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

In order to test the hypothesis that fish-eye disease (FED) is due to a def icient 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 activ ity was measured on a monomeric phospholipid enelogue, phospholipase A(2) a ctivity was measured on reconstituted high density lipoprotein (HDL), and a cyltransferase activity was measured both on rHDL and on low density lipopr otein (LDL). The natural FED mutants have decreased phospholipase Az activi ty on rHDL, which accounts for the decreased acyltransferase activity previ ously reported. All mutants engineered at positions 131 and 391 had decreas ed esterase activity on a monomeric substrate and decreased acyltransferase activity on LDL, In contrast, mutations at position 123 preserved these ac tivities and specifically decreased phospholipase A(2) and acyltransferase activites on rHDL, Mutations of hydrophilic residues in amphipathic helices ex 3-4 and a His to an alanine did not affect the mutants' activity on rHD L. Based upon the 3D model built for human LCAT, we designed a new mutant F 382A, which had a biochemical phenotype similar to the natural T123I FED mu tant. These data suggest that residues T123 and F382, located N-terminal of helices at 3-4 and a His, contribute specifically to the interaction of LC AT 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 ne cessary for the recognition of a lipoprotein substrate by the enzyme. C. La beur,J. Vandekerckhove,J. Tavernier, and M. Rosseneu. Relationship between structure and biochemical phenotype of lecithin:cholesterol acyltransferase (LCAT) mutants causing fish-eye disease.