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dc.contributor.authorTheuerle, James Douglas
dc.contributor.otherQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))en
dc.date2009-09-26 02:15:50.754en
dc.date.accessioned2009-09-27T19:06:36Z
dc.date.available2009-09-27T19:06:36Z
dc.date.issued2009-09-27T19:06:36Z
dc.identifier.urihttp://hdl.handle.net/1974/5233
dc.descriptionThesis (Master, Biochemistry) -- Queen's University, 2009-09-26 02:15:50.754en
dc.description.abstractElevated plasma concentrations of lipoprotein(a) [Lp(a)] have been identified as an independent risk factor for vascular diseases including coronary heart disease and stroke. In the current study, we have examined the binding and degradation of recombinant forms of apolipoprotein(a) [r-apo(a)], the unique kringle-containing moiety of Lp(a), using a cultured cell model. We found that the incubation of human hepatoma (HepG2) cells with an iodinated 17 kringle-containing (17K) recombinant form of apo(a) resulted in a two-component binding system characterized by a high affinity (Kd = 12 nM), low capacity binding site, and a low affinity (Kd = 249 nM), high capacity binding site. We subsequently determined that the high affinity binding site on HepG2 cells corresponds to the LDL receptor. In the HepG2 cell model, association of apo(a) with the LDL receptor was shown to be dependent on the formation of Lp(a) particles from endogenous LDL. Using an apo(a) mutant incapable of binding to the high affinity site through its inability to form Lp(a) particles (17KΔLBS7,8), we further demonstrated that the LDL receptor does not participate in Lp(a) catabolism. The low affinity binding component observed on HepG2 cells, familial hypercholesterolemia (FH) fibroblasts and human embryonic kidney (HEK) 293 cells may correspond to a member(s) of the plasminogen receptor family, as binding to this site(s) was decreased by the addition of the lysine analogue epsilon-aminocaproic acid. The lysine-dependent nature of the low affinity binding site was further confirmed in HepG2 binding studies utilizing r-apo(a) species with impaired lysine binding ability. We observed a reduction maximum binding capacity for 17K r-apo(a) variants lacking the strong lysine binding site (LBS) in kringle IV type 10 (17KΔAsp) and the very weak LBS in kringle V (17KΔV). Degradation of Lp(a)/apo(a) was found to be mediated exclusively by the low affinity component on both HepG2 cells and FH fibroblasts. Fluorescence confocal microscopy, using the 17K r-apo(a) variant fused to green fluorescent protein, further confirmed that degradation by the low affinity component on HepG2 cells does not proceed by the activity of cellular lysosomes. Taken together, these data suggest a potentially significant route for Lp(a)/apo(a) clearance in vivo.en
dc.format.extent2193810 bytes
dc.format.mimetypeapplication/pdf
dc.languageenen
dc.language.isoenen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectBiochemistryen
dc.subjectLipoprotein(a)en
dc.subjectApolipoprotein(a)en
dc.subjectCatabolismen
dc.subjectBindingen
dc.subjectLow-Density Lipoprotein Receptoren
dc.subjectLow-Density Lipoproteinen
dc.subjectPlasminogenen
dc.subjectPlasminogen receptoren
dc.titleAnalysis of Lipoprotein(a) Catabolismen
dc.typethesisen
dc.description.degreeMasteren
dc.contributor.supervisorKoschinsky, Marlysen
dc.contributor.departmentBiochemistryen


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