The major goal of the project is to establish that in addition to its pivotal role in cholesterol metabolism, lecithin-cholesterol acyltransferase (LCAT) has important functions in the pathophysiology of phosphatidyl choline (PC) species in plasma. Based on our preliminary results, we propose that LCAT, whose primary substrate is not cholesterol, but PC, has several non-cholesterol-related functions that include the metabolism of bioregulatory phospholipids such as oxidized PCs and platelet-activating factor. We further hypothesize that LCAT influences the risk of atherosclerosis (ATH) by altering its positional specificity in the presence of certain PC species, forming atherogenic saturated cholesteryl esters (CE).
In Specific Aim I, we will test the hypothesis that the oxidation of plasma results in a loss of the cholesterol-esterifying activity of LCAT, but not its capacity to hydrolyze and transesterify oxidized PC species. We will identify the products of PC oxidation and investigate their further metabolism by LCAT. Experiments in Specific Aim II will test the postulate that human LCAT, which normally transfers the sn-2 acyl group of PC, instead transfers the sn-1 acyl group, when the sn-2 position is occupied by certain long chain polyunsaturated fatty acids (PUFA) such as arachidonic acid. We will study the effect of structural changes in the sn-1 and sn-2 acyl groups of PC, as well as the nature of the acyl acceptor, on LCAT's positional specificity. Next (Specific Aim III) we will identify the specific domains and amino acid sequences in LCAT protein which determine its substrate and positional specificities, employing recombinant human/mouse chimeric LCATs, and site-directed mutations of human LCAT. We postulate (Specific Aim IV) that there are two classes of LCAT in nature with differing specificities: one, present in ATH-resistant animals, efficiently transfers long-chain PUFA from the sn-2 position of PC; and the other, from ATH-susceptible species, alters its positional specificity when certain PUFA (ex: 20:4, 22:6) are present at the sn-2 position of PC, and thereby produces saturated CE. To test this hypothesis, we will compare the substrate and positional specificities of LCATs from several ATH-susceptible and ATH-resistant animals, as well as study the plasma CE composition, and ATH-susceptibility of transgenic mice over-expressing human LCAT. These studies should not only provide evidence for the novel functions of LCAT, but also for its possible physiological significance in modulating the atherogenic risk.
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