Structural organization of lipid phase and protein-lipid interface in apolipoprotein-phospholipid recombinants: influence of cholesterol.

The complexes of individual human plasma apolipoproteins (apo) A-I, E and A-II with dipalmitoylphosphatidylcholine (DPPC) in the absence or in the presence of cholesterol (Chol) were prepared with initial DPPC/Chol/protein weight ratio as 3:0.15:1. ApoA-I/DPPC/Chol complexes with different protein content (initial DPPC/apoA-I weight ratios were changed from 10.5:1 to 2.6:1) but with a fixed initial DPPC/Chol weight ratio of 20:1 were also prepared. The complexes were isolated by gel-filtration and characterized by size and composition. ApoA-I- and apoA-II-complexes had the same size (80-84 A) and the complexes became more heterogeneous upon Chol inclusion; apoE-complexes were larger (97-100 A) and more homogeneous and Chol addition had no effect on their hydrodynamic properties. Chol seems to be excluded partially in the following manner for isolated complexes with different apo's: A-II > E > A-I. The possible existence of two lipid regions in the complexes differing in lipid dynamics - the lipid shell in the vicinity of apolipoprotein (boundary lipid) opposite to the remaining part of the lipid bilayer - has been studied by absorbance and fluorescence spectroscopy with cis-parinaric acid (cis-PA) and trans-parinaric acid (trans-PA) embedded into the complexes. Their application is based on a strong preference of trans-PA for solid lipid while cis-PA distributes more equally between co-existing fluid and solid lipid regions (Sklar et al. (1979) Biochemistry 18, 1707-1716). (1) For apoA-I-complexes, the partition of cis-PA between water and lipid phase at temperatures below and above the transition temperature of DPPC (T(t)) was insensitive to Chol and temperature, while partition of trans-PA into the lipid phase of Chol-containing complex was increased at high temperature and decreased at low temperature. These results seem to be related to trans-PA redistribution between Chol-rich and protein-rich lipid domains, the latter being more disordered at T < T(t) and more immobilized at T > T(t) compared to the bulk bilayer; cis-PA localizes preferentially in boundary lipid. This hypothesis was directly confirmed by measurements of energy transfer between apoA-I tryptophanyls and probe molecules. (2) The relative response of trans-PA fluorescence intensity to temperature-induced phase transition of DPPC in apoA-I/DPPC/Chol complexes was decreased as a function of apolipoprotein content in a non-monotonic fashion with a transition midpoint at a mol ratio DPPC/A-I of 250:1, probably indicating two different modes of apolipoprotein/DPPC interaction in different sized complexes. (3) The comparative study of lipid dynamics in apoA-I-, apoE- and apoA-II-containing complexes with temperature response to phospholipid phase transition with fluorescence parameters such as intensity and anisotropy of cis-PA and trans-PA revealed the presence of boundary lipid in all three complexes without Chol. In contrast to apoA-I-containing complexes, in apoA-II/DPPC/Chol complexes, trans-PA seems to move preferentially into boundary lipid and cis-PA to distribute between two different regions probably as a result of more ordering action induced by apoA-II compared to apoA-I on the nearest phospholipid molecules in Chol-containing complexes; the apoE action on trans-PA and cis-PA distribution could be intermediate. Based on these results, the degree of Chol exclusion from the boundary lipid region for complexes with different apo's increasing in the order A-II > E > A-I can be suggested. Different Chol distributions between two lipid regions in the complexes seems not to be a function of complex size, but rather is an inherent property of the particular apolipoprotein molecule.

In vitro interaction of human HDL with human apolipoprotein A-II. Synthesis of apolipoprotein A-II-rich HDL.

The aim of this study was to define the specific affinity of human apolipoproteins A-I and A-II for HDL lipids and to investigate the possible transfer of apolipoproteins from the HDL molecule. For this purpose we incubated human HDL with increasing amounts of isolated apolipoprotein A-II. After incubation the reaction products were separated by gel chromatography and apolipoproteins A-I and A-II were quantified separately by immunonephelometry and HDL lipids by thin-layer chromatography. According to our results, apolipoprotein A-II progressively displaces apolipoprotein A-I to generate an HDL-like particle with identical lipid composition, hydrodynamic properties and lipid fluidity. These data indicate that apolipoprotein A-II is able to displace quantitatively apolipoprotein A-I from HDL in vitro, and that such a mechanism might contribute to the regulation of the HDL2 in equilibrium or formed from HDL3 distribution in plasma.

Reassembly of human apoproteins A-I and A-II with unilamellar phosphatidylcholine-cholesterol liposomes. Association kinetics and characterization of the complexes.

The kinetics of association between the human apoprotein A-I and apoprotein A-II and cholesterol dimyristoyl phosphatidylcholine (DMPC) vesicles are compared in this study and the lipid-apoprotein complexes are characterized. The association kinetics are followed by turbidity measurements monitoring the decrease of the vesicular size and by fluorescence polarization measurements monitoring the decrease in the mobility of the phospholipid acyl chains during complex formation. The influence of the incubation temperature and of the cholesterol/DMPC ratio has been studied by both techniques. Under all incubation conditions the apoprotein A-II associates more readily with cholesterol-DMPC vesicles than apoprotein A-I, as the kinetics are faster and the complex yield larger. With both apoproteins optimal complex formation takes place around the phospholipid transition temperature and around 10 mol% cholesterol. The apoprotein A-I/lipid association seems restricted to this narrow range for the temperature and the cholesterol/DMPC ratio, while the apoprotein A-II still associates with vesicles containing 20 mol% cholesterol and at temperatures up to 32 degrees C. The lipid-apoprotein complexes were isolated by gradient ultracentrifugation and by gel chromatography. According to these data the apoprotein A-II associates more readily than apoprotein A-I with cholesterol-DMPC vesicles to form protein-rich complexes, whilst the optimal apoprotein A-I-lipid association requires a more disordered lipid structure.

Headgroup specificity of lecithin cholesterol acyltransferase for monomeric and vesicular phospholipids.

In this study, we investigated how the nature of the phospholipid head group and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin cholesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebutanoyl)-phosphatidylcholine, 1, 2-bis-(1-pyrenebutanoyl)-phosphatidylethanolamine and 1, 2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monomers or incorporated into small unilamellar vesicles consisting of dipalmitoylphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled phospholipids was determined both by fluorescence and by high performance liquid chromatography. V(max) and K(m) were calculated for the different substrates. The data show that V(max) is 10- to 30-fold higher for the hydrolysis of monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine (PE) and the phosphatidylalcohols, while K(m) values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholine ether vesicles, we observed a 4- to 10-fold increase of V(max) for PE and the phosphatidylalcohols, and no significant change for K(m). V(max) for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity on monomeric PC and PE. These data suggest that the activity of LCAT is determined both by the molecular structure of the phospholipid and by its macromolecular properties. The LCAT activity on monomeric substrates decreases as: phosphatidylcholine&z. Gt;phosphatidylethanolamine congruent withphosphatidylpropanol congruent withphosphatidylethanol congruent withphosphatidylethyleneglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospholipid head group.

Distribution and composition of HDL subclasses in students whose parents suffered prematurely from a myocardial infarction in comparison with controls.

The purpose of this study was to analyze the distribution and composition of the HDL subclasses in students whose parents suffered from a premature coronary heart disease (CHD), in comparison with a control group without any familial history of CHD. In the first part of this study, we observed significantly lower apo A-I concentrations in the cases than in the controls. Since there was a significant difference in smoking habits between cases and controls, and since we wanted to study the effect of a parental history of premature CHD on the composition of the HDL subclasses independently of smoking, the present study was conducted on the 17 pairs of non-smokers male cases and controls. In agreement with the results on total serum, we observed lower apo A-I concentrations in the HDL2b and HDL2a + 3a fractions in the cases compared to the controls. The apo A-II and cholesterol levels in these fractions were not significantly different between the 2 groups. The selective decrease of apo A-I in the HDL subclasses with the lowest density might be due to a decreased number of particles containing only apo A-I, with which a protective effect against atherosclerosis might be associated.

Metabolic effects of a biphasic oral contraceptive preparation containing ethinyloestradiol and desogestrel on serum lipoproteins and apolipoproteins.

The effect of the administration of a biphasic oral contraceptive containing ethinyloestradiol and desogestrel on the distribution and composition of serum lipoproteins was studied in a group of 17 healthy female volunteers. The women were treated for a period of 6 months and compared with a control group of ten untreated volunteers. The serum lipoproteins were fractionated by density gradient ultracentrifugation into very low density lipoproteins (VLDL), low density lipoproteins (LDL), and into the high density lipoprotein (HDL) subfractions 2 and 3 (HDL2, HDL3). Lipids and apolipoproteins were assayed in the various fractions. No modification of either the lipid or apolipoprotein concentrations was observed in the control group. In the treated group, sex hormone-binding globulin (SHBG) and cortisol-binding globulin (CBG), and the serum content of cholesterol, triglycerides, HDL-cholesterol, apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) increased significantly after 3 and 6 months. The cholesterol and apolipoprotein B (apo B) content of VLDL increased significantly after 3 and 6 months, but remained unchanged in LDL. High density lipoprotein subfraction 2 (HDL2)-cholesterol was significantly increased after 3 and 6 months but apo A-I only after 6 months. Since apo A-II did not change, the apo A-I/A-II ratio increased significantly after 6 months of treatment. In the HDL3 fraction, the apo A-I increase was significant after 3 and 6 months, while the increase of apo A-II was significant after 6 months. The apo A-I/A-II ratio remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

A screening procedure for dyslipoproteinemia in the newborn. Apoprotein quantitation on dried blood spots.

A new screening procedure for the detection of dyslipoproteinemia in the newborn is proposed, based on apoprotein quantitation. Blood is collected by heel-prick in infants 5--7 days after birth--when apoprotein and lipids have reached stable values--and adsorbed on filter paper. Blood spots are eluted with a detergent and apoprotein A-I and B are assayed by immunonephelometry. The apo A-I/B ratio is used as a screening parameter, given its high discriminative power between normals and individuals with cardiovascular disease. The quantitation of the apo A-I/B ratio in blood spots is independent of the volume of the blood spot and of the newborn hematocrit. In a follow-up study, plasma lipid and apoprotein concentrations were assayed in the infants with an apo A-I/B ratio lower than 1.0 at 5 days, and a family study was carried out. On screening 1500, 30 infants gave positive results at birth, and were controlled between 2 and 8 months. In eight families studied, six children had abnormally high cholesterol and apo B values and five children had abnormally low HDL cholesterol and apo A-I concentrations. This screening procedure is a new approach to the detection of familial dyslipoproteinemia in the newborn, as it is based on the quantitation of the apo A-I/B protein ratio, instead of cholesterol, LDL cholesterol or beta-lipoprotein quantitation. It enables a differentiation to be made between various forms of dyslipoproteinemia, leads to a better characterization of various diseases, and decreases the percentage of false positive cases.

Influence of dietary factors on the plasma lipoprotein composition and content in neonates.

Previous studies have shown that the cholesterol and apoprotein concentrations in newborn plasma are dependent on the degree of saturation (polyunsaturated/saturated fatty acids ratio) of the dietary fats. In the present study we compare the influence on the lipoprotein patterns of breast-feeding and of two adapted formulae with a similar P/S ratio in 30 infants. The lipoprotein distribution and composition were investigated by density-gradient ultracentrifugation at days 0, 7 and 30. The lipoprotein patterns were quite comparable at 0 and 7 days in the three groups. We found low VLDL and LDL and relatively elevated HDL concentrations with a high percentage of HDL2. A significant increase of both VLDL and LDL was observed between 0 and 7 days. The VLDL concentration in the breast-fed infants subsequently decreased between 7 and 30 days to a value close to that measured at birth. The infants receiving an adapted formula had significantly higher VLDL and lower LDL at 30 days compared to breast-fed children. HDL concentrations were not significantly different whereas the HDL2 percentage was significantly lower in the infants receiving the adapted formulae. These data further support the hypothesis that the lipoprotein patterns in infants are sensitive to the type of nutrition and that breast-feeding induces specific lipoprotein patterns compared to adapted milk formulae.

Plasma apoprotein and lipid patterns in newborns: influence of nutritional factors.

In this study the plasma lipid and apoprotein concentrations have been assayed in 80 full-term newborns, at 0, 7 and 30 days of life, and the data have been analyzed as a function of the composition of the diet. The total cholesterol, HDL cholesterol, the apo A-I, A-II and B protein concentrations were followed in 4 groups of infants receiving respectively breast-feeding, adapted formulae I, II with a P/S ratio close to that of maternal milk and a formula III enriched with polyunsaturated fatty acids. After 7 and 30 days the infants receiving the adapted formulae I and II have plasma lipid and apoprotein values similar to those of the breast-fed infants indicating a parallel evolution of the lipids and apoproteins in the three groups. The lipid and apoprotein patterns were significantly different in the group of infants receiving a diet enriched with polyunsaturated fatty acids. The total and VLDL-LDL cholesterol and the apo B protein concentrations are significantly lower than in the breast-fed infants after 7 days, and these differences become more pronounced after 30 days. These results suggest that the fatty acid composition of the diet influences the lipid and lipoprotein synthesis in newborns, specially by decreasing the lipid and apoprotein concentrations of the VLDL-LDL fraction.

Displacement of apo A-I by A-II in lipid-apoprotein complexes and human HDL.

The aim of this study was to define the specific affinity of human apo A-I and apo A-II for HDL lipids and to investigate the possible transfer of apoproteins from the HDL molecule. For this purpose we incubated apo A-I -- lipid complexes prepared "in vitro", as well as human HDL with increasing amounts of isolated apo A-II. After incubation the reaction products were separated by gradient ultracentrifugation and gel chromatography. The apoproteins were quantitated separately by immunonephelometry and the apo A-I content was monitored by measuring the intrinsic tryptophan fluorescence. These results suggest that apo A-II has a higher affinity than apo A-I for the lecithin-cholesterol vesicle and that 2 mol apo A-II are able to displace 1 mol apo A-I from the apo A-I lipid complexes. Analogous results were obtained with HDL where two mol apo A-II substitute to 1 mol apo A-I to yield en apo A-II - rich HDL with identical lipid composition, hydrodynamic properties and fluidity. Such a mechanism might contribute to the regulation of the HDL2 in equilibrium with HDL3 distribution in plasma.

Lipoprotein patterns in newborns. Influence of nutritional factors.

Plasma and apoprotein levels were assayed in 3 groups of newborns at 0, 7 and 30 days and analyzed as a function of the degree of fatty acid saturation of the diet. The levels of total and HDL cholesterol and the apo A-I, apo A-II and apo B proteins were quantitated in infants either breast-fed or given an adapted formula (I) with a P/S ratio close to that of mother's milk or a formula (II) enriched in polyunsaturated fatty acids. The lipid apoprotein levels are very similar in breast-fed infants and in those receiving the adapted formula I after 7 and 30 days. The addition of PUFA to formula II, significantly affects the plasma lipids and apoproteins of the infants after one week and one month of feeding, as total HDL cholesterol, apo A-I and apo B are significantly lower than in breast-fed infants. The results of this study therefore indicate that lipid and apoprotein synthesis in newborns is sensitive to the degree of fatty acid saturation of the diet. Plasma lipoprotein concentrations at 7 and 30 days are dependent on the type of nutrition.

Comparison of the aggregation properties, secondary structure and apoptotic effects of wild-type, Flemish and Dutch N-terminally truncated amyloid beta peptides.

The Dutch (E22Q) and Flemish (A21G) mutations in the betaAPP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three Abeta(12-42) variants [wild-type (WT), Dutch and Flemish] were tested. At a concentration of 5 microM the aggregation of these peptides followed the order: Abeta(1-42) WT > Abeta(12-42) WT > Abeta(12-42) Flemish > Abeta(12-42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their alpha-helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in beta-sheet and random coil content. Apoptosis was induced in neuronal cells by the Abeta(12-42) WT and Flemish peptides at concentrations as low as 1-5 microM, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase-3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N-truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length Abeta(1-42) peptide was weaker than that induced by its N-truncated variant. These data suggest that N-truncation enhanced the cytotoxic effects of Abeta WT and Flemish peptides, which may play a role in the accelerated progression of dementia.

Hydrolysis of very-low-density lipoproteins labeled with a fluorescent triacylglycerol: 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol.

The fluorescent triacylglycerol (DPBG) 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol was incorporated into plasma very-low-density lipoproteins (VLDL) to form DPBG-VLDL. In the presence of albumin, the addition of milk lipoprotein lipase to DPBG-VLDL hydrolyses DPBG together with the VLDL triacylglycerol and pyrenyl fatty acids are transferred to albumin. As a consequence the monomer fluorescence increases while that of the excimer decreases [Mantulin, W. W., Massey, J. B., Gotto, A. M., Jr & Pownall, H. J. (1981) J. Biol. Chem. 256, 10815-10819]. The relationship of the intensity of the excimer at 475 nm to that of the monomer at 396 nm was measured before and after lipolysis of VLDL by milk lipoprotein lipase. These fluorescent changes parallel the release of free fatty acids from VLDL and their uptake by albumin. The rate of increase of monomer to excimer fluorescence was dependent upon the enzyme, substrate and albumin concentration. The lipolysis reaction, as monitored by fluorescence changes, followed Michaelis-Menten kinetics with a Km of 1.7 M for milk lipoprotein lipase. The use of the fluorescent triacylglycerol probe increases the sensitivity of the technique by a factor 50-80 compared to a technique previously reported using a fluorescent phospholipid. The present method is applicable to 2-10 micrograms triacylglycerol corresponding to about 50-100 microliters of newborn plasma or 30-50 microliters normal adult plasma. The use of an Airfuge ultracentrifuge for VLDL isolation, in conjunction with that of DPBG as a fluorescent probe enables a rapid study of VLDL lipolysis on minimal sample amounts. It can therefore be easily applied to normal and dyslipoproteinemic samples and to the newborns.

Apolipoprotein and lipid composition of plasma lipoproteins in neonates during the first month of life.

In this study the lipid and apoprotein profiles were investigated in newborns at 0, 7, and 30 days of life. The plasma lipoproteins were separated both by ultracentrifugation and gel filtration in order to compare the patterns obtained by the two techniques. At birth, the apo E concentration is comparable to that measured in adults, but its distribution among lipoproteins is significantly different as more than 80% of the plasma apo E belongs to high-density lipoproteins (HDL). At 7 and 30 days the plasma apo E concentrations are close to the values at birth, but a significant redistribution occurs from HDL to very low-density lipoproteins. By analogy with apo B, the plasma apo CIII concentration is low at birth and increases between 0 and 7 days by a factor of about two. Plasma triglycerides increase significantly during the first week of life so that the apo CIII increase is most pronounced in very low-density lipoproteins. These lipoproteins therefore become enriched in apo E, apo CIII and triglycerides between 0 and 7 days. At birth, a distinct HDL fraction, enriched in apo E, apo AII and cholesterol (HDLE), could be detected. To compensate for the low LDL levels, this HDLE fraction might function as an additional source for cholesterol delivery to peripheral tissues via the apo (B, E) receptor. At later age, low-density lipoprotein synthesis is enhanced, apo E is transferred to very low-density lipoproteins, and cholesterol delivery via the HDLE becomes less important.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of lipoprotein patterns in newborns.

The plasma levels of total and high-density lipoprotein cholesterol and of the major apolipoproteins (apo B and apo A-I) were studied in 30 newborns, on cord blood and after 7 and 30 days of life. The evolution of these parameters during the first month of life shows that newborns have low LDL cholesterol and apo B levels at birth, which increase drastically during the first week and remain constant between 7 and 30 days. The HDL cholesterol and apo A-I levels are proportionally high at birth and keep increasing slowly up to 30 days. During this period, the cholesterol/apoprotein ratio remains constant in the LDL and HDL class. These data suggest that lipid and apoprotein levels at 7 days are more representative than cord-blood levels and more meaningful for a screening of congenital hypercholesterolemia. The cholesterol/apo B and apo B/apo A-I ratios, which are considered to be better predictive factors for atherosclerosis, should be included as screening parameters.

Fluorescence depolarization studies and phase transition in human apoprotein . phospholipid complexes.

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase.

Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT.

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.