Apolipoprotein A-IMilano and apolipoprotein A-IParis exhibit an antioxidant activity distinct from that of wild-type apolipoprotein A-I

Apolipoprotein A-I-Milano (apoA-I-Milano) and apoA-I-Paris are rare cysteine variants of apoA-I that produce a HDL deficiency in the absence of cardiovascular disease in humans. This paradox provides the basis for the hypothesis that the cysteine variants possess a beneficial activity not associated with wildtype apoA-I (apoA-I-WT). In this study, a unique antioxidant activity of apoA-I-Milano and apoA-I-Paris is described. ApoA-I-Milano was twice as effective as apoA-I-Paris in preventing lipoxygenase-mediated oxidation of phospholipids, whereas apoA-I-WT was poorly active. Antioxidant activity was observed using the monomeric form of the variants and was equally effective before and after initiation of oxidative events. ApoA-I-Milano protected phospholipid from reactive oxygen species (ROS) generated via xanthine/xanthine oxidase (X/Xo) but failed to inhibit X/Xo-induced reduction of cytochrome c. These results indicate that apoA-I-Milano was unable to directly quench ROS in the aqueous phase. There were no differences between lipid-free apoA-I-Milano, apoA-I-Paris and apoA-I-WT in mediating the efflux of cholesterol from macrophages, indicating that the cysteine variants interacted normally with the ABCA1 efflux pathway. The results indicate that incorporation of a free thiol within an amphipathic alpha helix of apoA-I confers an antioxidant activity distinct from that of apoA-I-WT. These studies are the first to relate gain of function to rare cysteine mutations in the apoA-I primary sequence.