Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that is responsible for the maturation of high density lipoproteins (HDL) and is the source of most plasma cholesterol esters. Familial LCAT deficiency (FLD) and fish eye disease (FED) are two inherited genetic diseases which result in abnormal LCAT function. In both diseases, LCAT no longer modifies HDL. Symptoms of FLD include nephropathy, renal insufficiency, proteinuria, hematuria, anemia and cloudy corneas. Cholesterol metabolism in the plasma is significantly altered resulting in increased cholesterol to cholesterol ester ratios. The most significant symptom of FED is severe corneal clouding. However, cholesterol to cholesterol ester ratios in the plasma are near normal, suggesting that LCAT is acting on another substrate such as low density lipoproteins (LDL). The proposed research will examine the hypothesis that LCAT mutations in FED alter LCAT substrate specificity; whereas mutations resulting in FLD decrease or abolish LCAT substrate binding. The overall goal of this proposal is to characterize the HDL and LDL substrate specificity of LCAT mutations associated with FED and FLD.
The specific aims are to create and express recombinant LCAT 'with recreated FED and FLD associated mutations and to measure binding affinity and capacity of these mutants to vesicles and recombinant HDL. The mutations will be created using mutagenic oligonucleotides to introduce site specific mutations into the LCAT cDNA. The altered cDNA will be transferred to a mammalian expression vector and used to create Chinese hamster ovary cell lines that stably express the mutant LCAT protein. Using this expression system, the LCAT can be metabolically labeled with 35 S-methionine and purified to homogeneity. The purified enzyme is mixed with phospholipid vesicles or recombinant HDL. The bound and free enzyme is separated on a pH7.5 native polyacrylamide gel. Quantitation of the radioactivity in each band allows the calculation of LCAT binding affinities and capacities for the substrate. The significance of this work is in its potential to increase understanding of the molecular defects which cause FED and FLD and to identity residues in LCAT that are important in substrate interfacial binding.
