IN-VITRO EXPRESSION OF STRUCTURAL DEFECTS IN THE LECITHIN-CHOLESTEROLACYLTRANSFERASE GENE

Classic LCAT deficiency (CLD) and fish eye disease (FED) are two clini cally distinct syndromes, associated with defects in the lecithin-chol esterol acyltransferase (LCAT) gene resulting in total (CLD) or partia l (FED) enzyme deficiency. In order to investigate the underlying mole cular mechanisms that lead to different phenotypic expression in CLD a nd FED, LCAT mutants associated with either CLD (LCAT(147), LCAT(156), and LCAT(228)) or FED (LCAT(10), LCAT(123), LCAT(158), LCAT(293), LCA T(300), and LCAT(347)) were expressed in vitro in human embryonic kidn ey 293 cells and characterized with respect to LCAT expression and enz yme 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 LCAT(147), LCAT(156),a nd LCAT(300) was reduced to less than 10% of normal, suggesting that t hese mutations disrupted LCAT secretion. Western blot analysis of cell culture media containing wild type or mutant LCAT demonstrated the pr esence of a single normal-sized band of 67 kDa. The ability of the dif ferent enzymes to esterify free cholesterol in high density lipoprotei n-like proteoliposomes (alpha-LCAT-specific activity) was reduced to l ess than 5% of normal for CLD mutants LCAT(147) and LCAT(228) and FED mutants LCAT(10) LCAT(123), LCAT(293), and LCAT(347), whereas that of LCAT(156), LCAT(158), and LCAT(300) ranged from 45 to 110% LCAT of con trol. Although most FED mutant LCAT enzymes retained the ability to es terify free cholesterol present in alpha- and beta-lipoproteins of hea t-inactivated plasma, esterification was undetectable in all CLD mutan ts (LCAT(147), LCAT(156), and LCAT(228)). In contrast, all mutant enzy mes retained the ability to hydrolyze the water soluble, short-chained fatty acid substrate p-nitrophenol-butyrate, In summary, our studies establish the functional significance of nine LCAT gene defects associ ated with either FED or CLD. Characterization of the expressed LCAT mu tants identified multiple, overlapping functional abnormalities that i nclude 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 functio nal domain mediating LCAT phospholipase activity, but alter structural domains involved in lipid binding or transesterification.