Properties of the retained N-terminal hydrophobic leader sequence in human serum paraoxonase/arylesterase.
Human serum paraoxonase/arylesterase (PON1) is HDL-associated and appears to protect low density lipoproteins (LDL) from oxidation. Mature PON1 retains its N-terminal hydrophobic signal sequence, which may be needed for binding to HDL. By site-directed mutagenesis, we created a mutant PON1 (A19A20) with a cleavable N-terminus to determine if this peptide mediated binding to lipoproteins. As a model system, we studied binding of mutant and wild type PON1s to lipoproteins in fetal bovine serum-containing expression medium and found that the wild type recombinant enzyme associated with lipoproteins whereas the A19A20 mutant did not. These results show that the N-terminus is required for binding to either apolipoproteins or phospholipids. Furthermore, we showed that wild type enzyme can bind to phospholipids directly without apolipoproteins. To determine if lipid binding is a requirement for PON1's protection against LDL oxidation, we used a copper ion-induced oxidation system and found that the wild type enzyme and A19A20 mutant showed similar reductions in both peroxide and aldehyde formation. We conclude that PON1 depends upon its N-terminal hydrophobic peptide for its association with serum lipoproteins.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109, USA.
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D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry; D043303:Aryldialkylphosphatase; D002265:Carboxylic Ester Hydrolases / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D002645:Chickens; D002784:Cholesterol / Q000737:chemistry; D003300:Copper / Q000494:pharmacology; D004950:Esterases / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry; D008077:Lipoproteins, LDL / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry / Q000235:genetics; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D021382:Protein Sorting Signals / Q000097:blood* / Q000737:chemistry / Q000235:genetics
D016632:Apolipoprotein A-I; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D010743:Phospholipids; D021382:Protein Sorting Signals; D003300:Copper; D002784:Cholesterol; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human