Lp[a] is a lipoprotein whose plasma concentration has been shown to have a strong correlation with coronary artery disease. The long term objective of the research proposed in this application is to elucidate several specific structural and functional properties of Lp[a] and to gain a clearer understanding of how its three major components (protein, carbohydrate, and lipid) interact. This objective will be approached through four specific aims.
The first aim i s to isolate apo[a] and apoB from Lp[a], and characterize these glycoproteins with respect to their individual chemical and physical properties. Their amino acid and carbohydrate composition wil be determined. Their secondary structure in the presence and absence of lipid will be studied by circular dichroism. The possibility of common immunological determinants on both apoproteins will be explored using a panel of monoclonal antibodies. Each apoprotein will be fragmented by chemical and enzymatic methods and the resulting fragments used to characterize these antibodies.
The second aim will be to reconstitute apo[a] and apoB separately and together into phospholipid/neutralipid pseudomicelles and to assess the effect of each apoprotein on the secondary structure, tertiary structure, and cell receptor bincing of the other using circular dichroic, intrinsic fluorescence, and cell culture techniques.
The third aim will be to determine how the carbohydrate present on the protein in Lp[a] affects the secondary structure of those proteins and the motion of lipids within the lipoprotein. Enzymatic and chemical reagents will be used to partially and completely remove the large amount of carbohydrate present on Lp[a] (20%) and apo[a] (8.5%). The effects of deglycosylation on polypeptide structure will be evaluated by circular dichroism, and on fatty acyl chain motions in the surface phospholipids and the core neutral lipids by spin-label electron paramagnetic resonance and 13C nuclear magnetic resonance.
The fourth aim will be focued on elucidating the number of disulfide bonds that link apo[a] to apoB within the Lp[a] particle. This will involve the isolation of unlinked apo[a] and apoB by immunoaffinity chromatography and quantitation of SH and SS groups by chemical and radiochemical techniques. Attainment of these aims should provide a fund of knowledge useful for discovering the properties of Lp[a] that are responsible for its atherogenicity.
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