Macrophage paraoxonase 1 (PON1) binding sites.
Paraoxonase 1 (PON1), an HDL-associated esterase, is known to possess anti-oxidant and anti-atherogenic properties. PON1 was shown to protect macrophages from oxidative stress, to inhibit macrophage cholesterol biosynthesis, and to stimulate HDL-mediated cholesterol efflux from the cells. The aim of the present study was to characterize macrophage PON1 binding sites which could be responsible for the above anti-atherogenic activities. Incubation of FITC-labeled recombinant PON1 with J774 A.1 macrophage-like cell line at 37 degrees C, resulted in cellular binding and internalization of PON1, leading to PON1 localization in the cell's cytoplasm compartment. In order to determine whether PON1 uptake is mediated via a specific binding to the macrophage, FITC-labeled recombinant PON1 was incubated with macrophages at 4 degrees C, followed by cell membranes separation. Macrophage membrane fluorescence was shown to be directly and dose-dependently related to the labeled PON1 concentration. Furthermore, binding assays performed at 4 and at 37 degrees C, using labeled and non-labeled recombinant PON1 (for competitive inhibition), demonstrated a dose-dependent significant 30% decrement in labeled PON1 binding to the macrophages, by the non-labeled PON1. Similarly, binding assays, using labeled PON1 and non-labeled HDL (the natural carrier of PON1 in the circulation) indicated that HDL decreased the binding of labeled PON1 to macrophages by 25%. Unlike HDL, LDL had no effect on labeled PON1 binding to macrophages. Finally, HDL were pre incubated without or with PON1 or apolipoprotein AI (apoAI) antibodies, in order to block PON1 or apoAI ability to bind to the cells. HDL incubation with antibody to PON1 or to apoAI significantly decreased HDL ability to inhibit macrophages-mediated LDL oxidation (by 32% or by 25%, respectively). A similar trend was also observed for HDL-mediated cholesterol efflux from macrophages, with an inhibitory effect of 35% or 19%, respectively. These results suggest that blocking HDL binding to macrophages through its apo A-I, and more so, via its PON1, results in the attenuation of HDL-PON1 biological activities. In conclusion, PON1 specifically binds to macrophage binding sites, leading to anti-atherogenic effects. Macrophage PON1 binding sites may thus be a target for future cardio protection therapy.
Efrat
Michal
M
The Lipid Research Laboratory, Technion Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Efron 1, Bat Galim, Haifa 31096, Israel.
Aviram
Michael
M