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J. Biol. Chem., Vol. 280, Issue 8, 6276-6284, February 25, 2005

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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^*

Kyriakos E. Kypreos¶¶, Ko W. van Dijk¶, Louis M. Havekes¶||**, and Vassilis I. Zannis

From the Molecular Genetics, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, the Department of Human Genetics, Leiden University Medical Center, 2333AL Leiden, The Netherlands, the ^¶Department of General Internal Medicine, Leiden University Medical Center, 2333AL, Leiden, The Netherlands, the ^||TNO-Preventie en Gezondheid Prevention and Health, Gaubius Laboratory, Leiden 2333CK, The Netherlands, the ^**Department of Cardiology, Leiden University Medical Center, 2333AL, Leiden, The Netherlands, and the Department of Biochemistry, Institute of Molecular Biology and Biotechnology, University of Crete Medical School, Heraklion, Crete 71110 Greece

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.

Received for publication, November 30, 2004

^* This work was supported by Grant HL68216 from the National Institutes of Health, Kos Pharmaceuticals, Grant LST.CLG.978402 from NATO (to V. I. Z.), and Grant MCIF 2000-02051 from the European Union Fifth Framework Program (to K. E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶¶ An EMBO Fellow.

To whom correspondence should be addressed: Section of Molecular Genetics, Whitaker Cardiovascular Institute, Dept. of Medicine, Boston University School of Medicine, 700 Albany St., W-509, Boston, Massachusetts, 02118. Tel.: 617-638-5085; Fax: 617-638-5141; E-mail: vzannis{at}bu.edu.

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