Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease

Abstract

In order to test the hypothesis that fish-eye disease (FED) is due to a deficient activation of lecithin:cholesterol acyltransferase (LCAT) by its co-factor apolipoprotein (apo) A-I, we overexpressed the natural mutants T123I, N131D, N391S, and other engineered mutants in Cos-1 cells. Esterase activity was measured on a monomeric phospholipid enelogue, phospholipase A 2 activity was measured on reconstituted high density lipoprotein (HDL), and acyltransferase activity was measured both on rHDL and on low density lipoprotein (LDL). The natural FED mutants have decreased phospholipase A 2 activity on rHDL, which accounts for the decreased acyltransferase activity previously reported. All mutants engineered at positions 131 and 391 had decreased esterase activity on a monomeric substrate and decreased acyltransferase activity on LDL. In contrast, mutations at position 123 preserved these activities and specifically decreased phospholipase A 2 and acyltransferase activites on rHDL. Mutations of hydrophilic residues in amphipathic helices ␣ 3-4 and ␣ His to an alanine did not affect the mutants' activity on rHDL. Based upon the 3D model built for human LCAT, we designed a new mutant F382A, which had a biochemical phenotype similar to the natural T123I FED mutant. These data suggest that residues T123 and F382, located N-terminal of helices ␣ 3-4 and ␣ His, contribute specifically to the interaction of LCAT with HDL and possibly with its co-factor apoA-I. Residues N131 and N391 seem critical for the optimal orientation of the two amphipathic helices necessary for the recognition of a lipoprotein substrate by the enzyme. -Vanloo, B., F.

Characterization and functional studies of lipoproteins, lipid transfer proteins, and lecithin:cholesterol acyltransferase in CSF of normal individuals and patients with Alzheimer's disease

“…As reference material we used the supernatant of a stable transfected human LCAT BHK cell line, which on average contains Ϯ 1.5 g LCAT/ml (24). The acyltransferase activity of the WT LCAT and mutants was determined by HPLC by measuring the cholesteryl-1-pyrenebutyrate formed by hydrolysis of 1,2-bis-(1-pyrenebutanoyl)-sn -glycero-3-phosphocholine and the transfer of the 1pyrenebutyric acid to the ␤ -hydroxyl of cholesterol by LCAT (27). The assay mixture contained approximately 2 m of 1,2-bis-(1-pyrene-butanoyl)-sn -glycero-3-phosphocholine, 4 m free cholesterol (FC) incorporated in the discoidal complexes, 4 m m 2-mercaptoethanol, and 400 l cell culture medium or CSF.…”

The Effect of Natural LCAT Mutations on the Biogenesis of HDL

“…On the basis of the computer derived 3D structure of LCAT, the enzyme contains seven conserved β-strands connected by loops to four α-helices . One of the helices of LCAT (helix α 4–5), located between residues 153–171, is close to the active site of the enzyme. , It has been suggested that electrostatic interactions between LCAT helices and helix 6 (residues 143–164) of apoA-I may draw the two proteins together on the surface of the lipoprotein particle and drive the activation of LCAT . The L159R mutation located within helix 6 of apoA-I apparently disrupts the apoA-I/LCAT interactions and inhibits the biogenesis of HDL …”

“…10,18,48 On the basis of the proposed 3D model of LCAT, it was proposed that the mutations associated with FLD, including the LCAT[P274S] mutant, are in the vicinity of the catalytic residues of the enzyme and the mutations associated with FED including the LCAT[T147I] mutant are on the hydrophilic surface of the amphipathic α helices of the enzyme that may interact with apoA-I and HDL. 49,50 In the present study, we have investigated the impact of two FLD (LCAT[P274S] and LCAT[C337Y]) and one FED (LCAT[T147I]) mutation on the in vitro and in vivo activities of LCAT and their ability to be secreted by the cells. The in vivo activities were assessed by the characteristics of the HDL formed in LCAT −/− mice that express the endogenous mouse apoA-I as well as their ability to correct the aberrant HDL phenotypes generated following expression of the apoA-I[L159R] FIN mutants in apoA-I −/− mice.…”

“…On the basis of the proposed 3D model of LCAT, it was proposed that the mutations associated with FLD, including the LCAT­[P274S] mutant, are in the vicinity of the catalytic residues of the enzyme and the mutations associated with FED including the LCAT­[T147I] mutant are on the hydrophilic surface of the amphipathic α helices of the enzyme that may interact with apoA-I and HDL. , …”

Πρωτεΐνες Που Ρυθμίζουν Τα Επίπεδα Και Τις Λειτουργίες Της HDL

“…Another study has examined the effect of FED mutations of LCAT which cause loss of α-LCAT activity only, possibly due to defective interactions with apoA-I. Mutations in the residues T123, N131, F382 and N391 of LCAT cause loss of α-LCAT activity and this makes them candidate residues that are involved in the interactions between apoA-I and LCAT (153). Furthermore, according to the 3D-model of the structure of LCAT, described previously, it has been suggested that LCAT mutations causing FLD are near the active site of LCAT while FED mutations are clustered in a different more hydrophilic side that may participate in the activation of LCAT by apoA-I (152;154).…”