The major goal of the project is to investigate the role of lecithin-cholesterol acyltransferase (LCAT) in the metabolism of phospholipid species, and its relation to atherogenesis. Studies performed in the present funding period established that, a) the positional specificity of human LCAT is altered in the presence of certain molecular species of phosphatidyl choline (PC), resulting in the increased synthesis of saturated cholesteryl esters (CE) and decreased formation of arachidonoyl CE (20:4 CE), and b) LCAT carries out several novel reactions not involving cholesterol, including the hydrolysis of oxidized PC generated during lipoprotein oxidation. In the next funding period, the physiological importance of the altered positional specificity of LCAT, and of the novel reactions carried out by it will be studied.
In Specific Aim I, evidence will be obtained for the altered positional specificity by identifying the positional isomers of lyso PC formed by 3'P NMR. Evidence for the in vivo alteration of positional specificity will be obtained by feeding rabbits long chain fatty acids known to alter the specificity in vitro, and by studying the specificity of LCAT in the plasma of transgenic mice expressing human LCAT.
In Specific Aim II, the physiological consequences of the altered positional specificity will be investigated by determining the effect of saturated CE on the transfer, and 'selective uptake' of HDL CE, and on the intracellular hydrolysis of CE in arterial cells. The consequence of impaired synthesis of 20:4 CE on the delivery of arachidonate to the cells, and on the synthesis of prostaglandins will be investigated.
In Specific Aim III, the physiological importance of the LCAT reactions not involving cholesterol will be investigated. These reactions include the hydrolysis of oxidized PC, the formation of antioxidant derivatives of steroid hormones and PAF, and the detoxification of oxysterols.
In Specific Aim I V, the structural domains of LCAT protein responsible for determining the positional specificity, acyl donor and acceptor specificity, and oxidative susceptibility will be investigated by site-directed mutagenesis. The hypothesis that some naturally occurring mutations of LCAT affect only the cholesterol esterification, and not its ancillary functions, will be tested. These studies should provide novel insights into the physiological importance of LCAT beyond its role in cholesterol esterification.
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