LCAT can Rescue the Abnormal Phenotype Produced by the Natural ApoA-I Mutations (Leu141Arg)_Pisa and (Leu159Arg)_FIN

Abstract

To explain the etiology and find a mode of therapy of genetically determined low levels of high-density lipoprotein (HDL), we have generated recombinant adenoviruses expressing apolipoprotein A-I (apoA-I)(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN and studied their properties in vitro and in vivo. Both mutants were secreted efficiently from cells but had diminished capacity to activate lecithin/cholesterol acyltransferase (LCAT) in vitro. Adenovirus-mediated gene transfer of either of the two mutants in apoA-I-deficient (apoA-I-/-) mice resulted in greatly decreased total plasma cholesterol, apoA-I, and HDL cholesterol levels. The treatment also decreased the cholesteryl ester to total cholesterol ratio (CE/TC), caused accumulation of prebeta1-HDL and small size alpha4-HDL particles, and generated only few spherical HDL particles, as compared to mice expressing wild-type (WT) apoA-I. Simultaneous treatment of the mice with adenoviruses expressing either of the two mutants and human LCAT normalized the plasma apoA-I, HDL cholesterol levels, and the CE/TC ratio, restored normal prebeta- and alpha-HDL subpopulations, and generated spherical HDL. The study establishes that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN inhibit an early step in the biogenesis of HDL due to inefficient esterification of the cholesterol of the prebeta1-HDL particles by the endogenous LCAT. Both defects can be corrected by treatment with LCAT.

In vivo tissue cholesterol efflux is reduced in carriers of a mutation in APOA1

“…Interestingly, plasma cholesterol esterifi cation fl uxes did not differ between cases and controls, indicating cholesterol esterifi cation independent of plasma apoA-I levels. In line , LCAT gene therapy has been demonstrated to correct low HDL-c levels in mice with mutations in APOA1 ( 38 ). Previous studies in humans have also suggested that plasma CE clearance is mostly mediated by apoB100-containing particles following CETP-mediated transfer, rather than by direct HDL-dependent CE removal ( 13,39,40 ).…”

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

“…The apoA-I gene lacking the BglII restriction site, which is present at nucleotide position 181 of the genomic sequence relative to the ATG codon of the gene, was cloned into the pCD-NA3.1 vector to generate the pCDNA3.1-apoA-I( ⌬ BglII) plasmid as described ( 11 ). This plasmid was used as a template to introduce the apoA-I mutations F225A/V227A/F229A/L230A using the QuickChange® XL mutagenesis kit (Stratagene, Santa Clara, CA) and the mutagenic primers shown in supplementary Table I.…”

“…Recombinant adenoviruses expressing the WT and the mutant apoA-I were constructed using the Ad-Easy-1 system in which the recombinant adenovirus construct is generated in bacteria BJ-5183 (purchased from Stratagene) ( 12 ). The recombinant adenovirus was packaged in 911 cells, amplifi ed in human embryonic kidney 293 (HEK 293) cells, purifi ed, and titrated as described ( 11 ).…”

“…These phenotypes were characterized by low HDL levels, preponderance of immature HDL subpopulations, or accumulation of discoidal HDL particles in plasma ( 1,10,11,15,18,19 ).…”

“…Potential abnormalities in the HDL phenotype in apoA-I Ϫ / Ϫ mice resulting from the expression of the apoA-I[225-230] mutant were verifi ed by two-dimensional gel electrophoresis of plasma that showed the formation of pre-␤ -and ␣ 4-HDL particles. Accumulation of such particles is indicative of defective maturation of HDL due to insuffi ciency of mouse LCAT ( 11,18 ). The LCAT insufficiency may originate from fast catabolism of the nascent HDL particles along with the endogenous LCAT bound to them ( 11 ).…”