to position 57 or C-terminal to 68, through combined W61F, F57W and W61L, L68W substitutions, respectively, while the oblique orientation of the corresponding peptides at a lipid/water interface was retained through additional permutations of the sequence (Table IGo). Another peptide was designed to lose the N->C hydrophobicity gradient along the helical axis, through a permutation of residues N65 and M66. As a consequence, this variant peptide has a predicted orientation of 0° at a lipid/water interface, compared with 40° for the wild-type peptide. This horizontal variant peptide has the highest hydrophobic moment, reflecting its increased amphipathicity (Table IGo).


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Table 1. Properties of the synthetic LCAT 56–68 peptides
 
Membrane-destabilizing properties of the synthetic LCAT 56–68 peptides

The intervesicular lipid mixing ability of the peptides was tested with PC–PE–cholesterol SUVs using a probe dilution assay (Pillot et al., 1996Go). In this assay, unlabelled SUVs are mixed with pyrene-labelled SUVs. Addition of a fusogenic peptide causes fusion of the labelled SUVs with unlabelled SUVs and leads to dilution of the pyrene label. This dilution of the pyrene probe leads to an increased monomer fluorescence and a decreased excimer fluorescence. These experiments were performed at pH 5.0, in order to increase the affinity of the LCAT peptide for lipids due to a partial protonation of D56 and D63, as performed previously for the NH2-terminal peptide of the influenza virus haemaglutinin (Rafalski et al., 1991Go). The LCAT-(56–68)WT peptide, the W61->68 and W61->57 variants decreased the excimer/monomer (E/M) ratio of mixed unlabelled and labelled SUVs as a function of time, whereas the LCAT-(56–68, 0°) variant had no effect (data not shown). The decrease of the E/M ratio after 10 min of incubation of increasing peptide concentrations with a fixed amount of lipid vesicles is depicted in Figure 3Go. These data show that the LCAT-(56–68) WT and W61->57 variant peptide decreased the E/M ratio by 40% after 10 min of incubation with PC–PE–cholesterol vesicles, compared with 50% for the W61–68 variant. The 0° variant peptide had little activity as the E/M ratio decreased by only 10%.



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Fig. 3. Influence of the peptide concentration on the extent of lipid mixing of PC–PE–cholesterol SUVs induced by the ({bullet}) LCAT-(56–68) WT, ({blacktriangleup}) LCAT-(56–68, 0°), ({blacksquare}) LCAT-(56–68, W61->68) and ({blacklozenge}) LCAT-(56–68, W61->57) peptides. Percentages of E/M ratio decrease were plotted versus peptide concentration, after 10 min of incubation of PC–PE–cholesterol SUVs and peptide.

 
Orientation of the peptides into the lipids

According to the modelling of the LCAT-(56–68) WT peptide at the lipid/water interface, the C-terminal end of the peptide should penetrate the bilayer and W61 should lie within the lipid phase. The same applies to the W61->68 variant peptide, whereas in the W61->57 variant, W57 should remain in the aqueous phase. In the 0° variant, W61 is located on the hydrophobic face of the amphipathic helix and should interact with lipids. This hypothesis was tested by measuring the maximum Trp fluorescence emission wavelength of the lipid-free and lipid-associated peptides. The W57, W61 and W68 residues are exposed to the aqueous solvent in the lipid-free peptides, with a maximum emission wavelength of ~354 nm (Table IGo). Upon lipid addition to the WT peptide and the 0° and W61->68 variants, the maximum fluorescence emission wavelength is blue-shifted to 340, 332 and 341 nm, respectively. In contrast, when the W61->57 variant is mixed with lipids, the maximum emission wavelength is shifted only to 350 nm, suggesting that W57 remains exposed to the aqueous phase. This observation thus supports the predicted orientation of this peptide relative to the lipid/water interface.

Enzymatic activity of the LCAT mutants

The mutants were transfected into Cos-1 cells and the LCAT mass and activity measured in the cellular media are listed in Table IIGo. The acyltransferase activity was measured on the r-HDL and LDL substrates, while the phospholipase activity was tested using the bispyrene-phospholipid. All mutants were expressed at significant levels, between 50 and 110%, compared with the wild-type enzyme. Deletion of residues 56–68 of the interfacial recognition domain abolished the LCAT activity on all substrates. The N65–M66 substitution was aimed at verifying the effect of the change of the peptide orientation on the LCAT activity. This double substitution, through which the synthetic peptide was orientated parallel to the lipid/water interface, decreased the LCAT activity on r-HDL and on the monomeric substrate to ~35% of that of the wild-type enzyme, while the activity on LDL was decreased to 8% (Table IIGo). The role of the W61 residue was investigated by mutation to a phenylalanine, tyrosine, leucine or glycine, in order to confirm the requirement for an aromatic residue at this position. The W61Y substitution is accompanied by a 3-fold increase in the phospholipase activity on the monomeric substrate, in agreement with the results of the W61F mutation. The acyltransferase activity on r-HDL is retained for the W61F and W61Y mutants, whereas there is only 20% residual activity on LDL. The W61L and W61G mutants lose activity on both an organized and a monomeric substrate (Table IIGo). These data thus suggest that an aromatic residue (W, F, Y) at position 61 of LCAT is required for the optimum activity of the enzyme with any of its substrates.


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Table 2. LCAT mass (µg/ml) and relative specific activity (% of wild-type) in cell media from mutant and wild-type LCAT transfectants
 
The contribution of the two basic residues R52 and K53 at the N-terminal end of the 50–74 loop to the LCAT catalytic activity was tested by mutating these residues, either separately or in concert, to an alanine residue. The double mutant retained little activity (±10%) on monomeric and organized substrates, whereas the individual mutants behaved differently. The R52A mutant retained 35 and 100% activity on an organized and a monomeric substrate, respectively, whereas the activities of the K53A mutant on HDL, LDL and the monomeric substrate were 78, 32 and 104%, respectively (Table IIGo). The lower activity of the R52A mutant on r-HDL suggests a more critical role for R52 compared with K53, in agreement with the highest level of conservation of the former residue among LCAT species.


    Discussion
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 References
 
We have investigated the structural and functional properties of a putative interfacial recognition domain (IRD) or `lid' in LCAT and especially the contribution of some critical residues in this domain. In previous papers (Brasseur et al., 1997Go; Peelman et al., 1998aGo), we showed that LCAT belongs to the {alpha} hydrolase fold family, like other lipolytic enzymes and especially lipases (Ollis et al., 1992Go). A common structural element in most of these enzymes is the existence of a lid, i.e. a loop that covers the active site and makes it inaccessible to the substrate, in the closed conformation of the enzyme. In the presence of the substrate, significant conformational rearrangements occur in this loop that enhance the access of the substrate to the catalytic triad. Opening of the lid exposes hydrophobic residues that are likely to interact with both the lipid interface and with the monomeric substrate (Grochulski et al., 1993Go, 1994Go). Opening of the lid had been proposed as the process underlying the interfacial activation in lipases; however, the Myocastor coypus pancreatic lipase has no interfacial activation in spite of a 23-residue lid, thus questioning this assumption (Thirstrup et al., 1994Go). The lipase lid has further been shown to influence substrate specificity. Relative to triacylglycerol hydrolysis, hepatic lipase displays higher phospholipase activity than lipoprotein lipase. A chimeric hepatic lipase, with the lid of lipoprotein lipase, becomes a preferential triglyceride hydrolase, whereas the chimeric lipoprotein lipase, with the lid of hepatic lipase, has increased phospholipase activity (Dugi et al., 1995Go).

In contrast to the core of lipases, the architecture of which is highly conserved, lids are less conserved elements found in variable positions in the various lipase structures. Their length varies from 15 residues in Rhizomucor miehei lipase (Derewenda et al., 1992Go) to 45 residues in Geotrichum candidum lipase (Schrag and Cygler, 1993Go). A common element in the lipase lids is the presence of one or more amphipathic helices. Opening of the lid involves different mechanisms, ranging from a hinged rigid-body type of movement of a small helix in Rhizomucor miehei lipase (Derewenda et al., 1992Go) to a movement involving drastic conformational changes in the pancreatic lipase lid (Egloff et al., 1995Go).

The 123D threading alignment suggests that LCAT and C.rugosa lipase have an {alpha}/ß hydrolase fold with the same general topology ( Peelman et al., 1998aGo). Threading alignment of the LCAT sequence on the C.rugosa lipase structure (Grochulski et al., 1993Go) matches the 50–74 loop of LCAT with the C.rugosa lipase lid. A comparison of this loop with the lids of the lipases with a known three-dimensional structure shows that the central part of this segment, at residues 56–68, resembles the amphipathic helix in the pancreatic and C.rugosa lipase lids. Brasseur and co-workers (Brasseur, 1991Go; Brasseur et al., 1997Go) hypothesized that the IRD of lipolytic enzymes should include a helical asymmetric hydrophobic peptide, able to destabilize the organized lipid substrate. Peptide modelling at a lipid/water interface (Brasseur, 1991Go) indicated that the LCAT 56–68 segment qualifies as an oblique membrane-destabilizing peptide. The oblique orientation of this peptide, due to the hydrophobicity gradient along the helical axis (Perez-Mendez et al., 1998Go), and its preferential interaction with a lipid phase through its C-terminal end are supported by tryptophan fluorescence measurements on the variant peptides. The membrane-destabilizing properties of the peptides were confirmed by their ability to induce vesicle fusion of PC–PE–cholesterol vesicles. A loss of the peptide obliquity in the parallel M65N–N66M variant was accompanied by a decrease in the fusogenic activity and the corresponding M65N–N66M LCAT mutant had decreased activity on all substrates and particularly on LDL. A specific decrease in the LCAT activity on HDL, with retained activity on LDL, leads to the clinical phenotype of fish-eye disease (Kuivenhoven et al., 1997Go). However, natural LCAT mutants with a specific decrease in LDL activity, combined with normal HDL activity, have never been reported so far. Such mutations might exist but remain undetected, as cholesterol esterification in plasma HDL, followed by cholesteryl ester transfer to LDL through the action of the cholesteryl ester transfer protein (CETP), would probably compensate for this defect. The differential effect of the M65N–N66M mutations on the LCAT activity on HDL and LDL might result from differences in the composition and organization of the HDL and LDL lipid surfaces. LDL contains more saturated phospholipids, cholesterol and sphingomyelin than HDL. As a result, the outer phospholipid monolayer of LDL is more condensed, thus restricting the access of LCAT to its lecithin and cholesterol substrates is (Ibdah et al., 1989Go). These data suggest that there is a correlation between the membrane-destabilizing properties of this segment and the LCAT activity of the corresponding mutant. The interaction of LCAT with the condensed LDL phospholipids probably requires the structural integrity of the proposed interfacial recognition site and the perturbation of the lipid organization by the oblique peptide. The primary site of interaction between LCAT and its lipid substrates might involve different segments of the enzyme.

The interaction of LCAT with its co-factor apo AI probably involves hydrogen bonding or salt bridges involving residues on helices {alpha} 3–4 (residues 116–129) and {alpha} His (residues 387–398) in LCAT, as suggested by the point mutations leading to fish-eye disease through the loss of activity on HDL (Kuivenhoven et al., 1998; Peelman et al., 1998bGo). We showed that the amphipathic helix at residues 154–171, which corresponds to helix {alpha} 4–5 in the 3D model, has lipid-binding activity (Peelman et al., 1997GoPeelman et al., 1998aGo). This helix is homologous with the C-terminal amphipathic segment of apolipoprotein E which associates with phospholipids. However, comparison of our LCAT model with the structures of lipases (Peelman et al.,1998aGo) and also site-directed mutagenesis experiments (Wang et al., 1998Go) do not suggest that this helix is involved in substrate binding, but rather represents a structural motif of the {alpha} hydrolase fold (Ollis et al., 1992Go).

Site-directed mutagenesis experiments show that the putative interfacial recognition domain between residues 50 and 74 in LCAT is important in substrate recognition. When the 56–68 sequence was deleted, the mutant enzyme was still secreted, but it became inactive on all substrates, consistent with the proposed role in substrate recognition. The loss of activity of the deletion mutant might further arise from the deletion of W61, which seems critical for the enzyme activity. The alignment of W61 in LCAT with W252 in the amphipathic helix of the pancreatic lipase lid suggests that this residue might be involved in interfacial recognition or in substrate binding. In the closed conformation of the pancreatic lipase lid, W252 penetrates into the active site cavity and partially impairs the access to the active site of the enzyme (Winkler et al., 1990Go). In the open conformation of the lid, W252 leaves the active site and forms part of the hydrophobic interfacial recognition site (Van Tilbeurgh et al., 1993Go; Egloff et al., 1995Go). Mutagenesis of a tryptophan residue in the lid of Humicola lanuginosa lipase demonstrated its contribution in determining the substrate chain length specificity (Martinelle et al., 1996Go). In LCAT, W61 was mutated to F, Y, L and G residues and the activity of these mutants was measured on r-HDL, LDL and a monomeric phospholipid analogue. An aromatic residue at this position seems required for full enzymatic activity, as the substitution of W61 by either a leucine or a glycine completely abolishes enzymatic activity, as reported previously (Peelman et al., 1998aGo). Replacement of W61 with a smaller aromatic residue (F, Y), causes a 3-fold increase in the LCAT activity on a monomeric phospholipid analogue. This may be due to a decrease in the steric hindrance created between W61 and the bulky side chains of the pyrene-phospholipid analogue. A similar activity increase towards monomeric substrates was reported when W89 was replaced by a smaller F residue in the lid of H.lanuginosa lipase (Martinelle et al., 1996Go). Substitution of W61 with a smaller aromatic residue retained the LCAT activity on HDL and decreased the activity on LDL. Wimley and White (1996) recently demonstrated that tryptophan residues have the highest affinity for a phospholipid interface; this might account for the requirement of W61 for an efficient interaction of LCAT with the tightly packed LDL phospholipids.

The two conserved basic residues R52 and K53 are found at the N-terminal extremity of the 50–74 loop and might form a salt bridge with residue D73. The single mutants R52A and K53A had decreased activity on HDL and LDL, but retained full activity on the monomeric substrate. The effect of the double mutation R52A–K53A was even more drastic, suggesting that these two residues contribute to the structural integrity and function of the LCAT lid.

In the Geotrichum candidum lipase/acetylcholinesterase family, and also in the pancreatic lipase family, the loop corresponding to the lid forms a highly variable element (Cygler et al., 1993Go; Hjorth et al., 1993Go; Soldatova et al., 1993Go). Both protein families contain lipolytic enzymes where the lid is replaced with a shorter loop that does not cover the active site (Withers-Martinez et al., 1996Go; Feaster et al., 1997Go). The 50–74 loop in LCAT is 13 residues shorter than the C.rugosa lid and could only be modelled in an open conformation in the 3D model we proposed for LCAT ( Peelman et al., 1998aGo). This loop might therefore not be an actual lid that covers the active site, but rather from a region that is important for substrate binding and/or interfacial recognition.

In summary, the alignment with lipase lids and the results obtained with the model peptides and with the mutants constructed by site-directed mutagenesis suggest an important contribution of the C50–C74 domain of LCAT for substrate recognition. In this domain, W61 specifically contributes to the binding of a phospholipid monomer. Measurement of the binding of some of these mutants to phospholipid micelles, to liposomes and to apolipoprotein–phospholipid complexes is currently in progress for a more precise evaluation of the significance of this region.


    Notes
 
5 To whom correspondence should be addressed. E-mail: maryvonne.rosseneu{at}rug.ac.be Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received July 3, 1998; revised September 24, 1998; accepted October 7, 1998.