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 its unique apoprotein, apo(a). this objective will be approached through several specific aims:
The first aim i s to reconstitute solubilized apo(a) and apoB separately and together into phospholipid/neutral lipid micro-emulsions and to assess the effect of each apoprotein on the secondary structure, tertiary structure, and cell receptor binding of the other using circular dichroism, intrinsic fluorescence, and cell culture techniques. The second and third aims will be to determine how the carbohydrate present on the proteins in Lp(a) affects the molecular weight and secondary structure of these proteins and the motions 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) (40%). 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 paramagnetic resonance and 13C nuclear magnetic resonance. The fourth and fifth aims will be to determine directly a significant portion of the amino acid sequence of apo(a) in order a) to establish the sequence position of N- or O-glycosylation sites, b) to establish the sequence position of half-cystines disulfide-linked to corresponding residues in apoB, and c) to confirm the alignment of its cDNA sequence and check its accuracy in selected regions. The sixth aim is to clone human apo(a) cDNA using the expression vector lambda gtll and specific poly- and mono-clonal antibodies , and to determine the complete nucleic acid sequence of human apo(a) cDNA, and thereby deduce the amino acid sequence of the apoprotein. Attainment of these aims should provide a fund of knowledge useful for discovering the properties of Lp(a) that are responsible for its apparent atherogenicity.
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