Evidence that apolipoprotein A-I(Milano) has reduced capacity, compared with wild-type apolipoprotein A-I, to recruit membrane cholesterol

Abstract

Human carriers of apolipoprotein (apo) A-I(Milano) are heterozygous for an Arg173→Cys substitution in the apoA-I primary sequence; despite severe reductions in HDL cholesterol concentrations, affected individuals do not develop coronary heart disease, suggesting that apoA-I(Milano) may possess antiatherogenic properties. As the beneficial effects of wild-type apoA-I are linked to its role in HDL cholesterol transport, we examined the capacity of apoA-I(Milano) to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-I(Milano) and wild-type apoA-I were expressed in Chinese hamster ovary cells, and their ability to recruit membrane phospholipid and cholesterol for the assembly of nascent HDL was compared. Both clonal cell lines exhibited similar levels of apolipoprotein accumulation in serum-free medium (≃2 μg/mg cell protein per 24 hours), and 15% of each apolipoprotein was associated with membrane lipids to form nascent HDL (d=1.063 to 1.21 g/mL). SDS-PAGE showed that a majority (66±12%) of the lipidated apoA-I(Milano) was in the homodimer form. Compositional analyses revealed that apoA-I(Milano) nascent HDL had a significantly lower (P