Effect of the cholesterol content of reconstituted LpA-I on lecithin:cholesterol acyltransferase activity

The production of cholesteryl ester (CE) by lecithin: cholesterol acyl transferase (LCAT) is elevated significantly in hyperlipidemic subjects at high risk for coronary artery disease. To elucidate the molecular events involved, the relationship between LCAT activation and apolipoprotein (apo) A-I charge and structure in high density lipoproteins (HDL) has been studied in both native HDL and homogeneous recombinant HDL (Lp2A-I) particles containing apoA-I, palmitoyloleoyl phosphatidylcholine and cholesterol. Increasing the cholesterol content of discoidal Lp2A-I from 4 to 26 molecules/particle raises the maximum rate of cholesterol esterification by LCAT (Vmax) from 3.1 to 9.2 nmol CE/h/unit of LCAT and increases the apparent Km from 0.5 to 3.5 microM cholesterol. Similarly, increasing the cholesterol content in triolein core-containing Lp2A-I (4-18 molecules/particle) and in native HDL3 (12-21 molecules/particle) also significantly increases the Vmax for LCAT (2.8-7.7 and 0.5-3.6 nmol CE/h, respectively) and raises the Km values (7.6-36.9 and 7.3-8.5 microM cholesterol, respectively). In contrast, changes in the cholesterol content of native and recombinant HDL have no significant effect on the apparent Km values when expressed in terms of the concentration of either apoA-I or palmitoyloleoyl phosphatidylcholine. This appears to indicate that interfacial cholesterol content has no effect on the binding affinity of LCAT to different LpA-I particles but directly affects catalysis by modulating the interaction of cholesterol molecules with the active site of LCAT. Increasing the cholesterol content of the different HDL particles progressively increases the particle net negative charge, and these changes in apoA-I charge are strongly correlated with both the Vmax and apparent Km values for LCAT. This suggests that the conformation and charge of apoA-I play a central role in LCAT activation and that these parameters are influenced by the amount of cholesterol in the surface of HDL particles.