Atherosclerosis 219 (2011) 116–123 Contents lists available at ScienceDirect Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis The aminoterminal 1–185 domain of human apolipoprotein E suffices for the de novo biogenesis of apoE-containing HDL-like particles in apoA-I deficient mice Peristera A. Petropoulou a , Donald L. Gantz b , Yanan Wang c , Patrick C.N. Rensen c , Kyriakos E. Kypreos a,∗ a Pharmacology Unit, University of Patras Medical School, Rio Achaias, TK 26500, Greece b Boston University School of Medicine, Department of Physiology and Biophysics, Boston, MA 02118, USA c Department of General Internal Medicine, Endocrinology, and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands article info Article history: Received 1 May 2011 Received in revised form 20 June 2011 Accepted 30 June 2011 Available online 14 July 2011 Keywords: Apolipoprotein E De novo biogenesis of HDL ABCA1 apoA-I deficient mice Truncated apoE forms Adenovirus-mediated gene transfer abstract Aims: Recently we showed that apolipoprotein E promotes the de novo biogenesis of apoE-containing HDL particles in a process that requires the function of the lipid transporter ABCA1. Here, we sought to identify the domain of apoE that is responsible for its functional interactions with ABCA1 and the formation of apoE-rich HDL-like particles. Methods and results: Recombinant attenuated adenoviruses expressing carboxy-terminal truncated forms of apoE4 (apoE4[1–259], apoE4[1–229], apoE4[1–202], and apoE4[1–185]) were administered to apoA-I- deficient mice at a low dose of 8 × 10 8 pfu and five days post-infection plasma samples were isolated and analyzed for HDL formation. Fractionation of plasma lipoproteins of the infected mice by density gradient ultracentrifugation and FPLC revealed that all forms were capable of promoting HDL formation. Negative staining electron microscopy analysis of the HDL density fractions confirmed that all C-terminal truncated forms of apoE4 promoted the formation of particles with diameters in the HDL region. Interestingly, apoE4[1–259], apoE4[1–229], and apoE4[1–202] led to the formation of spherical particles while plasma from apoE4[1–185] expressing mice contained a mixture of spherical and discoidal particles. Conclusions: Taken together, our data establish that the aminoterminal 1–185 region of apoE suffices for the formation of HDL particles in vivo. Our findings may have important ramifications in the design of new biological drugs for the treatment of dyslipidemia, atherosclerosis and coronary heart disease. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Apolipoprotein E (apoE) is a glycoprotein synthesized by the liver and other peripheral tissues. Lipoprotein-bound apoE is the ligand for the LDL receptor, LRP-1 and other scavenger receptors, and promotes the catabolism of apoE-containing lipoprotein remnants [1–5]. Mutations in apoE that prevent its binding to the LDL receptor and possibly other receptors and heparan sulfate Abbreviations: ABCA1, ATP-binding cassette A1 transporter; apoE, apolipopro- tein E; apoE4[1–259], truncated apoE4 form extending from aminoacids 1–259; apoE4[1–229], truncated apoE4 form extending from aminoacids 1–229; apoE4[1–202], truncated apoE4 form extending from aminoacids 1–202; apoE4[1–185], truncated apoE4 form extending from aminoacids 1–185; apoA-I, apolipoprotein A-I; Ad, adenovirus; FBS, fetal bovine serum; FPLC, fast pressure liq- uid chromatography; GFP, green fluorescent protein; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LCAT, lecithin:cholesterol acyl transferase; LDL, low density lipoprotein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; VLDL, very low density lipoprotein. ∗ Corresponding author. Tel.: +30 2610969120; fax: +30 2610994720. E-mail address: kkypreos@med.upatras.gr (K.E. Kypreos). proteoglycans are associated with type III hyperlipoproteinemia and premature atherosclerosis [6–13]. In humans, apolipoprotein E is found in three natural isoforms (apoE2, apoE3 and apoE4) which differ at aminoacid residues 112 and 158 [14]. In vitro binding studies showed that apoE3 and apoE4 bind to the LDLr with comparable affinities while apoE2 binds with a much lower affinity [15]. The importance of apoE in the mainte- nance of plasma cholesterol homeostasis was established in studies with human patients and animal models expressing no endogenous apoE or mutant apoE forms [16–24]. These studies showed that apoE is required for the clearance of cholesterol-rich lipoprotein remnants mainly VLDL and IDL [18–26] and this process depends on the expression of functional LDLr [27]. In humans, plasma apoE levels are directly proportional to plasma triglyceride levels [28]. This is probably a causal rela- tionship, as experiments in mouse models showed that increased plasma apoE levels inhibit lipolysis of triglyceride-rich lipoproteins in vivo, and that hepatic apoE promotes the rate of VLDL- triglyceride secretion, both of which result in hypertriglyceridemia [29–35]. In contrast, deficiency in apoE is associated with decreased VLDL-triglyceride secretion [36]. Based on the hypothesis that distinct domains of apoE mediate its functions, we reported in 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.06.057