PNNL

Abstract

The ability of high-density lipoprotein (HDL) to promote cellular cholesterol efflux is a more robust predictor of cardiovascular disease protection than its plasma quantity. Previously, we found that fully lipidated HDL containing apolipoprotein A-II (APOA2) promotes cholesterol efflux via the ATP-binding cassette transporter (ABCA1). This was surprising given that ABCA1 is thought to primarily interact with lipid-poor apolipoproteins. Having previously focused on isolated lipoproteins, we moved into human plasma to verify that APOA2 can enhance ABCA1-mediated cholesterol efflux in this more complex environment. Human plasma was incubated with increasing amounts of purified, human lipid-free APOA2, and the samples were assayed for cholesterol efflux from macrophages. We found that APOA2 dose-dependently increased whole plasma cholesterol efflux capacity (CEC). Next, we titrated increasing amounts of APOA2 onto human total HDL and directly tested the impact on the CEC of native particles. We found increasing amounts of APOA2 stimulated ABCA1-mediated cholesterol efflux to the fully lipidated HDLs and that cholesterol efflux occurred maximally when equal masses of APOA1 and APOA2 coexisted on the particles. Importantly, experiments using reconstituted HDL where we tightly controlled the number of molecules of APOA1 and APOA2 on the particles showed that enhanced CEC was only observed when both APOA1 and APOA2 were on the particle. To investigate the interaction of APOA1 and APOA2 on HDL, we used limited proteolysis and chemical cross-linking mass spectrometry to show that APOA2 induces a conformational change in APOA1 conformation at its N- and C-terminal helices. When HDL particles were reconstituted with APOA1 lacking either domain, APOA2 lost the ability to stimulate ABCA1 efflux to HDL when the C-terminal domain of APOA1 was deleted but not the N-terminus. Guided by our findings, we propose a model in which APOA2 displaces the C-terminal helix of APOA1 from the HDL surface which can then interact with ABCA1 - much like it does in lipid-poor APOA1. These findings suggest APOA2 may be a novel therapeutic target given this ability to open a large, high-capacity pool of HDL particles to ABCA1-mediated cholesterol efflux.