Abstract
Cardiovascular disease (CVD) is a major health problem and leading cause of death in America. High density lipoprotein (HDL) and its major protein, apolipoprotein A-I (apoA-I), are both correlated with lowered risk for heart disease. HDL removes cholesterol from peripheral cells in arterial walls and transports it to the liver for excretion. Despite its anti-atherogenic properties, apoA-I deposits as deleterious amyloid fibrils in various tissues and organs, including the heart, a process that is driven by mutation or influence of environmental factors during physiological processes. We and others have shown that wild-type (WT) apoA-I undergoes non-specific aggregation at acidic pH. Townsend and coworkers further demonstrated that heparin could stimulate the formation of amyloid in WT apoA-I at acidic pH, likely through an electrostatic interaction. Although the effect of heparin on acid-induced aggregation of full-length WT apoA-I has been examined, the effect of heparin on acid-induced aggregation of amyloidogenic variants has not been explored. The goal of this research is to examine potential synergistic effects between acidic pH, heparin, and mutations on amyloid formation in WT apoA-I and its two amyloidogenic mutants, G26R and L178H. Highly pure lipid-free apoA-I proteins were combined with Mcllvaine buffer in the presence and absence of heparin to a final protein and heparin concentration of 0.1 mg/mL at the desired pH. Samples were then incubated at 37°C with constant agitation up to 72 hours. Aliquots of 2 mL were removed at 1, 24, 48 and 72 hours for analysis. Protein conformational changes were examined using 8-Anilino-1-naphthalenesulfonic acid (ANS) and the intrinsic fluorescence of tryptophan residues. Aggregation was detected using turbidity measurements at 340 nm and amyloid formation was examined using a Thioflavin T (ThT) fluorescence assay. Solubility analysis of the protein was carried out using the Bradford assay and SDS-PAGE. Our findings reveal that both mutants formed mainly amorphous aggregates at mildly acidic pH, likely due to local instability and exposure of non-polar residues due to the mutation. All three proteins aggregated extensively without significant amyloid formation at low pH. Amyloid formation was observed in G26R at mildly acidic pH, demonstrating a greater susceptibility to amyloid formation than L178H. Heparin at acidic pH greatly stimulated amyloid formation in all three proteins, likely by providing an alternate aggregation pathway through electrostatic binding to positively charged clusters. Collectively, these results demonstrate a synergistic effect between acidic pH, heparin and mutation in promoting amyloid formation in apoA-I.