Generation of a Recombinant Apolipoprotein E Variant with Improved Biological Functions

Abstract

To identify the residues in the carboxyl-terminal region 260 -299 of human apolipoprotein E (apoE) that contribute to hypertriglyceridemia, two sets of conserved, hydrophobic amino acids between residues 261 and 283 were mutated to alanines, and recombinant adenoviruses expressing these apoE mutants were generated. Adenovirus-mediated gene transfer of apoE4-mut1 (apoE4 (L261A, W264A, F265A, L268A, V269A)) in apoE-deficient mice (apoE ؊/؊ ) corrected plasma cholesterol levels and did not cause hypertriglyceridemia. In contrast, gene transfer of apoE4-mut2 (apoE4 (W276A, L279A, V280A, V283A)) did not correct hypercholesterolemia and induced mild hypertriglyceridemia. ApoE-induced hyperlipidemia was corrected by co-infection with a recombinant adenovirus expressing human lipoprotein lipase. Both apoE4 mutants caused only a small increase in hepatic very low density lipoprotein-triglyceride secretion. Density gradient ultracentrifugation analysis of plasma and electron microscopy showed that wild-type apoE4 and apoE4-mut2 displaced apoA-I from the high density lipoprotein (HDL) region and promoted the formation of discoidal HDL, whereas the apoE4-mut1 did not displace apoA-I from HDL and promoted the formation of spherical HDL. The findings indicate that residues Leu-261, Trp-264, Phe-265, Leu-268, and Val-269 of apoE are responsible for hypertriglyceridemia and also interfere with the formation of HDL. Substitutions of these residues by alanine provide a recombinant apoE form with improved biological functions. ApoE1 is a polymorphic protein in humans (1). In vitro and in vivo studies have shown that apoE mutants that prevent binding of apoE-containing lipoproteins to the LDL receptor are associated with high plasma cholesterol levels and cause premature atherosclerosis in humans and experimental animals (2-4). ApoE promotes cholesterol efflux (5, 6) and thus may contribute to cell and tissue cholesterol homeostasis and protection from atherosclerosis (7,8). ApoE is also a risk factor for Alzheimer's disease (9, 10) and may contribute to lipid homeostasis in the brain (11).A series of recent studies used adenoviruses expressing fulllength and truncated genomic apoE sequences to correct the high cholesterol profile of the apoE-deficient (apoE Ϫ/Ϫ ) mice. It was shown that overexpression of full-length apoE (by infection of mice with 1-2 ϫ 10 9 pfu) did not correct the high cholesterol levels of the apoE Ϫ/Ϫ mice, in contrast, it increased VLDL triglyceride secretion and induced hypertriglyceridemia (12-16). Overexpression of apoE3 or apoE4 also aggravated the hypercholesterolemia in apoE2 knock-in mice (17). However the high cholesterol profile of apoE Ϫ/Ϫ mice or the apoE2 knock-in mice was corrected by infection with truncated apoE forms lacking different segments of the carboxyl-terminal domain (12-17). The hypertriglyceridemia induced by full-length apoE was independent of the apoE phenotype and mouse strain and could be corrected by overexpression of lipoprotein lipase (15). In normal C57BL6 mice overexpression...

A Dominant Negative Form of the Transcription Factor c-Jun Affects Genes That Have Opposing Effects on Lipid Homeostasis in Mice

“…In humans and experimental animals plasma apoE levels correlate with plasma triglyceride levels (57,58). On the basis of this information we hypothesized that expression of dn-c-Jun in apoE Ϫ/Ϫ mice would not affect the plasma lipid levels.…”

Apoprotein E as a lipid transport and signaling protein in the blood, liver, and artery wall

“…High expression levels of hepatic apoE result in a notable increase in VLDL triglyceride secretion (11,12). The C-terminal domain is required for the promotion of VLDL triglyceride secretion, particularly the hydrophobic amino acids between residues 260 and 270 (13). The multiple roles of apoE in VLDL homeostasis are concentration dependent with low levels of apoE sufficient to promote receptor-mediated lipoprotein clearance and higher concentrations required to induce hypertriglyceridemia.…”

Lipid homeostasis and apolipoprotein E in the development and progression of Alzheimer's disease

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