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Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

We identified two regions of human LCAT (hLCAT) that when mutated separately to the corresponding rat sequence (E149A and Y292H/W294F) and transiently expressed in COS-1 cells increased phospholipase A2 (PLA2) activity by 5.5- and 2.8-fold, respectively, and increased cholesteryl ester (CE) formation by 2.9- and 1.4-fold, respectively, relative to hLCAT using substrate particles containing 1-16:0,2-20:4-sn-glycero-3-phosphocholine (PAPC). In contrast, both activities with 1-16:0,2-18:1-sn-glycero-3-phosphocholine (POPC) substrate were similar among the three LCAT proteins. The triple mutant (E149A/Y292H/W294F) had increased PLA2 activity with PAPC similar to that observed with the E149A mutation alone; however, unlike E149A, the triple mutant demonstrated a 50% decrease in activity with POPC for both PLA2 activity and CE formation, suggesting an interaction between the two regions of LCAT. Additional mutagenesis studies demonstrated that W294F, but not Y292H, increased PLA2 activity by 3-fold with PAPC without affecting activity with POPC. The E149A/W294F double mutation mimicked the LCAT activity phenotype of the triple mutant (more activity with PAPC, less with POPC). In conclusion, separate mutation of two amino acids in hLCAT to the corresponding rat sequence increases activity with PAPC, whereas the combined mutations increase PAPC and decrease POPC activity, suggesting that these amino acids participate in the LCAT PC binding site and affect fatty acyl specificity.

We previously described a point mutation in human LCAT (E to A at residue 149; hE149A) that demonstrated greater activity with phosphatidylcholine (PC) substrate containing 20:4 in the sn-2 position compared with the wild-type enzyme [hLCAT; Wang et al. (1997) J. Biol. Chem. 272, 280-286], resulting in a human enzyme with the substrate specificity similar to that of rat LCAT. The purpose of the present study was to explore the molecular basis for the role of amino acid 149 in determining fatty acyl substrate specificity. In the first experiment, the reverse mutation in rat LCAT (rA149E) converted substrate specificity of rat LCAT toward that of the human enzyme, demonstrating that the mutation was context independent and reversible. In the second experiment, we found that hE149A compared with hLCAT demonstrated higher activity with PC species containing 20-carbon, but not 18-carbon, sn-2 fatty acyl chains. The increased activity of hE149A was due to an increase in apparent V(max) but not to apparent K(m) or LCAT binding to the PC surface. Substitution of different amino acids in the 149 position of hLCAT showed that activation of the enzyme with sn-2 20:4 containing PC substrate was only observed when the negative charge at residue 149 was removed. We conclude that the negative charge at amino acid 149 of LCAT is a critical determinant for the specificity of the enzyme for PC containing 18- vs 20-carbon sn-2 fatty acyl chains.

The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn-2 fatty acid of phospholipid to cholesterol. To facilitate the isolation of large quantities of LCAT and to assist in future structure;-function studies, LCAT containing a carboxy-terminal histidine-tag (H6) was expressed in Chinese hamster ovary cells (CHO). A high level of CHO-hLCATH6 expression ( approximately 15 mg L(-1)) was achieved over a 72-h period using 10 mm sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( approximately 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 +/- 26%. CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 +/- 6 and 26 +/- 3 nmol CE formed microg(-1) h(-1), respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4 degrees C in excess of 60 days. Substrate saturation studies, using rHDL composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indicated that the appK(m) for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at approximately 2 microm substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT.

The effects of polyunsaturated fatty acids (PUFA) on the structure of recombinant high density lipoprotein (rHDL) was investigated using homogeneous particles containing phosphatidylcholine (PC), [3H]cholesterol, and apolipoprotein A-I (apoA-I). The PC component of the rHDL contained sn -1 16:0 and sn -2 18:1 (POPC), 18:2 (PLPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC). The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.57-1.70 m) compared to those containing POPC (2.83 m). Intrinsic apoA-I tryptophan fluorescence emission intensity and lifetimes were decreased for rHDL containing long chain PUFA compared to POPC and PLPC rHDL. Monoclonal antibody binding studies demonstrated that apoA-I had decreased immunoreactivity with monoclonal antibodies spanning amino acid residues 115-147 in rHDL containing long chain PUFA. PC lipid fluidity, measured as diphenylhexatriene (DPH) fluorescence polarization, was increased in PUFA rHDL compared to POPC rHDL. There also was a strong correlation between the number of sn -2 double bonds in rHDL and DPH fluorescence lifetime (r 2 = 0. 89). LCAT reactivity of the homogeneous size rHDL was ordered POPC = PLPC>PAPC> PEPC>PDPC. We conclude that rHDL with long chain PUFA in the sn -2 position of PC contain apoA-I that is less stable and in a different conformation than that in POPC rHDL and have a fatty acyl region that is more fluid and hydrated. The weaker interaction of apoA-I with PC containing PUFA may lead to hypercatabolism of apoA-I in plasma explaining, in part, the decreased plasma HDL and apoA-I concentrations seen with PUFA diets.

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.

Rat lecithin:cholesterol acyltransferase (LCAT) cDNA was obtained by reverse transcriptase/polymerase chain reaction amplification of rat liver total RNA. A consensus sequence was derived from four independent clones from two strains of rats. In vitro expression of rat LCAT cDNA in COS cells resulted in secreted enzyme protein with the same fatty acyl specificity for phospholipase A2 activity and cholesterol esterification as rat plasma LCAT, but different from that of recombinant or human plasma LCAT.

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.

Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleoyl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmitoyl-2-arachidonoyl PC (PAPC). Six recombinant human LCAT cDNA clones were constructed with unique clusters of rat sequence substitutions in the human background spanning the region encoding amino acids 121-296. Media from transfected COS cells expressing each of the constructs were assayed for LCAT cholesterol esterification (CE) or phospholipase A2 (PLA2) activity using substrate particles containing POPC or PAPC. The PAPC/POPC CE activity ratio of the cluster 1 construct (amino acids 149-158) was 1.3, resembling rat LCAT, whereas cluster 2-5 clones produced CE activity ratios <0.3, unchanged from human LCAT. The cluster 6 clone (Y292H/W294F) had an intermediate ratio (0.6). Similar results were observed for LCAT PLA2 activity. In additional studies, position 149 of human LCAT was changed to the rat sequence (hE149A) and compared to a triple mutation containing the remainder of the cluster 1 changes (G151R/E154D/R158Q). CE and PLA2 activity ratio for the hE149A construct was >1.7, similar to rat LCAT, whereas the triple mutation construct retained a ratio similar to human LCAT (<0.6). Thus, a single amino acid substitution (E149A) was sufficient to alter the fatty acyl specificity of human LCAT to that of rat LCAT, with an increase in activity toward PAPC. This is the first example of a point mutation in an enzyme with PLA2 activity that results in an increase in activity toward arachidonic acid.

Plasma cholesteryl ester (CE) synthesis by lecithin cholesterol acyltransferase (LCAT) is activated by apolipoprotein (apo)A-I. We studied the effect of plasma apoA-I concentration on LCAT activation, using normal, heterozygous or homozygous apoA-I-deficient mice made by gene targeting. Plasma esterified cholesterol concentrations of mice fed chow diets were ordered (mean +/- SEM): 105 +/- 7 (normal) > 70 +/- 5 (heterozygotes) > 26 +/- 2 (homozygotes) mg/dl. Plasma free cholesterol concentrations were similar among the three genotypes. Endogenous LCAT activity, measured as the decrease in plasma free cholesterol after a 1 h incubation at 37 degrees C, was ordered: 44 +/- 3 (normal) > 21 +/- 2 (heterozygotes) > 5 +/- 1 (homozygotes) nmol CE formed/h per ml plasma. Using a recombinant exogenous substrate consisting of egg yolk phospholipid, [14C]cholesterol, and apoA-I, CE formation of normals and heterozygotes was similar (27.4 +/- 0.6 and 28.8 +/- 1.3 nmol/h per ml plasma, respectively), but was significantly less for homozygotes (19.2 +/- 1.7 nmol/h per ml plasma). However, using a small unilamellar vesicle substrate particle containing phospholipid and [14C]cholesterol, CE formation was ordered: 1.6 +/- 0.1 (normal) = 1.6 +/- 0.1 (heterozygotes) > 0.6 +/- 0.1 (homozygotes) nmol/h per ml plasma; addition of apoA-I to the plasma of homozygous animals restored CE formation to normal levels (1.6 +/- 0.1). CE fatty acid analysis demonstrated that plasma from homozygous mice contained significantly more saturated and monounsaturated and fewer polyunsaturated fatty acids compared to normal and heterozygous mice.(ABSTRACT TRUNCATED AT 250 WORDS)