Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) - containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined th e effect: of apoA-I on PON' s enzymatic activity and its ability to associa te with HDL. Additionally, we examined whether changes in apoA-I primary st ructure (cysteine substitution mutations) could modulate these effects. Chi nese hamster ovary cells stably transfected with human PON1A cDNA were incu bated in the presence and absence of recombinant wild-type apoA-I (ApoA-I-W T) and specific Cys substitution mutations. Extracellular accumulation of P ON activity in the presence of ApoA-I-WT was 0.095 +/- 0.013 unit/mg of cel l protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in th e absence of apoA-I (n 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting th at maximal PON activity is lipid-dependent and requires coassembly of PON a nd apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C signi ficantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9% , and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations i n the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and th at the N-terminal region of apoA-I may be important in the assembly process .