The specific aims of the application are 1) to synthesize peptides of apolipoprotein E making amino acid substitutions into the putative receptor binding region, 2) to identify additional lipid binding region of apoE, and 3) to model the apoE receptor binding region using a model lipid associating peptide previously shown to display many of the properties of the apolipoproteins. The peptides of interest will be synthesized by solid phase methodology. They will be purified by ion exchange and reversed phase high performance liquid chromatography. The purity of the peptides will be determined by isoelectric focusing and analytical reversed phase HPLC. The purified peptides will be tested for lipid binding using fluorescence (where appropriate) and C.D. spectroscopy and by density gradient ultracentrifugation. The peptides will be mixed with cholesterol-rich phospholipid dispersions and tested for hepatocyte receptor binding in competition with apoE containing dispersions. They will be added to thrombin treated hypertriglyceridemic VLDL and/or intralipid and evaluated for binding and uptake by human skin fibroblasts. Elucidating the relationship between receptor binding, cellular uptake and lipid binding will further our understanding of how cholesterol is taken up and metabolized by the liver. A knowledge of the protein determinants for receptor binding could clarify the difference in hepatic uptake of various lipoprotein remnants in disease states and could probably lead to effective therapy for Type III hyperlipoproteinemia where a defect has been demonstrated in apolipoprotein E receptor binding due to a substitution of cystiene for argine in the amino acid sequence.
