1 |
|
Generation of a recombinant apolipoprotein E variant with improved biological functions: Hydrophobic residues (LEU-261, TRP-264, PHE-265, LEU-268, VAL-269) of apoE can account for the apoE-induced hypertriglyceridemia
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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Chemicals / CAS: alanine, 56-41-7, 6898-94-8; amino acid, 65072-01-7; leucine, 61-90-5, 7005-03-0; lipoprotein lipase, 83137-80-8, 9004-02-8; phenylalanine, 3617-44-5, 63-91-2; tryptophan, 6912-86-3, 73-22-3; valine, 7004-03-7, 72-18-4; Apolipoproteins E; Cholesterol, 57-88-5; Lipoproteins, HDL; Lipoproteins, VLDL; Peptide Fragments; Recombinant Proteins; Triglycerides; very low density lipoprotein triglyceride
|
[PDF]
[Abstract]
|
2 |
|
ApoC-III deficiency prevents hyperlipidemia induced by apoE overexpression
Adenovirus-mediated overexpression of human apolipoprotein E (apoE) induces hyperlipidemia by stimulating the VLDL-triglyceride (TG) production rate and inhibiting the LPL-mediated VLDL-TG hydrolysis rate. Because apoC-III is a strong inhibitor of TG hydrolysis, we questioned whether Apoc3 deficiency might prevent the hyperlipidemia induced by apoE overexpression in vivo. Injection of 2 × 109 plaque-forming units of AdAPOE4 caused severe combined hyperlipidemia in Apoe-/- mice [TG from 0.7 ± 0.2 to 57.2 ± 6.7 mM; total cholesterol (TC) from 17.4 ± 3.7 to 29.0 ± 4.1 mM] that was confined to VLDL/intermediate density lipoprotein-sized lipoproteins. In contrast, Apoc3 deficiency resulted in a gene dose-dependent reduction of the apoE4-associated hyperlipidemia (TG from 57.2 ± 6.7 mM to 21.2 ± 18.5 and 1.5 ± 1.4 mM; TC from 29.0 ± 4.1 to 16.4 ± 9.8 and 2.3 ± 1.8 mM in Apoe-/-, Apoe -/-.Apoc3+/-, and Apoe-/-.Apoc3-/- mice, respectively). In both Apoe-/- mice and Apoe -/-.Apoc3-/- mice, injection of increasing doses of AdAPOE4 resulted in up to a 10-fold increased VLDL-TG production rate. However, Apoc3 deficiency resulted in a significant increase in the uptake of TG-derived fatty acids from VLDL-like emulsion particles by white adipose tissue, indicating enhanced LPL activity. In vitro experiments showed that apoC-III is a more specific inhibitor of LPL activity than is apoE. Thus, Apoc3 deficiency can prevent apoE-induced hyperlipidemia associated with a 10-fold increased hepatic VLDL-TG production rate, most likely by alleviating the apoE-induced inhibition of VLDL-TG hydrolysis. Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc. Chemicals / CAS: cholesterol, 57-88-5; lipid, 66455-18-3; lipoprotein lipase, 83137-80-8, 9004-02-8; Apolipoprotein C-III; Apolipoprotein E4; Apolipoproteins C; Apolipoproteins E; Lipids; Lipoprotein Lipase, EC 3.1.1.34; Lipoproteins; Lipoproteins, VLDL; Triglycerides
|
[PDF]
[Abstract]
|
3 |
|
Domains of apolipoprotein E contributing to triglyceride and cholesterol homeostasis in vivo. Carboxyl-terminal region 203-299 promotes hepatic very low density lipoprotein-triglyceride secretion
Apolipoprotein (apo) E has been implicated in cholesterol and triglyceride homeostasis in humans. At physiological concentration apoE promotes efficient clearance of apoE-containing lipoprotein remnants. However, high apoE plasma levels correlate with high plasma triglyceride levels. We have used adenovirus-mediated gene transfer in apoE-deficient mice (E-/-) to define the domains of apoE required for cholesterol and triglyceride homeostasis in vivo. A dose of 2 × 109 plaque-forming units of apoE4-expressing adenovirus reduced slightly the cholesterol levels of E -/- mice and resulted in severe hypertriglyceridemia, due to accumulation of cholesterol and triglyceride-rich very low density lipoprotein particles in plasma. In contrast, the truncated form apoE4-202 resulted in a 90% reduction in the plasma cholesterol levels but did not alter plasma triglyceride levels in the E-/- mice. ApoE secretion by cell cultures, as well as the steady-state hepatic mRNA levels in individual mice expressing apoE4 or apoE4-202, were similar. In contrast, very low density lipoprotein-triglyceride secretion in mice expressing apoE4, but not apoE4-202, was increased 10-fold, as compared with mice infected with a control adenovirus. The findings suggest that the amino-terminal 1-202 region of apoE4 contains the domains required for the in vivo clearance of lipoprotein remnants. Furthermore, the carboxyl-terminal 203-299 residues of apoE promote hepatic very low density lipoprotein-triglyceride secretion and contribute to apoE-induced hypertriglyceridemia. Chemicals/CAS: cholesterol, 57-88-5; Apolipoprotein E4; Apolipoproteins E; Cholesterol, 57-88-5; DNA Primers; RNA, Messenger; Triglycerides
|
[PDF]
[Abstract]
|
4 |
|
Hyperlipidemia in APOE2 transgenic mice is ameliorated by a truncated apoE variant lacking the C-terminal domain
article |
2003
|
Author: |
Gerritsen, G.
·
Kypreos, K.E.
·
Zee, A. van der
·
Teusink, B.
·
Zannis, V.I.
·
Havekes, L.M.
·
Dijk, K.W. van
|
Keywords: |
Biology · Adenoviridae · Animals · Apolipoprotein E2 · Apolipoprotein E4 · Apolipoproteins E · Cholesterol · Humans · Hyperlipidemias · Lipids · Lipoproteins · Lipoproteins, VLDL · Liver · Mice · Mice, Transgenic · Protein Isoforms · Protein Structure, Tertiary · Triglycerides
|
Familial dysbetalipoproteinemia associated with the apolipoprotein E2 (APOE2) genotype is a recessive disorder with low penetrance. We have investigated whether additional expression of full-length APOE3, APOE4, or a truncated variant of APOE4 (APOE4-202) can reduce APOE2-associated hyperlipidemia. This was achieved using adenovirus-mediated gene transfer to mice transgenic for human APOE2 and deficient for endogenous Apoe (APOE2.Apoe-/- mice). The hyperlipidemia of APOE2.Apoe-/- mice was readily aggravated by APOE3 and APOE4 overexpression. Only a very low dose of APOE4 adenovirus was capable of reducing the serum cholesterol and triglyceride (TG) levels. Expression of higher doses of APOE4 was associated with an increased VLDL-TG production rate and the accumulation of TG-rich VLDL in the circulation. In contrast, a high dose of adenovirus carrying APOE4-202 reduced both the cholesterol and TG levels in APOE2.Apoe-/- mice. Despite the absence of the C-terminal lipid-binding domain, APOE4-202 is apparently capable of binding to lipoproteins and mediating hepatic uptake. Moreover, overexpression of APOE4-202 in APOE2.Apoe-/- mice does not aggravate their hypertriglyceridemia. These results extend our previous analyses of APOE4-202 expression in Apoe-/- mice and demonstrate that apoE4-202 functions even in the presence of clearance-defective apoE2. Thus, apoE4-202 is a safe and efficient candidate for future therapeutic applications. Chemicals/CAS: Apolipoprotein E2; Apolipoprotein E4; Apolipoproteins E; Cholesterol, 57-88-5; Lipids; Lipoproteins; Lipoproteins, VLDL; Protein Isoforms; Triglycerides; very low density lipoprotein triglyceride
|
[PDF]
[Abstract]
|