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 the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the 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 that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly 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 in 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 that the N-terminal region of apoA-I may be important in the assembly process.
Oda
M N
MN
Department of Molecular Medicine, Life Sciences Division MS 315A, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Bielicki
J K
JK
Berger
T
T
Forte
T M
TM
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043303:Aryldialkylphosphatase; D015153:Blotting, Western; D016466:CHO Cells; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics; D004950:Esterases / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D043303:Aryldialkylphosphatase; D003545:Cysteine