Properties of the retained N-terminal hydrophobic leader sequence in humanserum paraoxonase arylesterase

Human serum paraoxonase/arylesterase (PON1) is HDL-associated and appears t o protect low density lipoproteins (LDL) from oxidation. Mature PON1 retain s its N-terminal hydrophobic signal sequence, which may be needed for bindi ng to HDL. By site-directed mutagenesis, we created a mutant PON1 (A19A20) with a cleavable N-terminus to determine if this peptide mediated binding t o lipoproteins. As a model system, we studied binding of mutant and wild ty pe PON1s to lipoproteins in fetal bovine serum-containing expression medium and found that the wild type recombinant enzyme associated with lipoprotei ns whereas the A19A20 mutant did not. These results show that the N-terminu s is required for binding to either apolipoproteins or phospholipids. Furth ermore, 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 oxid ation system and found that the wild type enzyme and A19A20 mutant showed s imilar reductions in both peroxide and aldehyde formation. We conclude that PON1 depends upon its N-terminal hydrophobic peptide for its association w ith serum lipoproteins. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.