Comparison of lipid-binding and lecithin:cholesterol acyltransferase activation of the amino- and carboxyl-terminal domains of human apolipoprotein E3.
To extend the characterization of the functional domains of apolipoprotein E (apoE), the amino-(residues 1-191, 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 approximately 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 alpha-helices of the various apoE forms were parallel to the acyl chains of the phospholipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
De Pauw
M
M
Department of Biochemistry, Faculty of Medicine, University of Gent, Belgium.
Vanloo
B
B
Weisgraber
K
K
Rosseneu
M
M
D053318:Apolipoprotein E3; D001057:Apolipoproteins E / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D004951:Esterification; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D009391:Nephelometry and Turbidimetry; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013696:Temperature
D053318:Apolipoprotein E3; D001057:Apolipoproteins E; D010743:Phospholipids; D007862:Phosphatidylcholine-Sterol O-Acyltransferase