Characterization of ligand binding to the low density lipoprotein receptor-related protein (LRP)

Work presented in here focuses on the characterization of three soluble LDL-like domains (sLRP2, 3 and 4) found within the low density lipoprotein receptor-related protein (LRP1). Studies focus on the expression, purification and characterization of these domains. Each purified domain was tested for functionality and was used to study the binding of a number of different ligands. Specifically, the examination of the apolipoprotein E/LRP1 interaction was studied and characterized.; In Chapter II, the construction, expression, purification and characterization of each sLRP was performed. Pichia pastoris was used to express large quantities of each sLRP. The sLRPs were purified in 1–10 mg/L quantities and characterized. Each was found to be >95% pure by N-terminal sequencing and was found to correspond with their respective expected molecular weight. Finally each sLRP and was found to react with a polyclonal antibody raised to native LRP1.; In Chapter III, each sLRP was tested for binding to a number of ligands. Once functionality was established, binding studies of each sLRP and two thrombin-SEPRIN complexes, receptor-associated protein (RAP), lactoferrin, α ₂-macroglobulin and apolipoprotein E-enriched β-VLDL particles was studied. Binding studies included competition with EDTA and RAP.; In Chapter IV, an examination of the apolipoprotein E/LRP1 interaction was studied using two bioactive apolipoprotein E mimetic peptides. Each sLRP was shown to bind to each peptide with similar characteristics as full length apolipoprotein E. Using these peptides, binding kinetics were calculated using surface plasmon resonance. Two mutant apolipoprotein E peptides, apoE(130–149, Arg142Glu) and apoE(130–149, Lys143Glu) were also studied and binding kinetics were obtained.; In Chapter V, each apolipoprotein E mimetic peptide was structurally characterized using analytical ultracentrifugation, circular dichroism spectroscopy and nuclear magnetic resonance. Each peptide existed in an α-helical conformation and this helical conformation was found to be essential for sLRP binding. Interestingly, this helical conformation was found to exist in the absence of lipid, something not previously reported. Finally, each sLRP was further truncated to produce clusters of three complement-type domains and were tested for binding to each peptide.; In Chapter VI, an outlook to future studies is presented.