Evidence That Apolipoprotein A-I Milano Has Reduced Capacity, Compared With Wild-Type Apolipoprotein A-I, to Recruit Membrane Cholesterol

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Abstract
Human carriers of apolipoprotein (apo) A-I Milano are heterozygous for an Arg 173 →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 <.001) unesterified cholesterol/phospholipid mole ratio (0.47±0.10) than wild-type apoA-I complexes (1.29±0.14), indicating that apoA-I Milano had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-I Milano nascent HDL consisted mostly of small 7.4-nm particles compared with wild-type apoA-I, in which 11- and 9-nm particles predominated. Despite these changes in nascent HDL particle size and composition, apoA-I Milano activated LCAT normally. We conclude that, even though apoA-I Milano is a normal activator of LCAT, it is less efficient than wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-I Milano may be unrelated to the initial stages of reverse cholesterol transport.

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