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

Abstract

AbstractBackgroundCholesterol 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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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.Clinical PerspectiveUsing chemical crosslinking and molecular dynamics simulations, we showed that the C-termini of APOA1, HDL’s major protein, have increased mobility and conformational freedom in small HDL particles.The enhanced mobility of the C-termini of APOA1 in small HDLs allows the C-termini to ‘flip’ off a particle’s surface, activating ABCA1 thereby stimulating cholesterol removal from cells.Because of small HDLs’ vital role in cholesterol efflux, quantification of HDL-P (the size and concentration of HDL subspecies) might be a better metric for gauging cardiovascular disease risk than HDL-cholesterol levels.Therapeutic interventions that increase small HDL levels, with or without increasing HDL-cholesterol levels, may be cardioprotective.