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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL052597-04
Application #
2750424
Study Section
Pathology A Study Section (PTHA)
Project Start
1995-08-01
Project End
1999-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Rush University Medical Center
Department
Type
DUNS #
City
Chicago
State
IL
Country
United States
Zip Code
60612
Subbaiah, Papasani V; Sowa, Jennifer M; Singh, Dev K (2008) Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity. Arch Biochem Biophys 474:32-8
Huang, Fei; Subbaiah, Papasani V; Holian, Oksana et al. (2005) Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk. Am J Physiol Lung Cell Mol Physiol 289:L176-85
Subbaiah, Papasani V; Sowa, Jennifer M; Davidson, Michael H (2004) Evidence for altered positional specificity of LCAT in vivo: studies with docosahexaenoic acid feeding in humans. J Lipid Res 45:2245-51
Sargis, Robert M; Subbaiah, Papasani V (2003) Trans unsaturated fatty acids are less oxidizable than cis unsaturated fatty acids and protect endogenous lipids from oxidation in lipoproteins and lipid bilayers. Biochemistry 42:11533-43
Liu, Lijuan; Bortnick, Anna E; Nickel, Margaret et al. (2003) Effects of apolipoprotein A-I on ATP-binding cassette transporter A1-mediated efflux of macrophage phospholipid and cholesterol: formation of nascent high density lipoprotein particles. J Biol Chem 278:42976-84
Lum, Hazel; Qiao, Jing; Walter, Robert J et al. (2003) Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells. Am J Physiol Heart Circ Physiol 285:H1786-9
Francone, Omar L; Subbaiah, Papasani V; van Tol, Arie et al. (2003) Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1. Biochemistry 42:8569-78
Subbaiah, Papasani V; Billington, Stephen J; Jost, B Helen et al. (2003) Sphingomyelinase D, a novel probe for cellular sphingomyelin: effects on cholesterol homeostasis in human skin fibroblasts. J Lipid Res 44:1574-80
Wang, Kewei; Subbaiah, Papasani V (2002) Role of the interfacial binding domain in the oxidative susceptibility of lecithin:cholesterol acyltransferase. Biochem J 365:649-57
Gesquiere, Laurence; Cho, Wonhwa; Subbaiah, Papasani V (2002) Role of group IIa and group V secretory phospholipases A(2) in the metabolism of lipoproteins. Substrate specificities of the enzymes and the regulation of their activities by sphingomyelin. Biochemistry 41:4911-20

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