Characterization of apolipoprotein E7 (Glu

Previously, a mutant apolipoprotein (apo) E, apolipoprotein E7 (Glu 244 ➝ Lys, Glu 245 ➝ Lys), has been identified in association with hyperlipidemia and atherosclerosis. To investigate the effects of its structural changes on lipoprotein metabolisms and its correlation with atherosclerosis, we characterized this mutant apoE with respect to its receptor-binding, heparin-binding, and lipoprotein association. In a competitive binding assay, apoE7 ? dimyristoylphosphatidylcholine displayed a defective binding to the low density lipoprotein (LDL) receptor. The concentration of apoE7 required for 50% displacement of 125 I-labeled LDL was 0.223 m g/ml, while that for apoE3 was 0.048 m g/ml. ApoE7 possesses only 23% of normal binding activity. To investigate the lipoprotein preference of apoE7, we determined the relative amounts of apoE7 in plasma lipoprotein fractions obtained by ultracentrifugation or gel filtration. Like human apoE4, apoE7 was preferentially associated with the very low density lipoproteins (VLDL). For determination of heparin-binding activity, apoVLDL was applied to a heparin-Sepharose affinity column and the bound materials were eluted with a salt gradient. The apoE7 was eluted at a higher NaCl concentration (157 m m ) than apoE3 (126 m m ), indicating that this mutant has a higher affinity for heparin than does apoE3. While the reduced receptor-binding activity indicates delayed clearance of the triglyceride-rich lipoproteins, the preferential association of apoE7 with larger-size lipoproteins and the stronger interaction with heparin may compensate, to some extent, for the delayed clearance of triglyceride-rich lipoproteins. The strong interaction with proteoglycans in the arterial wall seems to be one of the possible explanations for the association of apoE7 with atherosclerosis.— Yamamura, T., L-M. Dong, and A. Yamamoto. Characterization of apolipoprotein E7 (Glu 244 ➝ Lys, Glu 245 ➝ Lys), a mutant apolipoprotein E associated with hyperlipidemia and atherosclerosis. J. Lipid Res. 1999. 40: 253–259. Supplementary key words apolipoprotein mutant • heparin binding • lipoprotein • LDL receptor • receptor binding Apolipoprotein (apo) E is a polymorphic protein (1, 2). Three common apoE isoforms in human are designated as apoE2 (Cys-112, Cys-158), apoE3 (Cys-112, Arg-158), and apoE4 (Arg-112, Arg-158) (1, 2). The polymorphism of apoE influences the plasma cholesterol and low density lipoprotein (LDL) concentrations. ApoE4 is known to be associated with higher cholesterol and LDL concentrations, and has been implicated as one of the risk factors for cardiovascular disease (3, 4). Although apoE2 is generally associated with lower cholesterol and LDL levels, under certain circumstances, homozygosity of apoE2 is an underlying cause of type III hyperlipoproteinemia, a genetic disorder characterized by elevated cholesterol and triglyceride levels and accelerated coronary artery disease (3, 4). Apolipoprotein E has several important biological functions, including lipoprotein receptor binding, heparin binding, and lipoprotein association (for review, see ref. 5). The structural changes influence these functions. The wild-type apoE3 is known to preferentially associate with high density lipoprotein (HDL) when with cysteine at position 112, whereas apoE4 is known to preferentially associate with very low density lipoprotein (VLDL) when with arginine at position 112 (6, 7). The apoE4 preference for VLDL has been suggested to at least partially explain the elevated cholesterol and LDL levels in apoE4-carrying subjects (7). With respect to the lipoprotein receptor binding, apoE3 and apoE4 bind equally, whereas apoE2 with cysteine at position 158 is defective (less than 1% of apoE3 or apoE4 activity) (8). As a consequence of this defective binding, subjects homozygous for apoE2 allele have a delayed clearance of remnant lipoproteins (9). This, combined with genetic, environmental, or hormonal influences, may result in recessive expression of type III hyperlipoproteinemia (9). Several other identified apoE variants with defective receptor-binding ability have been shown to have associations with impaired catabolism of Abbreviations: apo, apolipoprotein; DMPC, dimyristoylphosphatidylcholine; ELISA, enzyme-linked immunosorbent assay; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein. 1 To whom correspondence should be addressed. 2 Present address: Research Scientist, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, CA 94304 USA. by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom