Apolipoprotein (apo) E is a major cholesterol transport protein in the plasma and brain of humans, with the APOE ε4 allele (coding for R112) associated with a higher risk for cardiovascular and Alzheimer's diseases (CVD and AD, respectively) compared to APOE ε3 (coding for C112). The molecular basis underlying the link between APOE ε4 and CVD/AD is poorly understood. Here apoE from Cavia porcellus (guinea pig, GP), which is 72% identical to human apoE4 but lacking residues 193-197 and 246-252, a feature noted in all hystricomorph apoE, was used as a model to understand the role of apoE4. Western blot with anti-human apoE antibody revealed cross reactivity with bacterially expressed recombinant GP apoE. GP apoE solubilized phospholipids far more efficiently than apoE3/E4 but promoted macrophage cholesterol efflux to a similar extent. The overall secondary structure and tetrameric organization of GP apoE were broadly similar to those of apoE3/E4. Guanidine HCl-induced denaturation revealed a biphasic unfolding pattern indicative of a two-domain architecture for GP apoE. Hydrogen-deuterium exchange coupled to mass spectrometry of GP apoE revealed mixed EX1/EX2 kinetics similar to that noted for apoE4, with peak broadening indicative of the presence of partially folded intermediate states. Limited proteolysis reveals more resistance to cleavage compared to apoE3/E4. Taken together, the findings suggest that the CT domain modulates the lipid-binding ability of apoE and attenuates the overall dynamics of the protein, which bears direct relevance in regulation of lipoprotein metabolism with implications in amyloid-related neurodegeneration.
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Jul 3, 2025
G L Iverson + 6
Just Published
Open Access