Minimal lipidation of pre-β HDL by ABCA1 results in reduced ability to interact with ABCA1

Objectives - The aim of this study was to determine the role of ATP binding cassette transporter A1 (ABCA1) on generation of different-sized nascent HDLs. Methods and Results - HEK293 cells stably-transfected with ABCA1 (HEK293-ABCA1) or non-transfected ( control) cells were incubated with lipid free I-125-apoA-I for 24 hours. Incubation of apoA-I with HEK293-ABCA1 cells, but not control cells, led to the formation of heterogeneous-sized, pre-beta migrating nascent HDL subpopulations ( pre-beta 1 to - 4) that varied in size (7.1 to 15.7 nm), lipid, and apoA-I content. Kinetic studies suggested that all subpopulations were formed simultaneously, with no evidence for a precursor-product relationship between smaller and larger-sized particles. When isolated nascent pre-beta HDLs (pre-beta 1 to - 4) were added back to HEK293- ABCA1 cells, their ability to bind to ABCA1 and efflux lipid was severely compromised. Heat-denaturation of pre-beta 1 HDL resulted in partial recovery of ABCA1 binding, suggesting that initial interaction of apoA-I with ABCA1 results in a constrained conformation of apoA-I that decreases subsequent binding. Conclusions - Interaction of apoA-I with ABCA1 results in the simultaneous generation of pre-beta HDLs of discrete size and chemical composition. These nascent particles are poor substrates for subsequent lipidation by ABCA1 and presumably require additional non-ABCA1-mediated lipidation for further maturation.