Decreased stability of the M54 isoform of paraoxonase as a contributory factor to variations in human serum paraoxonase concentrations

There are considerable variations in serum concentrations of the high densi ty lipoprotein (HDL)-associated enzyme, paraoxonase (PON), which is an impo rtant determinant of the antioxidant capacity of HDL. The present study exa mined the hypothesis that differences in the stability of isoforms arising from the coding region L54M polymorphism could contribute to such variation s. A model system was developed using transfected Chinese hamster ovary cel ls to secrete recombinant PON corresponding to human L or M isoforms, The r ecombinant peptides exhibited the molecular properties of human serum PON, They formed complexes with Lipoproteins in culture medium, notably binding to apolipoprotein A-I-containing particles. The enzymatic properties of the recombinant isoforms were comparable to those of human serum PON. The reco mbinant M isoform lost activity more rapidly and to a greater extent than t he recombinant L isoform [26.0 +/- 3.0% vs. 14.0 +/- 1.0% (phenylacetate su bstrate) and 36.1 +/- 2.0% vs. 19.3 +/- 2.0% (paraoxon substrate) over 96 h (P < 0.01)] in medium containing fetal calf serum or PON-free human serum, Addition of a protease inhibitor resulted in retention of activity by both isoforms, Parallel results were obtained in incubation studies of human se rum from donors homozygous LL or MM for the L54M polymorphism, Enzyme activ ity was lost more rapidly and to a greater extent from MM than LL sera (P < 0.01), A parallel loss of PON peptide mass was also observed, with a signi ficantly greater loss from MM homozygotes (P < 0.001). It corresponded to t he appearance of a smaller molecular mass band on SDS-PAGE analysis. Direct analysis of the proteolytic effect using HDL isolated from homozygotes and incubated with purified kallikrein confirmed the greater loss of activity from MM homozygotes and the protective effect of proteolysis inhibitor. The results provide evidence for lesser stability of the M54 isoform of PON, a pparently involving Beater susceptibility to proteolysis, It provides one m echanism to explain variations in serum levels of PON and has implications for the antioxidant capacity of HDL.