Flipped C-Terminal Ends of APOA1 Promote ABCA1-dependent Cholesterol Efflux by Small HDLs

BACKGROUND: Cholesterol efflux capacity (CEC) predicts cardiovascular disease (CVD) independently of HDL cholesterol (HDL-C) levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 pathway, but the underlying mechanisms are unclear. METHODS: We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 in the different particles, and the CECs of plasma and isolated HDLs. RESULTS: We quantified macrophage and ABCA1 CEC of four distinct sizes of reconstituted HDL (r-HDL). CEC increased as particle size decreased. MS/MS analysis of chemically crosslinked peptides and molecular dynamics simulations of APOA1 (HDL’s major protein) indicated that the mobility of that protein’s C-terminus was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs–like r-HDLs–are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3–5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. CONCLUSIONS: We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the two antiparallel molecules of APOA1 are flipped off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased CVD risk. Thus, extra-small and small HDLs may be key mediators and indicators of HDL’s cardioprotective effects.