Date of Award

5-10-2018

Degree Type

Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

First Advisor

Koschinsky, Marlys

Second Advisor

Boffa, Michael

Rights

CC-BY-NC-ND

Abstract

Elevated plasma levels of lipoprotein(a) (Lp(a)) is considered to be the strongest genetic risk factor for cardiovascular diseases (CVDs). Lp(a) is similar in composition to low density lipoprotein (LDL), but is distinguishable by its unique polymorphic glycoprotein, apolipoprotein(a) (apo(a)). Lp(a) plasma levels are primarily determined by allelic variations in the gene that encodes apo(a). Rate of apo(a) secretion from hepatocytes and the efficiency of Lp(a) assembly influences plasma levels. Lp(a) is assembled by a non-covalent interaction between its two components, that precedes formation of a single disulfide bond between apo(a) and apolipoproteinB-100 (apoB-100) of the LDL moiety. Single nucleotide polymorphisms (SNPs) identified in the apo(a) domains that are implicated in this two-step assembly process have been associated with plasma Lp(a) levels. G17R, P52L, S37F, and T23P, have been associated with decreased Lp(a) levels, whereas R18W has been associated with increased levels. The work in this study uses site-directed mutagenesis to introduce the aforementioned SNPs into a physiologically relevant 17K apo(a) isoform to establish potential causation for the observed associative Lp(a) levels followed by in vitro assays of Lp(a) formation. Pulse-chase experiments in transiently transfected human hepatoma cells (HepG2) with mutants and wildtype (WT) 17K apo(a) was used to study the secretion of apo(a) variants and a recombinant Lp(a) assay with purified components was used to study the assembly of Lp(a). Computational chemistry was used to determine conformational changes introduced by the SNPs in their respective apo(a) domains. Interpretation of the presented data suggests that R18W enhances the rate of covalent Lp(a) assembly and P52L decreases efficiency of covalent Lp(a) assembly. S37F, G17R, and T23P, did not present data that correlated to their effects on human Lp(a) plasma concentration.

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