COMPARISON OF LIPID-BINDING AND LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVATION OF THE AMINO-TERMINAL AND CARBOXYL-TERMINAL DOMAINS OF HUMAN APOLIPOPROTEIN E3

To extend the characterization of the functional domains of apolipoprotein E(apoE), the amino(residues 1-198, 22-kDa) and carboxyl-terminal (residues 216-299, 10-kDa) fragments were tested for Lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation, A disulfide bond linking helices 2 and 3 of the four-helix bundle amino-terminal domain was introduced by mutating threonine-57 to cysteine (Thr57 --> Cys) in apoE3 (cysteine at position 112) to determine the influence of the disulfide bond on the properties of this domain. Lipid-binding properties were determined by the ability to form complexes with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine, assessed by measuring decreases in turbidity as a function of temperature. The results demonstrate that the relative Lipid binding efficiencies were intact apoE3 similar to 10-kDa fragment > 22-kDa fragment > Thr57 --> Cys variant. In addition, free, non-lipid-associated protein was observed with the two 22-kDa fragments but not with intact apoE3 or the 10-kDa fragment. The transition temperatures determined by fluorescence polarization were higher for the DMPC complexes with intact apoE3 and with 22- and 10-kDa fragments (25.5 degrees C) than with the 22-kDa Thr57 --> Cys variant (23.5 degrees C), suggesting that the variant fragment possessed the lowest affinity for lipid. Attenuated total reflection infrared measurements of the complexes indicated that the long axes of the a-helices of the various apoE forms were parallel to the acyl chains of the phospholipid bilayer. The substrate efficiency (V-max/K-m) for LCAT activation was higher for the palmitoyllinoleoylphosphatidylcholine-cholesterol complexes with the 10-kDa fragment (1.5 nmol of cholesteryl esters h(-1) mu M(-1)) than with intact apoE3 (0.5 nmol of cholesteryl esters h(-1) mu M(-1)). The reaction rates for the 22-kDa fragment and the Thr57 --> Cys variant were too low for an accurate determination of the kinetic parameters. Collectively, these data indicate that the 22-kDa fragment associates less with lipids than do intact apoE3 and the 10-kDa fragment. Moreover, the Lipid binding is further. decreased when two of the four helical segments of the 22-kDa fragment are linked by a disulfide bond. These observations support the concept that an antiparallel orientation of 17-residue helices provides an optimal configuration and a general mode of association for amphipathic helices within a discoidal apoprotein-lipid complex. The LCAT-activating properties of intact apoE3 are contained within the carboxyl-terminal domain.