PHOSPHOLIPID-BINDING AND LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVATION PROPERTIES OF APOLIPOPROTEIN-A-I MUTANTS

Recombinant human apolipoprotein A-I (ape A-I) and three deletion mutants: apo A-I(Delta Leu(44)-Leu(126)), apo A-I(Delta Glu(139)-Leu(170)), and apo A-I(Delta Ala(190)-Gln(243)), purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(Delta Ala(190)-Gln(243)) with dimyristoylphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding. The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A-I(Delta Leu(44)-Leu(126)), 6.1 nm for apo A-I(Delta Glu(139)-Leu(170)), and 6.5 nm for apo A-I(Delta Ala(190)-Gln(243))] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(Delta Leu(44)-Leu(126)), 6 per apo A-I(Delta Glu(139)-Leu(170)), and 4 per apo A-I(Delta Ala(190)-Gln(243)) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively. The catalytic efficiencies for activation of lecithin-cholesterol acyltransferase (LCAT) were 2-fold lower for apo A-I(Delta Leu(44)-Leu(126)), 11-fold lower for apo A-I(Delta Glu(139)-Leu(170)), and 8-fold lower for apo A-I(Delta Ala(190)-Gln(243)) than for intact apo A-I, as a result of a significant decrease of V-max but not of K-m values. In aggregate, these data suggest that deletion of the amino-terminal or of the central domains of apo A-I has little effect on lipid binding, whereas deletion of the carboxy-terminal domain reduces the rate but not the extent of lipid binding. Furthermore, deletion of the central domain [in apo A-I(Delta Glu(139)-Leu(170))] or conformational modifications in the central domain [lin apo A-I(Delta Ala(190)-Glu(243))] result in a decrease of the rate of LCAT activity that is independent of the binding of LCAT to rHDL.