Point mutations in apolipoprotein A-I mimic the phenotype observed in patients with classical lecithin: Cholesterol acyltransferase deficiency

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks flanking helices 4 and 6 of apoA-I and of the deletion of helix 6 on the in vivo activity of LCAT and the biogenesis of HDL. The LCAT activation capacity of apoA-I in vitro was nearly abolished by the helix 6 point (helix 6P-apoA-I[R160V/HI62A]) and deletion {helix 6 Delta-apoA-I[Delta(l44-165)]} mutants, but was reduced to 50% in the helix 4 point mutant (helix 4P-apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-1 deficient mice, the level of plasma HDL cholesterol was greatly reduced in helix 6P and helix 6 Delta mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 6P mutant formed predominantly high levels of apoA-1 containing discoidal particles and had an increased pre beta 1-HDL/alpha-HDL ratio. The helix 6 Delta mutant formed few spherical particles and had an increased prep beta 1-HDL/alpha-HDL ratio. Mice infected with adenovirus expressing the helix 4P mutant or wild-type apoA-1 had normal HDL cholesterol and formed spherical alpha-HDL particles. Coinfection of mice with adenoviruses expressing human LCAT and the helix 6P mutant dramatically increased plasma HDL and apoA-1 levels and converted the discoidal into spherical HDL, indicating that the LCAT activity was rate-limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL cholesterol and apoA-1 levels and in a-HDL particle numbers in the helix 6 Delta mutant. The findings indicate a critical contribution of residue 160 of apoA-1 to the in vivo activity of LCAT and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects carrying this mutation.