PubMed1995
Classic LCAT deficiency (CLD) and fish eye disease (FED) are two clinically distinct syndromes, associated with defects in the lecithin-cholesterol acyltransferase (LCAT) gene resulting in total (CLD) or partial (FED) enzyme deficiency. In order to investigate the underlying molecular mechanisms that lead to different phenotypic expression in CLD and FED, LCAT mutants associated with either CLD (LCAT147, LCAT156, and LCAT228) or FED (LCAT10, LCAT123, LCAT158, LCAT293, LCAT300, and LCAT347) were expressed in vitro in human embryonic kidney 293 cells and characterized with respect to LCAT expression and enzyme activity. Evaluation of mutant LCAT gene transcription by Northern blot analysis demonstrated LCAT mRNA of normal size and concentration. Although all constructs gave rise to similar intracellular LCAT mass, the amount of enzyme present in the media for LCAT147, LCAT156, and LCAT300 was reduced to less than 10% of normal, suggesting that these mutations disrupted LCAT secretion. Western blot analysis of cell culture media containing wild type or mutant LCAT demonstrated the presence of a single normal-sized band of 67 kDa. The ability of the different enzymes to esterify free cholesterol in high density lipoprotein-like proteoliposomes (alpha-LCAT-specific activity) was reduced to less than 5% of normal for CLD mutants LCAT147 and LCAT228 and FED mutants LCAT10, LCAT123, LCAT293, and LCAT347, whereas that of LCAT156, LCAT158, and LCAT300 ranged from 45 to 110% of control. Although most FED mutant LCAT enzymes retained the ability to esterify free cholesterol present in alpha- and beta-lipoproteins of heat-inactivated plasma, esterification was undetectable in all CLD mutants (LCAT147, LCAT156, and LCAT228). In contrast, all mutant enzymes retained the ability to hydrolyze the water soluble, short-chained fatty acid substrate p-nitrophenolbutyrate. In summary, our studies establish the functional significance of nine LCAT gene defects associated with either FED or CLD. Characterization of the expressed LCAT mutants identified multiple, overlapping functional abnormalities that include defects in secretion and/or disruption of enzymic activity. All nine LCAT mutants retained the ability to hydrolyze the water-soluble PNPB substrate, indicating intact hydrolytic function. Based on these studies we propose that mutations in LCAT residues 147, 156, 228 (CLD) and 10, 123, 158, 293, 300, and 347 (FED) do not disrupt the functional domain mediating LCAT phospholipase activity, but alter structural domains involved in lipid binding or transesterification.