Accumulating data suggest that paraoxonase-1 (PON1) is a primary determinant of the antioxidant and anti-inflammatory capacities of high-density lipoproteins (HDLs). Variations in HDLs and PON1 have been shown to influence the functions of both. There is a wide spectrum of serum PON1 mass in humans, to which promoter polymorphisms make an important contribution. The present studies attempted to define: (i) the relevance in vivo of promoter polymorphisms by analysing haplotype structure; and (ii) molecular mechanisms implicated in promoter activity. Highly significant differences (P<0.0001) in serum mass and activity were observed as a function of haplotype sequence. Of three promoter polymorphisms (−107, −824 and −907), the −107 site was shown to be of predominant importance to serum PON1. Significant increases in serum PON1 mass and activities between haplotype subgroups could be explained by unit increases in the number of high-expresser variants of the −107 site (−107C) alone. No significant contribution was observed for the −824 and −907 sites. The coding-region Leu55→Met (L55M) polymorphism made an independent contribution to serum PON1 mass, which may account for variations in serum PON1 mass and activity within haplotype subgroups defined by the −107 site. A molecular basis for the effect of the −107 polymorphism on serum PON1 was indicated by the greater affinity of the high-expresser variant (−107C) for hepatocyte nuclear extracts, indicating higher affinity for transcription factors. Competition studies with oligonucleotides representing the consensus (and mutated) sequence for Sp1, and the use of Sp1 antibodies, confirmed formation of complexes between the transcription factor and the PON1 promoter during incubation with nuclear extracts. The data underline the importance of the region containing the C(−107)T polymorphism for gene expression in vivo. Differences in the affinity of the −107C and −107T polymorphic fragments for nuclear extracts have been demonstrated, and coincide with their impact on gene expression. A potential role for the transcription factor Sp1 has been demonstrated, which is consistent with the disruption of an Sp1 recognition sequence by the −107 polymorphism.
Abbreviations used: ASO, allele-specific oligonucleotides; EMSA, electrophoretic mobility-shift assay; HDL, high-density lipoprotein; L55M polymorphism, etc., Leu55→Met polymorphism, etc.; PON1, paraoxonase-1; SREBP, sterol-regulatory-element-binding protein.