Headgroup specificity of lecithin cholesterol acyltransferase for monomeric and vesicular phospholipids

• Published in: Biochimica et biophysica acta 2000-07, Vol.1486 (2), p.321-327
• Main Authors: Christiaens, Bart, Vanloo, Berlinda, Gouyette, Catherine, Van Vynckt, Inge, Caster, Hans, Taveirne, Josee, Verhee, Annick, Labeur, Christine, Peelman, Frank, Vandekerckhove, Joël, Tavernier, Jan, Rosseneu, Maryvonne
• Format: Article
• Language: English
• Subjects: 1,2-Dipalmitoylphosphatidylcholine - chemistry; Animals; Catalysis; Cell Line; Chromatography, High Pressure Liquid; Cricetinae; Esterase activity; Kinetics; Lecithin cholesterol acyltransferase; Mutagenesis, Site-Directed; Phosphatidylcholine-Sterol O-Acyltransferase - chemistry; Phosphatidylcholine-Sterol O-Acyltransferase - genetics; Phosphatidylethanolamines - chemistry; Phospholipid; Phospholipids - chemistry; Pyrenes - chemistry; Recombinant Proteins - chemistry; Substrate; Substrate Specificity; Transfection; Vesicle
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/S1388-1981(00)00075-5

In this study, we investigated how the nature of the phospholipid head group and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin cholesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebutanoyl)-phosphatidylcholine, 1,2-bis-(1-pyrenebutanoyl)-phosphatidylethanolamine and 1,2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monomers or incorporated into small unilamellar vesicles consisting of dipalmitoylphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled phospholipids was determined both by fluorescence and by high performance liquid chromatography. V max and K m were calculated for the different substrates. The data show that V max is 10- to 30-fold higher for the hydrolysis of monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine (PE) and the phosphatidylalcohols, while K m values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholine ether vesicles, we observed a 4- to 10-fold increase of V max for PE and the phosphatidylalcohols, and no significant change for K m. V max for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity on monomeric PC and PE. These data suggest that the activity of LCAT is determined both by the molecular structure of the phospholipid and by its macromolecular properties. The LCAT activity on monomeric substrates decreases as: phosphatidylcholine≫phosphatidylethanolamine≅phosphatidylpropanol≅phosphatidylethanol≅phosphatidylethyleneglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospholipid head group.