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PubMed1996

Glycosylation structure and enzyme activity of lecithin:cholesterol acyltransferase from human plasma, HepG2 cells, and baculoviral and Chinese hamster ovary cell expression systems.

The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT), media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.

Amidohydrolases↗
PubMedDate not supplied

Time course of changes in rat serum lecithin-cholesterol acyl-transferase activity and high-density-lipoprotein composition during the consumption of two different low-protein diets followed by a balanced diet.

We studied the effects of low-protein diets on high-density-lipoprotein (HDL) composition and checked whether the changes observed were correlated with lecithin-cholesterol acyl-transferase (LCAT) activity. We also studied whether HDL lipid and protein compositions and LCAT activity were modified differently in growing rats during the consumption of two low-protein diets [2% casein (C) and 5% gluten (GI)] for 28 days, followed by the refeeding of a balanced diet containing 15% casein for 14 days. The control group was fed the balanced diet for 42 days. LCAT activity was determined by conversion of 3H-cholesterol into 3H-esterified cholesterol. The consumption of both protein-depleted diets highly decreased LCAT activity. At the end of the period of protein malnutrition, LCAT activity was only 22% and 13% of that of the control group in the C and GI groups, respectively. There was no significant difference between the two depleted diets. At day 3 of refeeding, values of both C and GI groups returned to control values. Despite the reduction in LCAT activity with both types of protein-depleted diets, HDL metabolism was not significantly impaired. This might be partly due to the maintenance of higher apolipoprotein A-I levels. The reduced LCAT activity could be attributable to reduced synthesis of LCAT in the liver during both protein-depleted diets.

Animals↗
PubMed1996

Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates.

Comparative studies indicate that plasma cholesteryl ester (CE) composition is correlated with susceptibility to atherosclerosis. We previously showed that the lecithin:cholesterol acyltransferases (LCATs) of susceptible species such as rabbit, pig, and chicken (group I) differ in their substrate and positional specificities from the LCATs of resistant species such as rat and mouse (group II). However, the relative importance of enzyme specificity and substrate phosphatidylcholine (PC) composition in determining the CE composition is not known. To address this, we analyzed the molecular species composition of plasma PC in the same 14 vertebrates in which we previously studied the CE composition and LCAT specificity. The utilization of native PC species by LCAT was studied by determining the loss of each PC after incubation of plasma at 37 degrees C. The major contributor for LCAT reaction was either 16:0-18:2 PC or 18:0-18:2 PC in all species except dog, in which it was 18:0-20:4 PC. The formation of 20:4 CE correlated more with the consumption of 18:0-20:4 PC in group I, and with the consumption of 16:0-20:4 PC in group II. The group II enzymes exhibited higher selectivity for sn-2-20:4 PCs, whereas the group I enzymes showed preference for sn-2-18:2 PCs. The synthesis of high percentage of 20:4 CE in dog plasma was found to be due to the presence of unusually high concentration of 18:0-20:4 PC, rather than due to enzyme selectivity. These results show that the PC molecular species composition, especially the concentrations of sn-2-20:4 phosphatidylcholines has profound influence on plasma CE composition, and possibly on atherogenic risk.

Animals↗
PubMed1996

Effects of carboxy-terminal truncation on human lecithin:cholesterol acyltransferase activity.

Mutagenesis was carried out in human lecithin:cholesterol acyltransferase (LCAT) to generate mutants with stop codons at positions corresponding to amino acids 315, 341, 359, 375, 388, 394, and 398 of the 416-amino acid sequence of the mature enzyme protein. Deletion of the 18 terminal amino acids of the protein was without effect on LCAT phospholipase or acyltransferase activity, or the stability of the protein to denaturation at 37 degrees C. Further deletion led to loss of most of the activity, associated with a 10-fold increase in the rate of denaturation at 37 degrees C. These data indicate that the proline-rich C-terminus of LCAT is not required for effective enzyme activity. The loss of activity that accompanied deletion of residues 394-398 suggests a structural role for these residues, part of a series of predicted beta-sheet sequences in the C-terminal third of the LCAT primary sequence.

Animals↗
PubMed1996

Remodelling of reconstituted high density lipoproteins by lecithin: cholesterol acyltransferase.

Discoidal reconstituted high density lipoproteins (rHDL) with a diameter of 7.9 nm, a molar ratio of egg phosphatidylcholine (PC): unesterified cholesterol (UC): cholesteryl esters (CE): apolipoprotein (apo) A-I of 33:7:0:1 and containing two molecules of apoA-1 per particle were incubated with lecithin:cholesterol acyltransferase (LCAT) in the presence of low density lipoproteins as a source of additional UC and PC for the LCAT reaction. After 24 h of incubation, the rHDL had a diameter of 8.8 nm, a molar ratio of PC:UC:CE:apoA-I of 16:3:23:1 and contained three rather than two molecules of apoA-I per particle. The fact that there was no change in the concentration of rHDL-associated apoA-I indicated that the increase from two to three molecules of apoA-I per particle was achieved at the expense of a one-third reduction in the number of rHDL particles in a process that must have involved particle fusion. When the incubations were repeated in the presence of exogenous, lipid-free apoA-I, the resulting rHDL were identical in size and composition to those generated in its absence. Under these conditions, however, the increase from two to three molecules of apoA-I per rHDL particle coincided with a 50% increase in the concentration of rHDL-associated apoA-I. Thus, when lipid-free apoA-I is available, the LCAT-mediated increase in number of apoA-I molecules per rHDL particle is achieved by a direct incorporation of lipid-free apolipoprotein without any need for particle fusion and therefore without a reduction in the number of rHDL particles.

Apolipoprotein A-I↗
PubMed1977

Liver tissue lecithin: cholesterol acyltransferase activity in patients with liver disease.

Lecithin: Cholesterol acyltransferase (LCAT) activity was measured in serum and liver tissue from patients with parenchymal liver disease. Serum LCAT activity was within normal limits and it was probably related to the absence of clinical and laboratory evidence of a decompensated liver function. Liver tissue LCAT activity is about tenfold lower than that in serum. The relative proportion of cholesterol esters in liver tissue was much higher that could be expected according the low tissue LCAT activity. This findings suggest that LCAT is a "plasma specific" enzyme and that cholesterol esters in parenchyma may be considered as a storage form of cholesterol esterified in plasma pool.

Acyltransferases↗
PubMed1996

Localization of apolipoprotein A-I epitopes involved in the activation of lecithin:cholesterol acyltransferase.

Eight murine monoclonal antibodies (Mab) to apolipoprotein A-I were characterized for their epitopes and for their ability to interfere with lecithin:cholesterol acyltransferase (LCAT) activation mediated by apo apoA-I using a synthetic substrate. Using overlapping synthetic peptides we have identified six continuous epitopes that span amino acids 1-10 (Mab A-I-19), 96-101 (Mab A-I-15), 133-141 (Mab A-I-5), 140-145 (Mab A-I-9), 144-148 (Mab A-I-8), and 167-174 (Mab A-I-57). Furthermore, antibodies A-I-11 and A-I-16 recognized discontinuous epitopes, namely amino acids 124-128 and 144-148. When antibodies were tested for their ability to inhibit LCAT activation, an inhibitory effect was observed with those whose epitopes covered the area of apoA-I encompassing amino acids 96-174. From these data we conclude that several areas of apoA-I spanning the middle region of the apolipoprotein act in concert to stimulate LCAT activity, possibly by cooperative interaction with the enzyme.

Animals↗
PubMed1997

[Acylation of 3beta-(2-hydroxyethoxy)cholest-5-ene in the presence of lecithin:cholesterol acyltransferase from blood plasma].

3 beta-(2-Hydroxyethoxy)cholest-5-ene in the composition of the micellar complexes with apolipoprotein A1 and palmitoyloleoylphosphatidylcholine was acylated in the presence of lecithin-cholesterol acyltransferase from human plasma. The initial rate of acylation was 6-8 times lower than the rate of cholesterol acylation under the same conditions. The presence of 3 beta-(2-hydroxyethoxy)cholest-5-ene did not affect the rate of the enzymic acylation of cholesterol.

Acylation↗
PubMed1997

Expression of human lecithin:cholesterol acyltransferase in transgenic mice: effects on cholesterol efflux, esterification, and transport.

Human lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol and is postulated to participate in a physiologic process called reverse cholesterol transport. We have used transgenic mice expressing the human LCAT transgene to study the effect of increased plasma levels of LCAT in each of the proposed steps involved in the reverse cholesterol transport pathway. High density lipoprotein (HDL) from LCAT transgenic mice was 44% more efficient than control mouse HDL in the efflux of cholesterol from human skin fibroblasts. Esterification of cell-derived cholesterol was also markedly increased in mice expressing the human LCAT transgene. The rate of plasma clearance of HDL cholesteryl ester was virtually the same in both types of animals whereas the HDL cholesteryl ester transport rate was significantly increased in mice expressing the human LCAT transgene (152.3 +/- 16.9 micrograms/h vs. 203.1 +/- 30.9 micrograms/h in control and transgenic mice, respectively). Liver cholesteryl ester uptake was significantly increased in mice expressing human LCAT (58.0 +/- 1.4 micrograms/h/g liver vs. 77.9 +/- 1.7 micrograms/h/g liver in control and transgenic mice, respectively). These studies indicate that LCAT modulates the rate by which cholesterol is effluxed from cell membranes onto HDL, esterified, and transported to the liver.

Animals↗
PubMed1977

Rotational relaxation of the "microviscosity" probe diphenylhexatriene in paraffin oil and egg lecithin vesicles.

The rotational relaxation of the widely used "microviscosity" probe, 1,6-diphenyl-1,3,5-hexatriene, was examined by the technique of nanosecond time-resolved fluorescence depolarization. The decays of the emission anisotropy were determined at five temperatures in the range 3-31 degrees both in a reference paraffin oil and in sonicated egg lecithin vesicles. These decays were complex in both media. Marked qualitative as well as quantitative differences were observed in the rotational behavior of the probe in the complex bilayer medium as opposed to the homogeneous reference solvent. The results are discussed in relation to the structure of the hydrophobic bilayer membrane interior and the concept of its "microviscosity".

Mathematics↗
PubMed1997

Long-chain polyunsaturated fatty acids in the sn-2 position of phosphatidylcholine decrease the stability of recombinant high density lipoprotein apolipoprotein A-I and the activation energy of the lecithin:cholesterol acyltransferase reaction.

The lecithin:cholesterol acyltransferase (LCAT) kinetics and activation energy and the stability of apolipoprotein A-I (apoA-I) were investigated using recombinant HDL (rHDL) containing phosphatidylcholine (PC), [3H]cholesterol, and apo A-I. The PC component of the rHDL contained sn-1 16:0 and sn-2 18:1 (POPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC) or 10% of the respective PC species and 90% sn-1 18:1, sn-2 16:0 PC ether (OPPC ether). The appVmax of the rHDL containing 100% PC varied 10-fold and was ordered POPC > PEPC > PAPC > PDPC, whereas the appKm values varied 19-43 microns PC. The ether-containing rHDL had appVmax values 17-40% of their respective 100% PC rHDL, but maintained the same rank order. The activation energy of LCAT was lower for rHDL containing long chain polyunsaturated fatty acids (PUFA) compared to rHDL containing 100% POPC or 10% PC/90% OPPC ether. The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.2-16 M) compared to those containing 100% POPC or 10% PC/90% OPPC (2.2-2.4 M) and there was a strong correlation (r = 0.71) between LCAT activation energy and the stability of apoA-I (i.e., D1/2). We conclude that long chain PUFA in the sn-2 position of PC decreases the catalytic efficiency of LCAT, the activation energy of the LCAT reaction and the stability of apoA-I on the rHDL particles. The strong association between rHDL apoA-I stability and LCAT activation energy suggests that the temperature-dependent step of the LCAT reaction may be sensitive to the strength of the interaction of apoA-I with rHDL PC.

Apolipoprotein A-I↗
PubMed1997

Biochemical and biophysical characterization of human recombinant lecithin: cholesterol acyltransferase.

We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.

Animals↗
PubMed1997

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.

Animals↗
PubMed1976

[Determination of lecithin: cholesterol acyltransferase activity by a radioisotopic assay (author's transl)].

A method for determining Lecithin: Cholesterol Acyltransferase (LCAT) activity is presented. Trace amounts of labelled cholesterol are added to innactivated homologous substrate from a pool of normal serum. LCAT activity is determined measuring the cholesterol esterification, and is expressed as micrograms of cholesterol esters formed per milliliter plasma per hour. LCAT time-course, reproducibility, activity after serum storage at 4 degrees C, and normal values from 20 healthy subjects are studied.

Acyltransferases↗
PubMed1998

Lecithin-bound superoxide dismutase in the prevention of neutrophil-induced damage of corneal tissue.

PURPOSE: To evaluate the effects of a lipophilic analog of superoxide dismutase (SOD) in the prevention of polymorphonuclear leukocyte (PMN)-induced damage to corneal epithelial cells in vitro and in bacterial corneal ulcers in vivo. METHODS: Immortalized human corneal epithelial cells (T-HCEC) were cocultured with human PMNs activated with N-formyl-methionyl-leucyl-phenylalanine for 18 hours, after which lactate dehydrogenase (LDH) activity of the supernatant was measured as a marker of cellular damage. The inhibitory effects of lecithin-bound SOD (PC-SOD) and unmodified SOD, as well as PMNs pretreated with anti-CD 18 monoclonal antibody, were compared with untreated control. The retention of each drug on the ocular surface of healthy volunteers was measured by flow cytometry using brush cytology samples. The protective effects of a 0.1% solution of PC-SOD on Pseudomonas aeruginosa corneal infection in guinea pigs were assessed by inflammatory grading scores and histology. RESULTS: Both PC-SOD and SOD effectively suppressed PMN-induced LDH release in T-HCEC in a dose-dependent manner. LDH release was also attenuated when PMNs were pretreated with anti-CD 18 antibodies, suggesting that adhesion molecules were involved in the process. Brush cytology of conjunctival samples showed that PC-SOD was retained longer on the ocular surface compared with unmodified SOD. PC-SOD significantly prevented excessive tissue damage by infiltrating PMNs in P. aeruginosa corneal infection, whereas in control eyes, perforation of the cornea occurred by 6 days. CONCLUSIONS: PC-SOD was effective in attenuating PMN-related tissue damage to corneal tissue both in vitro and in P. aeruginosa infection in guinea pigs.

Animals↗
PubMed1997

Overexpression of human lecithin:cholesterol acyltransferase in cholesterol-fed rabbits: LDL metabolism and HDL metabolism are affected in a gene dose-dependent manner.

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme well known for its involvement in the intravascular metabolism of high density lipoproteins; however, its role in the regulation of apolipoprotein (apo) B-containing lipoproteins remains elusive. The present study was designed to investigate the metabolic mechanisms responsible for the differential lipoprotein response observed between cholesterol-fed hLCAT transgenic and control rabbits. 131I-labeled HDL apoA-I and 125I-labeled LDL kinetics were assessed in age- and sex-matched groups of rabbits with high (HE), low (LE), or no hLCAT expression after 6 weeks on a 0.3% cholesterol diet. In HE, the mean total cholesterol concentration on this diet, mg/dl (230 +/- 50), was not significantly different from that of either LE (313 +/- 46) or controls (332 +/- 52) due to the elevated level of HDL-C observed in HE (127 +/- 19), as compared with both LE (100 +/- 33) and controls (31 +/- 4). In contrast, the mean nonHDL-C concentration for HE (103 +/- 33) was much lower than that for either LE (213 +/- 39) or controls (301 +/- 55). FPLC analysis of plasma confirmed that HDL was the predominant lipoprotein class in HE on the cholesterol diet, whereas cholesteryl ester-rich, apoB-containing lipoproteins characterized the plasma of LE and, most notably, of controls. In vivo kinetic experiments demonstrated that the differences in HDL levels noted between the three groups were attributable to distinctive rates of apoA-I catabolism, with the mean fractional catabolic rate (FCR, d-1) of apoA-I slowest in HE (0.282 +/- 0.03), followed by LE (0.340 +/- 0.01) and controls (0.496 +/- 0.04). A similar, but opposite, pattern was observed for nonHDL-C levels and LDL metabolism (h-1), such that HE had the lowest nonHDL-C levels with the fastest rate of clearance (0.131 +/- 0.027), followed by LE (0.057 +/- 0.009) and controls (0.031 +/- 0.001). Strong correlations were noted between LCAT activity and both apoA-I (r= -0.868, P < 0.01) and LDL (r = 0.670, P = 0.06) FCR, indicating that LCAT activity played a major role in the mediation of lipoprotein metabolism. In summary, these data are the first to show that LCAT overexpression can regulate both LDL and HDL metabolism in cholesterol-fed rabbits and provide a potential explanation for the prevention of diet-induced atherosclerosis observed in our previous study.

Animals↗
PubMedDate not supplied

Antioxidant activity of flavones from Scutellaria baicalensis in lecithin liposomes.

The antioxidant effect of a trihydroxyflavone extract from Scutellaria baicalensis on oxidation induced by ultraviolet light, was studied with phosphatidylcholine liposome membrane. Also, as standards, the antioxidative activity of baicalin, wogonin, baicalein and butylated hydroxytoluene (BHT) was investigated. Comparison of the protective effects of the compounds studied against photoinduced lipid peroxidation in lecithin liposome membranes showed that: (1) the inhibitory effect of those compounds (at 1.2 mol% antioxidant content in liposomes) on TBA reactive materials from lipid peroxidation decreased in the order of baicalin > BHT approximately equal to Scutellaria baicalensis. These were found much greater than wogonin and baicalein; (2) the depressed effect of those compounds (at 1.1 mol% compounds content in liposomes) on the production of conjugated dienes (proportional to oxidation index) could be classified as follows: Scutellaria baicalensis approximately equal to baicalin > BHT, these three were found more active much greater than baicalein and wogonin. Results obtained by ESR measurement confirm that Scutellaria baicalensis extract and the BHT compound significantly depressed the effect of liposome oxidation. It was found that the new trihydroxyflavones of Scutellaria baicalensis, ensured a very satisfactory concentration-dependent protection of the liposome membrane against UV-induced oxidation. These findings suggest that some of the beneficial effects of the extract of the Scutellaria baicalensis can be mediated in certain diseases (for example in skin diseases) by their ability to scavenge free radicals and by their protective effect on lipid peroxidation caused by sunlight irradiation.

Antioxidants↗
PubMed1998

Role of glutamic acid residues 154, 155, and 165 of lecithin:cholesterol acyltransferase in cholesterol esterification and phospholipase A2 activities.

Previous studies have shown that cholesterol esterification activity by lecithin:cholesterol acyltransferase (LCAT) is progressively inhibited as up to three acidic acid residues are chemically modified. The purpose of this study was to determine whether three glutamic acid residues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid binding region of apoE, were necessary for enzymatic activity. Site-directed mutagenesis was used to generate mutant constructs of LCAT in which glutamic acid residues 154, 155, and 165 were replaced with glutamine or lysine. Media harvested from transiently transfected COS cells was used as a source of LCAT for cholesterol esterification and phospholipase A2 (PLA2) assays. Cholesterol esterification for all mutant constructs (11-26 nmol CE/h/microg) was similar to or greater than that of wild type LCAT (16 nmol CE/h/microg), except for a triple mutant, in which glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/microg). PLA2 activity followed a similar trend. There was a significant decrease in the cholesterol esterification to PLA2 activity ratio when residue 165 was mutated from its wild type negative charge (E) to an uncharged (Q) or positive (K) charged residue (10.2 vs. 6.0 vs. 4.3, respectively). We conclude that glutamic acid residues 154, 155, and 165 individually or collectively are not necessary for LCAT activity and that residue 165 may be in a region of LCAT that is involved with cholesterol binding or is sensitive to cholesterol binding at the active site of the enzyme.

Amino Acid Sequence↗