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
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Abstract |
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Keywords: cholesterol/interfacial recognition domain/lecithin/lecithin cholesterol acyltransferase/lipoprotein/membrane destabilization
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Introduction |
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In this work, we investigated more closely the structural and functional properties of the 5074 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, 1991; Brasseur et al., 1997
) 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 5668, with sequence similarity to the amphipathic helical segment of the lipase lids. We investigated the lipid-destabilizing properties of the synthetic LCAT 5668 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., 1993
; Martinelle et al., 1996
). 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 5074 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.
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Materials and methods |
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L--Phosphatidylcholine (PC) from egg yolk, L-
-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, 1991; Brasseur and Ruysschaert, 1986
). 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., 1998). 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., 1989
).
Lipid-mixing experiments
Small unilamellar vesicles (SUV) were prepared from a mixture of PCPEFC (50:25:7.5, w/w), when necessary in the presence of 2.5 mol% of Pyr-PC (Pillot et al., 1996). Fusion of pyrene-labelled and unlabelled vesicles was measured using a fluorescence probe dilution assay (Pillot et al., 1996
; Perez-Mendez et al., 1998
). 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 0.22 µM.
Tryptophan fluorescence measurements
The environment of the single tryptophan residue of the lipid-free and lipid-associated LCAT 5668 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 PCPEFC vesicles (Perez-Mendez et al., 1998). 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., 1998a) 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., 1998a). Reconstituted HDL (rHDL) consisting of 1-palmitoyl-2-linoleoylphosphatidylcholinecholesterolapo AI complexes at a molar ratio of 100:10:1 were prepared by the cholate-dialysis method (Matz and Jonas, 1982
). The percentage of cholesteryl esters formed during the enzymatic reaction was determined by HPLC (Vercaemst et al., 1989
; Vanloo et al., 1992
; Peelman et al., 1998a
). LDL was purified from plasma by sequential ultracentrifugation at densities between 1.007 and 1.063 g/ml and dialysed against 10 mM TrisHCl 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, 1971
). 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 TrisHCl buffer, pH 8). The enzymatic reaction was stopped after 3 h by addition of 4 ml of hexanepropan-2-ol (3:2, v/v). Unesterified cholesterol and cholesteryl esters were separated by thin-layer chromatography on silica gel plates developed with hexanediethyl etheracetic 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, 1992
). 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 0.52 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 TrisHCl 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 chloroformmethanol (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 acetonitrilewatertrifluoroacetic 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 109 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., 1998a
).
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Results |
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Threading alignment of the LCAT sequence against a library of solved three-dimensional protein structures showed that LCAT has an /ß hydrolase fold (Peelman et al., 1998a
). 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 C60C97 of C.rugosa lipase and residues C50C74 of LCAT. Both segments are closed by a disulphide bridge (Yang et al., 1987
; Grochulski et al., 1993
). An amphipathic
-helix was identified between residues 74 and 84, in the closed lid of C.rugosa lipase (Figure 1A
), which is shifted N-terminal towards residues 7282, upon opening of the lid (Grochulski et al., 1994
). The analysis of the lid of the crystallized human pancreatic lipase identified an
-helix at residues 248253 in the closed lid. This helix is shifted to residues 250259 in the open conformation of the lid (Egloff et al., 1995
) (Figure 1A
). Figure 1B
illustrates the similarity between residues 5668 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 7282 segment of C.rugosa lipase and the 5668 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 5668 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 5668 LCAT segment (Figure 1C
). The proposed interfacial recognition domain at residues 5668 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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Computer modelling and characterization of the LCAT-(5668) peptides.
Brasseur and co-workers (Brasseur, 1991; Brasseur et al., 1997
) 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 5668 peptide was verified using the programs developed by Brasseur (1991). According to these calculations, residues 5668 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 2
, showing that the 5668 WT LCAT is tilted at ~40° at the lipid/water interface.
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The intervesicular lipid mixing ability of the peptides was tested with PCPEcholesterol SUVs using a probe dilution assay (Pillot et al., 1996). 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., 1991
). The LCAT-(5668)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-(5668, 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 3
. These data show that the LCAT-(5668) WT and W61
57 variant peptide decreased the E/M ratio by 40% after 10 min of incubation with PCPEcholesterol vesicles, compared with 50% for the W6168 variant. The 0° variant peptide had little activity as the E/M ratio decreased by only 10%.
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According to the modelling of the LCAT-(5668) 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 W6168 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 I
). 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 II. 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 5668 of the interfacial recognition domain abolished the LCAT activity on all substrates. The N65M66 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 II
). 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 II
). 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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Discussion |
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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., 1992) to 45 residues in Geotrichum candidum lipase (Schrag and Cygler, 1993
). 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., 1992
) to a movement involving drastic conformational changes in the pancreatic lipase lid (Egloff et al., 1995
).
The 123D threading alignment suggests that LCAT and C.rugosa lipase have an /ß hydrolase fold with the same general topology ( Peelman et al., 1998a
). Threading alignment of the LCAT sequence on the C.rugosa lipase structure (Grochulski et al., 1993
) matches the 5074 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 5668, resembles the amphipathic helix in the pancreatic and C.rugosa lipase lids. Brasseur and co-workers (Brasseur, 1991
; Brasseur et al., 1997
) 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, 1991
) indicated that the LCAT 5668 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., 1998
), 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 PCPEcholesterol vesicles. A loss of the peptide obliquity in the parallel M65NN66M variant was accompanied by a decrease in the fusogenic activity and the corresponding M65NN66M 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., 1997
). 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 M65NN66M 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., 1989
). 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 34 (residues 116129) and
His (residues 387398) 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., 1998b
). We showed that the amphipathic helix at residues 154171, which corresponds to helix
45 in the 3D model, has lipid-binding activity (Peelman et al., 1997
Peelman et al., 1998a
). 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.,1998a
) and also site-directed mutagenesis experiments (Wang et al., 1998
) do not suggest that this helix is involved in substrate binding, but rather represents a structural motif of the
/ß hydrolase fold (Ollis et al., 1992
).
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 5668 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., 1990). 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., 1993
; Egloff et al., 1995
). 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., 1996
). 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., 1998a
). 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., 1996
). 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 5074 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 R52AK53A 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., 1993; Hjorth et al., 1993
; Soldatova et al., 1993
). 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., 1996
; Feaster et al., 1997
). The 5074 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., 1998a
). 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 C50C74 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 apolipoproteinphospholipid complexes is currently in progress for a more precise evaluation of the significance of this region.
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Notes |
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Received July 3, 1998; revised September 24, 1998; accepted October 7, 1998.