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