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-IMilano 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-IMilano 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-IMilano to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-IMilano 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 (approximately 2 micrograms/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-IMilano was in the homodimer form. Compositional analyses revealed that apoA-IMilano 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-IMilano had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-IMilano 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-IMilano activated LCAT normally. We conclude that, even though apoA-IMilano is a normal activator of LCAT, it is less efficient that wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-IMilano may be unrelated to the initial stages of reverse cholesterol transport.

Cysteine mutants of human apolipoprotein A-I: a study of secondary structural and functional properties

“…In our study, A-I(K107C) exhibited a greater efficiency in recruiting cholesterol from THP-1 macrophages, whereas A-I M (R173C) and A-I(K195C) showed decreased efflux abilities compared with wtapoA-I. The decreased efflux capacity of A-I M (R173C) was also observed by other research groups (40,41). Conversely, A-I M /A-I M , distinct from its monomer, was a more efficient acceptor of cellular cholesterols after mixing with phospholipid (42).…”

Exploiting the Vascular Protective Effects of High-Density Lipoprotein and its Apolipoproteins

“…The absence of vascular disease in the carriers of apolipoprotein A-I Milano has been attributed to the increased efficiency of the mutant in promoting reverse cholesterol transport. This conjecture is supported by in vitro data from our laboratory and by data reported by Franceschini et al 42 However, opposite results have been reported by Bielicki et al 43 using a different in vitro model system.…”

Wild-Type ApoA-I and the Milano Variant Have Similar Abilities to Stimulate Cellular Lipid Mobilization and Efflux

“…Because the ability of the Milano variant of apoA-I to promote greater cholesterol efflux than apoA-I wt is controversial, 6,37 we performed comparative experiments to address the efflux potential of lipid-free and lipid-associated apoA-I Milano (rHDL). In addition, serum and HDL from mice expressing the apoA-I Milano and apoA-I wt were compared.…”

“…1,3,4 Lipid efflux studies are also contradictory with some reporting apoA-I Milano promotes greater cholesterol efflux, 5 and others indicating that apoA-I Milano does not enhance cholesterol mobilization. 6 A number of mechanisms have been identified by which free cholesterol (FC) moves from cells to extracellular acceptors. These pathways include aqueous diffusion, 7 SR-BImediated efflux, 8 ABCA1-mediated efflux, 9 and ABCG1-mediated efflux.…”