标题：Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis
出版社：American Society for Biochemistry and Molecular Biology
摘要：It is well accepted that both apolipoprotein A-I (apoA-I) andABCA1 play crucial roles in HDL biogenesis and in the humanatheroprotective system. However, the nature and specifics ofapoA-I/ABCA1 interactions remain poorly understood. Here, wepresent evidence for a new cellular apoA-I binding site havinga 9-fold higher capacity to bind apoA-I compared with the ABCA1site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoicacid. This new cellular apoA-I binding site was designated "high-capacitybinding site" (HCBS). Glyburide drastically reduced 125I-apoA-Ibinding to the HCBS, whereas 125I-apoA-I showed no significantbinding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore,reconstituted HDL exhibited reduced affinity for the HCBS. Deletionof the C-terminal region of apoA-I (187-243) drastically reducedthe binding of apoA-I to the HCBS. Interestingly, overexpressingvarious levels of ABCA1 in BHK cells promoted the formationof the HCBS. The majority of the HCBS was localized to the plasmamembrane (PM) and was not associated with membrane raft domains.Importantly, treatment of cells with phosphatidylcholine-specificphospholipase C, but not sphingomyelinase, concomitantly reducedthe binding of 125I-apoA-I to the HCBS, apoA-I-mediated cholesterolefflux, and the formation of nascent apoA-I-containing particles.Together, these data suggest that a functional ABCA1 leads tothe formation of a major lipid-containing site for the bindingand the lipidation of apoA-I at the PM. Our results providea biochemical basis for the HDL biogenesis pathway that involvesboth ABCA1 and the HCBS, supporting a two binding site modelfor ABCA1-mediated nascent HDL genesis.Supplementary key words ATP binding cassette transporter A1 • high density lipoprotein • atherosclerosis
Abbreviations: apoA-I, apolipoprotein A-I; 9CRA, 9-cis-retinoic acid; 2D-PAGGE, two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis; DSP, dithiobis(succinimidylpropionate); HCBS, high-capacity binding site; ICC, intracellular compartment; LpA-I, nascent apolipoprotein A-I-containing particle; 22OH, rLpA-I, reconstituted high density lipoprotein particle; 22-(R)-hydroxycholesterol; PC-PLC, phosphatidylcholine-specific phospholipase C; PM, plasma membrane; SMase, sphingomyelinase; WT, wild-type
187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of 125I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.
Supplementary key words ATP binding cassette transporter A1 • high density lipoprotein • atherosclerosis
Abbreviations: apoA-I, apolipoprotein A-I; 9CRA, 9-cis-retinoic acid; 2D-PAGGE, two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis; DSP, dithiobis(succinimidylpropionate); HCBS, high-capacity binding site; ICC, intracellular compartment; LpA-I, nascent apolipoprotein A-I-containing particle; 22OH, rLpA-I, reconstituted high density lipoprotein particle; 22-(R)-hydroxycholesterol; PC-PLC, phosphatidylcholine-specific phospholipase C; PM, plasma membrane; SMase, sphingomyelinase; WT, wild-type
