Influence of Isoforms and Carboxyl-Terminal Truncations on the Capacity of Apolipoprotein E To Associate with and Activate Phospholipid Transfer Protein Ioannis Dafnis, † Jari Metso, ‡ Vassilis I. Zannis, § Matti Jauhiainen,* ,‡,# and Angeliki Chroni* ,†,# † Institute of Biosciences and Applications, National Center for Scientific Research “Demokritos”, Agia Paraskevi 15310, Athens, Greece ‡ Genomics and Biomarkers Unit, Biomedicum, National Institute for Health and Welfare, Helsinki 00290, Finland § Departments of Medicine and Biochemistry, Molecular Genetics, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118, United States * S Supporting Information ABSTRACT: Phospholipid transfer protein (PLTP), a main protein in lipid and lipoprotein metabolism, exists in high-activity (HA-PLTP) and low-activity (LA-PLTP) forms in human plasma. Proper phospholipid transfer activity of PLTP is modulated by interactions with various apolipoproteins (apo) including apoE. The domains of apoE involved in interactions with PLTP are not known. Here we analyzed the capacity of recombinant apoE isoforms and apoE4 mutants with progressive carboxyl-terminal deletions to bind to and activate HA-PLTP and LA-PLTP. Our analyses demonstrated that lipid-free apoE isoforms bind to both HA-PLTP and LA-PLTP, resulting in phospholipid transfer activation, with apoE3 inducing the highest PLTP activation. The isoform-specific differences in apoE/PLTP binding and PLTP activation were abolished following apoE lipidation. Lipid-free apoE4[Δ(260-299)], apoE4[Δ(230-299)], apoE4[Δ(203-299)], and apoE4[Δ(186-299)] activated HA-PLTP by 120- 160% compared to full-length apoE4. Lipid-free apoE4[Δ(186-299)] also activated LA-PLTP by 85% compared to full-length apoE4. All lipidated truncated apoE4 forms displayed a similar effect on HA-PLTP and LA-PLTP activity as full-length apoE4. Strikingly, lipid-free or lipidated full-length apoE4 and apoE4[Δ(186-299)] demonstrated similar binding capacity to LA-PLTP and HA-PLTP. Biophysical studies showed that the carboxyl-terminal truncations of apoE4 resulted in small changes of the structural or thermodynamic properties of lipidated apoE4, that were much less pronounced compared to changes observed previously for lipid-free apoE4. Overall, our findings show an isoform-dependent binding to and activation of PLTP by lipid-free apoE. Furthermore, the domain of apoE4 required for PLTP activation resides within its amino-terminal 1-185 region. The apoE/PLTP interactions can be modulated by the conformation and lipidation state of apoE. P hospholipid transfer protein (PLTP) is an important protein in lipid and lipoprotein metabolism. PLTP transfers phospholipids, diacylglycerol, free cholesterol, α-tocopherol, and lipopolysaccharides among lipoproteins and between lipopro- teins and cells. 1,2 Plasma PLTP has been shown to be involved in high-density lipoprotein (HDL) metabolism. More specifically, PLTP remodels HDL particles in a process whereby small HDL3 particles are fused leading to the generation of large fused HDL particles and preβ-HDL. 1, 2 In addition, PLTP transfers postlipolytic very low density lipoprotein (VLDL) surface phospholipids to HDL. 1,2 Functions of PLTP which may influence the formation of atherosclerotic lesions include the regulation of plasma HDL levels, generation of acceptors for lipid efflux from cells, modulation of proteins involved in cellular lipid efflux process, protection of lipoproteins from oxidation, and regulation of production of atherogenic lipoproteins. 1,2 Received: June 18, 2015 Revised: August 31, 2015 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A DOI: 10.1021/acs.biochem.5b00681 Biochemistry XXXX, XXX, XXX-XXX