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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1637-1643.)
© 1997 American Heart Association, Inc.

Articles

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

John K. Bielicki; Mark R. McCall; Lori J. Stoltzfus; Amir Ravandi; Arnis Kuksis; Edward M. Rubin; ; Trudy M. Forte

From the Life Sciences Division 1-213, Department of Molecular and Nuclear Medicine, Ernest Orlando Lawrence Berkeley National Laboratory, University of California at Berkeley.

Correspondence to John K. Bielicki, Life Sciences Division 1-213, Department of Molecular and Nuclear Medicine, Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720.

Abstract Human carriers of apolipoprotein (apo) A-I_Milano are heterozygous for an Arg₁₇₃Cys 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.

Key Words: nascent HDL assembly • apoA-I_Milano • cellular cholesterol recruitment • reverse cholesterol transport • LCAT activation

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