Binding Of Lecithin-cholesterol Acyltransferase Research Articles

Systematic evaluation of lecithin:Cholesterol acyltransferase binding sites in apolipoproteins via peptide based nanodiscs: Regulatory role of charged residues at positions 4 and 7.

Lecithin:cholesterol acyltransferase (LCAT) exhibits α-activity on high-density and β-activity on low-density lipoproteins. However, the molecular determinants governing LCAT activation by different apolipoproteins remain elusive. Uncovering these determinants would offer the opportunity to design and explore advanced therapies against dyslipidemias. Here, we have conducted coarse-grained and all-atom molecular dynamics simulations of LCAT with nanodiscs made with α-helical amphiphilic peptides either derived from apolipoproteins A1 and E (apoA1 and apoE) or apoA1 mimetic peptide 22A that was optimized to activate LCAT. This study aims to explore what drives the binding of peptides to our previously identified interaction site in LCAT. We hypothesized that this approach could be used to screen for binding sites of LCAT in different apolipoproteins and would provide insights to differently localized LCAT activities. Our screening approach was able to discriminate apoA1 helixes 4, 6, and 7 as key contributors to the interaction with LCAT supporting the previous research data. The simulations provided detailed molecular determinants driving the interaction with LCAT: the formation of hydrogen bonds or salt bridges between peptides E4 or D4 and LCAT S236 or K238 residues. Additionally, salt bridging between R7 and D73 was observed, depending on the availability of R7. Expanding our investigation to diverse plasma proteins, we detected novel LCAT binding helixes in apoL1, apoB100, and serum amyloid A. Our findings suggest that the same binding determinants, involving E4 or D4 -S236 and R7-D73 interactions, influence LCAT β-activity on low-density lipoproteins, where apoE and or apoB100 are hypothesized to interact with LCAT.

Abstract 1025: Structural Basis Of Apolipoprotein A1 Activation Of Lecithin:Cholesterol Acyl Transferase In High Density Lipoproteins

Lecithin:cholesterol acyl transferase (LCAT) is responsible for the formation of extracellular cholesteryl ester, which is critical for high density lipoprotein (HDL) maturation. It also modulates numerous HDL physiological functions by impacting particle subspeciation patterns. LCAT is activated by apolipoprotein A1 (APOA1) which forms antiparallel dimers around the edge of nascent HDL. Previous work has suggested that the interface of APOA1 helices 4 and 6, when properly aligned, creates two symmetrical sites for productive LCAT binding. Size exclusion chromatography was used to isolate reconstituted HDL discs containing two molecules of APOA1 chemically crosslinked to 2 LCAT molecules. Mass spectrometry using multiple crosslinkers, including a novel application of formaldehyde, indicated that LCAT interacts with both APOA1 helices 4 and 6. Cryo-electron microscopy (cryoEM) was used to determine a 10 Å structure of the complex. Using the HADDOCK suite of molecular docking tools, we developed a structural model of LCAT bound to APOA1 discs that is consistent with the preponderance of data accumulated so far. The model shows that LCAT may bind at the two symmetrical sites in different orientations, possibly reflecting different steps of the LCAT reaction. Work to improve the resolution of the cryoEM datasets is ongoing. Further work, using strategically designed cysteine mutations in APOA1 to lock helices 4 and 6 together, suggests that the helices do not move apart during the LCAT reaction process. Determination of the structural interactions between LCAT and its activator, APOA1, is crucial to understanding HDL maturation, which has significant clinical implications. This work provides the highest resolution structural model available for this important interaction.

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT–HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.

A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein

The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu(159)-Leu(170)) in nascent HDL, the so-called "solar flare" (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861-868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro(165), Tyr(166), Ser(167), and Asp(168)) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr(166) was further supported using reconstituted HDL generated from apoA-I mutants (Tyr(166) → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr(166)-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr(166)-apoA-I, after subcutaneous injection into hLCAT(Tg/Tg), apoA-I(-/-) mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency.

Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques

The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 μM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 μM −1 and for UV excitation at 295 nm was 3.2 μM −1. In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.

Interaction of lecithin:cholesterol acyltransferase (LCAT).alpha 2-macroglobulin complex with low density lipoprotein receptor-related protein (LRP). Evidence for an alpha 2-macroglobulin/LRP receptor-mediated system participating in LCAT clearance.

The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of critical importance in reverse cholesterol transport, but the structural and functional pathways involved in the regulation of LCAT have not been established. We present evidence for the direct binding of LCAT to alpha(2)-macroglobulin (alpha(2)M) in human plasma to form a complex 18.5 nm in diameter. Forty percent of plasma LCAT-HDL was associated with alpha(2)M; moreover, most of the LCAT in cerebrospinal fluid and in the medium of cultured human hepatoma cell line was associated with alpha(2)M. Purified recombinant human LCAT (rLCAT) labeled with (125)I bound to native and methylamine-activated alpha(2)M (alpha(2)M-MA) in vitro in a time- and concentration-dependent manner, and this binding did not depend on the presence of lipid. rLCAT bound to alpha(2)M-MA with greater affinity than to alpha(2)M. Furthermore, rLCAT did not activate alpha(2)M as phosphatidylcholine-specific phospholipase C does. Reconstituted HDL particles (LpA-I) inhibited the binding of rLCAT to alpha(2)M more efficiently than native HDL(3) did. LCAT associated with alpha(2)M was enzymatically inactive under both endogenous and exogenous assay conditions. Purified rLCAT alone did not bind to low density lipoprotein receptor-related protein (LRP) as lipoprotein lipase (LPL) does; however, when rLCAT was combined with alpha(2)M-MA to form a complex, binding, internalization, and degradation of rLCAT took place in LRP-expressing cells (LRP (+/+)) but not in cells deficient in LRP (LRP (-/-)). It is concluded that the binding of LCAT to alpha(2)M inhibits its enzymatic activity. Furthermore, the finding supports the possibility that the LRP receptor can act in vivo to mediate clearance of the LCAT-alpha(2)M complex and may significantly influence the bioavailability of LCAT.

Surface plasmon resonance biosensor studies of human wild-type and mutant lecithin cholesterol acyltransferase interactions with lipoproteins.

Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.

Effect of the surface lipid composition of reconstituted LPA-I on apolipoprotein A-I structure and lecithin:cholesterol acyltransferase activity

Characterization of the factors that regulate plasma cholesterol esterification shows that the increased activity of lecithin:cholesterol acyltransferase (LCAT) in the plasma of hyperlipidemic subjects is due to enhanced interactions with a preferred substrate. The details of how the physical properties of high density lipoproteins (HDL) may affect their ability to stimulate cholesterol esterification by LCAT have been investigated in homogeneous reconstituted HDL particles containing two molecules of apolipoprotein (apo) A-I (Lp2A-I) and palmitoyl-oleoyl phosphatidylcholine (POPC). Increasing the POPC or sphingomyelin (SPH) content in an Lp2A-I complex increases particle size and stability but decreases the negative surface charge of apoA-I. Increasing Lp2A-I POPC or SPH content also significantly inhibits cholesterol esterification by LCAT. Increase in the maximum rate of CE production ( V max) by LCAT is directly related to an increased negative charge on the different Lp2A-I particles and to a reduced amount and stability of amphipathic α-helices in apoA-I. In contrast, increasing the Lp2A-I complex negative charge directly by addition of a charged lipid, phosphatidylinositol (PI), has minimal effect on apoA-I conformation and LCAT activation. While variations in Lp2A-I PI content have little effect on the interfacial binding of LCAT, increasing POPC content appears to directly increase the binding affinity of LCAT for the different Lp2A-I particles. These results show that LCAT is stimulated by an apoA-I conformation-dependent increase in negative charge but is less sensitive to electrostatic changes in the lipid interface of discoidal Lp2A-I. The activation of LCAT appears to be dependent on the exposure of both central (residues 98–132) and N-terminal (residues 2–8) domains in apoA-I. A strong relationship between the immunoreactivity of two specific mAbs, 4H1 and A11, and LCAT reactivity suggests that the N-terminus of apoA-I may interact with a central domain in a manner that may regulate the accessibility of LCAT to the edge of the disc. This indicates that the conformation and charge of apoA-I are sensitive to the surface-lipid composition of HDL particles and play a central role in regulating LCAT activation. Since alterations in the surface lipid composition of HDL particles from hyperlipidemic subjects also modify the charge and structure of these particles, this may stimulate the rates of cholesterol esterification by making these lipoproteins preferred LCAT substrates.

The Effect of Apolipoprotein A-II on the Structure and Function of Apolipoprotein A-I in a Homogeneous Reconstituted High Density Lipoprotein Particle

In this study we examined the effects of apoA-II on the structure and function of apoA-I in homogeneous reconstituted HDL (rHDL). First, we measured the binding of apoA-II to apoA-I-rHDL, containing dipalmitoylphosphatidylcholine or palmitoyloleoylphosphatidylcholine, and the degree of apoA-I displacement at various ratios of apolipoproteins. Using fluorescence methods, we determined that apoA-II binding is rapid, irreversible, and associated with apoA-I displacement only when the molar ratio of apoA-II/apoA-I is greater than 1:2. Next, we used the stable apoA-II/apoA-I-rHDL complex at the apoA-II/apoA-I ratio of 1:2 to examine its physical properties, apoA-I structure, and reactivity with lecithin:cholesterol acyltransferase (LCAT). Using chemical cross-linking in conjunction with fluorescence and electrophoretic methods, we demonstrated that the conformation of apoA-I must be flexible to allow apoA-II binding to the apoA-I-rHDL particles and showed that the hybrid particles have an unchanged Stokes diameter. Fluorescence and circular dichroism measurements revealed little or no change in the secondary structure or in the N-terminal domain of apoA-I, but showed a marked destabilization of apoA-I to denaturation by guanidine hydrochloride. Limited tryptic digestion indicated that the central region of apoA-I becomes accessible to proteolysis in the hybrid particles. Together, these results suggest that amphipathic alpha-helices of apoA-II replace four central helices of one apoA-I molecule (residues approximately 99-187) in the complex and in the process destabilize apoA-I. Thus, apoA-II binding at physiologic ratios may not completely displace apoA-I from HDL, but may provide a reservoir of easily exchangeable apoA-I. Finally, we showed that the reaction of the hybrid HDL with LCAT was inhibited 2-5-fold, relative to apoA-I-rHDL, due to a corresponding increase in the apparent Km value. This suggests that LCAT binding to the hybrid particles is sterically hindered by the excess protein (portions of apoA-I and apoA-II not bound to lipid). Therefore, apoA-II can modulate the reaction of HDL with LCAT by decreasing LCAT binding to hybrid particles and making the enzyme available for reaction with other substrates.

Influence of vesicle surface composition on the interfacial binding of lecithin:cholesterol acyltransferase and apolipoprotein A-I

Interfacial binding affinities and capacities of lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein A-I (apoA-I) for surfaces of different phosphatidylcholine (PC) composition, cholesterol content, and apolipoprotein content were measured with a vesicle model system. Native polyacrylamide gel electrophoresis was used to separate free protein from vesicle-bound protein. ApoA-I was isolated from human plasma and radiolabeled with iodine, whereas radiolabeled LCAT was purified from the media of Chinese hamster ovary cells that were transfected with human LCAT cDNA and incubated in the presence of [35S] cysteine and methionine. Bound and free radiolabeled LCAT and apoA-I were quantified by phosphorimage analysis. ApoA-I binding was not influenced by cholesterol content (14 mole%) but was influenced by the PC fatty acyl composition of the vesicle. PC species containing long chain, polyunsaturated fatty acids (PUFA) in the sn-2 position resulted in increased binding affinity (Kd = 75-177 nM) but reduced capacity (0.1-0.3 apoA-I/ 1000 PC) in comparison to sn-1 palmitoyl, sn-2 oleoyl PC (POPC, 750 nM and 1.4 apoA-I/1000 PC). LCAT binding affinity to POPC (2190 nM) was stronger in the presence of cholesterol (530 nM), and LCAT binding capacity was reduced (2.63 and 0.6 molecules LCAT/1000 PC, respectively). In comparison to POPC, LCAT binding affinity to sn-1 palmitoyl, sn-2 arachidonyl PC was stronger (611 nM) and binding capacity was reduced (0.7 LCAT/1000 PC). LCAT binding affinity and capacity to sn-1 palmitoyl, sn-2 eicosapentaneoyl PC (2041 nM, and 2.5 LCAT/1000 PC) were similar to those observed for POPC. We conclude that vesicle surface PC fatty acyl composition and cholesterol content significantly influence LCAT and apoA-I interfacial binding and therefore may alter LCAT enzymatic activity.

Identification of a Domain of Lecithin–Cholesterol Acyltransferase That Is Involved in Interfacial Recognition

Lecithin–cholesterol acyltransferase (LCAT) is an interfacial enzyme that acts on lipid substrates on the surface of high density lipoproteins (HDL). Based on observations with other interfacial lipases, we propose that LCAT contains a surface region of 25 amino acids linked by a disulfide bond (C50-C74) that is involved in the binding of LCAT to lipoproteins. Using LCAT cDNA, we have deleted most of this region (Δ53-71) and expressed the mutant enzyme (LCATΔ53-71) in COS-1 cells. The deletion mutant is expressed and secreted at levels similar to wildtype LCAT, suggesting that the deleted region is located on the surface of the enzyme and is not required for folding. The enzymatic activity of the mutant was tested using two interfacial substrates, reconstituted HDL (rHDL) and low density lipoprotein (LDL), as well as a water soluble substrate, p-nitrophenyl butyrate (PNPB). There was no reaction with rHDL and LDL, but 30% of the activity with PNPB was retained. This suggests that the deleted region plays a role in interfacial binding, while the active site core is not disrupted. We thus conclude that this region (C50-C74) forms part of the interfacial binding domain of LCAT.

Analysis of human lecithin–cholesterol acyltransferase activity by carboxyl-terminal truncation

Lecithin–cholesterol acyltransferase (LCAT) is a key enzyme in reverse cholesterol transport and catalyzes the esterification of cholesterol in human plasma. Human LCAT is a glycosylated protein, containing 416 amino acids and a proline-rich region at the C-terminus. To address the function of the C-terminal region of LCAT as well as that of the proline-rich region, we constructed and expressed LCAT mutants with C-terminal truncations at different positions. The expression of wild-type LCAT in COS-1 cells resulted in an enzymatically active protein that was secreted by the cells. The mutants lacking the proline-rich region at the C-terminus were expressed and secreted at levels comparable to those of wild-type (∼50% of wild-type concentrations in cell media). The proline-deletion mutants were similar to wild-type LCAT in terms of phospholipase or transferase activities with various interfacial substrates, including reconstituted HDL, proteoliposomes, LDL, and micelles of platelet activating factor. Thus, the binding of LCAT to the diverse interfaces is not affected by the removal of its C-terminal region. Also, the activation by apolipoproteins and access of water-insoluble substrates to the active site are not significantly affected by the deletion of the proline-rich region. However, deletions of the proline-rich region, including the five amino acids nearest to the C-terminus, resulted in approximately an 8-fold increase in the specific activity of LCAT towards the water-soluble substrate, p-nitrophenylbutyrate. This suggests that the C-terminal proline-rich region may interfere with the access of this water-soluble substrate to the active site of LCAT, and may form part of a protective covering of the active site of LCAT while in solution. Further deletions at the C-terminus, beyond the proline-rich region, impaired the secretion of the enzyme, implying that this region may play a critical role in either the secretion or folding of LCAT in COS-1 cells.

Sphingomyelin Inhibits the Lecithin-Cholesterol Acyltransferase Reaction with Reconstituted High Density Lipoproteins by Decreasing Enzyme Binding

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the formation of cholesterol esters on high density lipoproteins (HDL) and plays a critical role in reverse cholesterol transport. Sphingomyelin, an important constituent of HDL, may regulate the activity of LCAT at any of the key steps of the enzymatic reaction: binding of LCAT to the interface, activation by apo A-I, or inhibition at the catalytic site. In order to clarify the role of sphingomyelin in the regulation of the LCAT reaction and its effects on the structure of apolipoprotein A-I, we prepared reconstituted HDL (rHDL) containing egg phosphatidylcholine, cholesterol, apolipoprotein A-I, and up to 22 mol % sphingomyelin. Because the interfacial properties of substrate particles can dramatically affect LCAT binding and kinetics, we also prepared and analyzed proteoliposome substrates having the same components as the rHDL, except for a 4-fold higher ratio of phospholipid to apolipoprotein A-I. The reaction kinetics of LCAT with the rHDL particles revealed no significant change in the apparent Vmax but showed a concentration-dependent increase in slope of the reciprocal plots and in the apparent Km values with sphingomyelin content. The dissociation constant (Kd) for LCAT with these particles increased linearly with sphingomyelin content up to 22 mol %, changing in parallel with the apparent Km values. No structural changes of apolipoprotein A-I were detected in the particles with increasing content of sphingomyelin, but fluorescence results with lipophilic probes revealed that significant changes in the acyl chain, backbone, and head group regions of the lipid bilayer of the particles are introduced by the addition of sphingomyelin. On the other hand, the proteoliposome substrates also had increased Kdvalues for LCAT at high sphingomyelin contents but compared with the rHDL particles had a 6-10-fold lower affinity for LCAT binding and exhibited kinetics consistent with competitive inhibition by sphingomyelin at the active site. These results show conclusively that the dominant mechanism for the inhibition of LCAT activity with rHDL particles by sphingomyelin is the impaired binding of the enzyme to the interface. The results also underscore the significant differences in the enzyme reaction kinetics with different substrate particles.

Binding of lecithin:cholesterol acyltransferase to reconstituted high density lipoproteins is affected by their lipid but not apolipoprotein composition.

The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of critical importance in reverse cholesterol transport. We studied the relationship between LCAT binding and HDL composition using two assays to determine LCAT binding affinity for discoidal reconstituted HDL (rHDL). We prepared rHDL with egg phosphatidylcholine (egg-PC), cholesterol, and either apolipoprotein (apo) A-I or apoA-II. The rHDL, identical in lipid content, had sizes of 96 and 100 A, respectively. Binding constants (Kd) determined by the activity-inhibition method were 2.2 +/- 0.3 x 10(-7) and 1.1 +/- 0.3 x 10(-16) M, whereas those determined by a solid-phase method were 3.3 +/- 0.9 x 10(-7) and 3.7 +/- 0.9 x 10(-7) M for apoA-I and apoA-II rHDL, respectively. The stoichiometry was 1 LCAT bound/rHDL. The appVmax/appKm for apoA-I was 80-fold higher than for apoA-II rHDL. The large difference between LCAT-binding constants and enzymatic activity measurements for the two particles suggests that LCAT binding and activation by apolipoproteins are independent events. To determine the effects of phospholipid headgroup on LCAT binding affinity, we tested rHDL containing up to 16 mol % of egg phosphatidylethanolamine (egg-PE), egg phosphatidic acid (egg-PA), or bovine phosphatidylserine (PS). Kd was unchanged for PS rHDL, but increased 6-fold with increasing PE content. AppVmax increased with PE content, but decreased with PS or PA relative to egg-PC controls. AppKm increased in PE rHDL, but remained unchanged in rHDL with PA or PS. Fluorescence characterization of the lipid domains of rHDL shows small differences in the polarity of the headgroup region of PE rHDL. Thus, LCAT binding is influenced by the lipid, but not the protein composition of rHDL. AppVmax values reflect active site preferences, while appKm values reflect interfacial binding affinity.

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: Cholesterol acyltransferase: An energy transfer study

1. 1. To investigate whether a direct protein-protein interaction between apoA-I and lecithin: cholesterol acyltransferase (LCAT) is necessary for the activation of the enzyme, apoA-I was labelled with N-methylisatoic anhydride at lysine residues. The intermolecular resonance energy transfer from tryptophan residues of LCAT (donor) to N-methyl-anthraniloyl (NMA)-labelled apoA-I (NMA-apoA-I) (acceptor) was used as a sensitive fluorescence method for studying molecular interactions. 2. 2. In the absence of lipids no fluorescence energy transfer was measurable. 3. 3. Fluorescence energy transfer occurred from LCAT to NMA-apoA-I in the presence of liposomes with phospholipid/cholesterol ratios ranging from 5:1 to 18:1 and regardless whether only 1 or up to 5 NMA-apoA-I molecules resided at the liposome surface. 4. 4. This indicates a preferred binding of the enzyme directly to or in spatial proximity to the activator protein NMA-apoA-I even if enough space at the liposome surface is available to allow LCAT binding at a distance, where no energy transfer is measurable.

Two different allelic mutations in the lecithin-cholesterol acyltransferase gene associated with the fish eye syndrome. Lecithin-cholesterol acyltransferase (Thr123----Ile) and lecithin-cholesterol acyltransferase (Thr347----Met).

We have elucidated the genetic defect in a 66-yr-old patient with fish eye syndrome (FES) presenting with severe corneal opacities and hypoalphalipoproteinemia. The patient's plasma concentration of high density lipoprotein (HDL) cholesterol was reduced at 7.7 mg/dl (35.1-65.3 mg/dl in controls) and the HDL cholesteryl ester content was 31% (60-80% in controls); however, total plasma cholesteryl esters were similar to normal (60% of total cholesterol vs. a mean of 66% in controls). The patient's plasma cholesterol esterification rate was slightly reduced at 51 nmol/ml per h (control subjects: 61-106 nmol/ml per h), whereas lecithin-cholesterol acyltransferase (LCAT) activity, assayed using a HDL-like exogenous proteoliposome substrate, was virtually absent (0.9 nmol/ml per h vs. 25.1-27.9 nmol/ml per h in control subjects). DNA sequence analysis of the proband's LCAT gene revealed two separate C to T transitions resulting in the substitution of Thr123 with Ile and Thr347 with Met. The mutation at codon 347 created a new restriction site for the enzyme Nla III. Analysis of the patient's polymerase chain reaction-amplified DNA containing the region of the Thr347 mutation by digestion with Nla III confirmed that the proband is a compound heterozygote for both defects. The patient's daughter, who is asymptomatic despite a 50% reduction of LCAT activity, is heterozygous for the Thr123----Ile mutation. Our data indicate that the regions adjacent to Thr123 and Thr347 of LCAT may play an important role in HDL cholesterol esterification, suggesting that these regions may contain a portion of the LCAT binding domain(s) for HDL.