Apolipoprotein A-II alters the proteome of human lipoproteins and enhances cholesterol efflux from ABCA1

HDLs are a family of heterogeneous particles that vary in size, composition, and function. The structure of most HDLs is maintained by two scaffold proteins, apoA-I and apoA-II, but up to 95 other “accessory” proteins have been found associated with the particles. Recent evidence suggests that these accessory proteins are distributed across various subspecies and drive specific biological functions. Unfortunately, our understanding of the molecular composition of such subspecies is limited. To begin to address this issue, we separated human plasma and HDL isolated by ultracentrifugation (UC-HDL) into particles with apoA-I and no apoA-II (LpA-I) and those with both apoA-I and apoA-II (LpA-I/A-II). MS studies revealed distinct differences between the subfractions. LpA-I exhibited significantly more protein diversity than LpA-I/A-II when isolated directly from plasma. However, this difference was lost in UC-HDL. Most LpA-I/A-II accessory proteins were associated with lipid transport pathways, whereas those in LpA-I were associated with inflammatory response, hemostasis, immune response, metal ion binding, and protease inhibition. We found that the presence of apoA-II enhanced ABCA1-mediated efflux compared with LpA-I particles. This effect was independent of the accessory protein signature suggesting that apoA-II induces a structural change in apoA-I in HDLs. HDLs are a family of heterogeneous particles that vary in size, composition, and function. The structure of most HDLs is maintained by two scaffold proteins, apoA-I and apoA-II, but up to 95 other “accessory” proteins have been found associated with the particles. Recent evidence suggests that these accessory proteins are distributed across various subspecies and drive specific biological functions. Unfortunately, our understanding of the molecular composition of such subspecies is limited. To begin to address this issue, we separated human plasma and HDL isolated by ultracentrifugation (UC-HDL) into particles with apoA-I and no apoA-II (LpA-I) and those with both apoA-I and apoA-II (LpA-I/A-II). MS studies revealed distinct differences between the subfractions. LpA-I exhibited significantly more protein diversity than LpA-I/A-II when isolated directly from plasma. However, this difference was lost in UC-HDL. Most LpA-I/A-II accessory proteins were associated with lipid transport pathways, whereas those in LpA-I were associated with inflammatory response, hemostasis, immune response, metal ion binding, and protease inhibition. We found that the presence of apoA-II enhanced ABCA1-mediated efflux compared with LpA-I particles. This effect was independent of the accessory protein signature suggesting that apoA-II induces a structural change in apoA-I in HDLs. HDLs are circulating complexes of protein and lipid best known for mobilizing cholesterol in the periphery and transporting it to the liver for excretion. The term HDL actually refers to a family of diverse subspecies that share similar hydrated densities. These can vary widely in size, protein/lipid composition, and charge [reviewed in (1Shah A.S. Tan L. Long J.L. Davidson W.S. Proteomic diversity of high density lipoproteins: our emerging understanding of its importance in lipid transport and beyond.J. Lipid Res. 2013; 54: 2575-2585Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar)]. Recent MS studies across different laboratories have found up to 95 different proteins in HDL preparations (2.Davidson, W. S., 2017. Davidson Lab Webpage: Lipoprotein Proteome Watch. Accessed March 15, 2017, at www.DavidsonLab.com.Google Scholar). This compositional heterogeneity mirrors a well-known functional heterogeneity (3Gordon S.M. Hofmann S. Askew D.S. Davidson W.S. High density lipoprotein: it's not just about lipid transport anymore.Trends Endocrinol. Metab. 2011; 22: 9-15Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) in that HDLs can attenuate the oxidation of LDLs (4Navab M. Hama S.Y. Anantharamaiah G.M. Hassan K. Hough G.P. Watson A.D. Reddy S.T. Sevanian A. Fonarow G.C. Fogelman A.M. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3.J. Lipid Res. 2000; 41: 1495-1508Abstract Full Text Full Text PDF PubMed Google Scholar), reduce vascular cell-mediated inflammation (5Barter P.J. Rye K.A. High density lipoproteins and coronary heart disease.Atherosclerosis. 1996; 121: 1-12Abstract Full Text PDF PubMed Scopus (340) Google Scholar), regulate proteolytic and thrombotic pathways [reviewed in (6Nofer J.R. Brodde M.F. Kehrel B.E. High-density lipoproteins, platelets and the pathogenesis of atherosclerosis.Clin. Exp. Pharmacol. Physiol. 2010; 37: 726-735Crossref PubMed Scopus (74) Google Scholar)], reduce cellular apoptosis (7Muller C. Salvayre R. Negre-Salvayre A. Vindis C. HDLs inhibit endoplasmic reticulum stress and autophagic response induced by oxidized LDLs.Cell Death Differ. 2011; 18: 817-828Crossref PubMed Scopus (80) Google Scholar), and affect insulin secretion from pancreatic cells (8Fryirs M.A. Barter P.J. Appavoo M. Tuch B.E. Tabet F. Heather A.K. Rye K.A. Effects of high-density lipoproteins on pancreatic beta-cell insulin secretion.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1642-1648Crossref PubMed Scopus (216) Google Scholar). Despite advances in cataloging the lipoproteome, progress in molecularly characterizing lipoprotein subspecies in human plasma has been more limited. In the case of HDLs, a significant obstacle is the isolation of individual subspecies, most of which have similar physicochemical properties. For example, HDLs can be subdivided by density into HDL2 and HDL3 using sequential ultracentrifugation (UC) (9De Lalla O.F. Gofman J.W. Ultracentrifugal analysis of serum lipoproteins.Methods Biochem. Anal. 1954; 1: 459-478Crossref PubMed Google Scholar); however, each subclass retains significant compositional heterogeneity (10Blanche P.J. Gong E.L. Forte T.M. Nichols A.V. Characterization of human high-density lipoproteins by gradient gel electrophoresis.Biochim. Biophys. Acta. 1981; 665: 408-419Crossref PubMed Scopus (443) Google Scholar). Gel filtration chromatography similarly suffers from a lack of resolution with the added drawback of contaminating nonlipoprotein entities of similar size (11Gordon S.M. Deng J. Lu L.J. Davidson W.S. Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography.J. Proteome Res. 2010; 9: 5239-5249Crossref PubMed Scopus (171) Google Scholar). Two-dimensional gel electrophoresis techniques have identified numerous size and charge subpopulations of HDLs (12Asztalos B.F. Sloop C.H. Wong L. Roheim P.S. Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations.Biochim. Biophys. Acta. 1993; 1169: 291-300Crossref PubMed Scopus (216) Google Scholar), but there are obstacles with recovering them for functional characterization. Thus, only a handful of HDL subspecies have been defined at the molecular level, with even fewer defined functionally. The prototypical example is trypanosome lytic factor (13Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Characterization of a novel trypanosome lytic factor from human serum.Infect. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar), a HDL subparticle containing apoA-I, apoL-I, and haptoglobin-related protein. This protein triad can mediate the lysis of the parasite, Trypanosoma brucei, through a series of cooperative actions (14Shiflett A.M. Bishop J.R. Pahwa A. Hajduk S.L. high density lipoproteins are for the of immune Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). widely HDL subspecies is the HDL identified by and of cholesterol into high-density PubMed Scopus Google Scholar), This a in cholesterol efflux S. Characterization and of particles by ABCA1-mediated cellular lipid efflux to Lipid Res. Full Text Full Text PDF PubMed Scopus Google of formation and PubMed Scopus Google A. A.K. of HDL by in to with Thromb. Vasc. Biol. PubMed Scopus Google Scholar). J.W. protein in human plasma with proteins to and 2010; PubMed Scopus Google Scholar) chromatography to plasma complexes containing protein. found with apoA-I, and and Unfortunately, a functional has not been for these particles. not a of the between and coronary heart 1: Google Scholar) that HDL particles more with coronary heart than those containing These the of molecularly and distinct HDL subspecies in and the of HDL proteins, there are more functional subspecies to be can isolation of lipoprotein for functional and Characterization of lipoprotein particles isolated by containing and and particles containing but no Biol. Full Text PDF PubMed Google Scholar) HDLs into with apoA-I, but no apoA-II and those with both apoA-I and apoA-II (LpA-I/A-II). The of these particles was in with K. J. M. M.F. L. of and high density lipoproteins containing in and PubMed Scopus Google Scholar), protein R. K. S. S. and functional differences of subspecies of lipoprotein in with plasma protein Lipid Res. Full Text PDF PubMed Google Scholar), 2 F. C. C. R. cholesterol transport in on and LpA-I Thromb. Vasc. Biol. PubMed Scopus Google Scholar), C. S. on the of the protein in lipoprotein Lipid Res. Full Text PDF PubMed Google Scholar), and coronary heart B.F. Roheim P.S. M. J.R. of HDL subpopulations in with coronary heart Thromb. Vasc. Biol. 2000; PubMed Scopus Google Scholar). with to In of on high density lipoprotein particles LpA-I and Lipid Res. Full Text PDF PubMed Google lipid apoA-I and apoA-II Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar), cholesterol efflux R. efflux from cells is by particles but not by Biophys. Res. PubMed Scopus Google S. E.L. Effects of particles containing with on cholesterol Biophys. Acta. 1993; PubMed Scopus Google Scholar), and and of and in A-I-containing Lipid Res. Full Text PDF PubMed Google R. M. A. S. effect of subspecies of lipoprotein containing on cholesterol efflux from functional with cholesterol Biophys. Acta. PubMed Scopus Google Scholar), and of in the liver L. A. the and lipid of high density lipoprotein by Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). 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The effect of on the structure and of in a high density lipoprotein Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and particles were using the A. complexes of human with and cholesterol from Biol. Full Text PDF PubMed Google Scholar). LpA-I particles were using of LpA-I/A-II particles were from a of three independent apoA-I by apoA-II at a of and for at preparations were on a in at were and by to were using J. M. of the structure of in structural to lipid Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). were at a of for at efflux was using LpA-I and LpA-I/A-II Davidson W.S. The of and of in lipid and ABCA1-mediated cholesterol Biophys. 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We that of apoA-I in the preparations enhanced ABCA1-mediated was on in to the of protein in the not a gel efflux were with protein of plasma apoA-I into the particles significantly the containing the apoA-I a for contaminating to the from the we the to the the LpA-I/A-II particles the LpA-I particles the was on particles this of the from significant of cholesterol from particles to the apoA-II can apoA-I from lipoprotein particles A. The effect of on the structure and of in a high density lipoprotein Biol. Full Text Full Text PDF PubMed Scopus Google A.M. In of the composition of high density of from high density lipoprotein by Biol. Full Text PDF PubMed Google C. M. A.M. the of the of by from the high density lipoprotein of and molecular of Biol. Full Text PDF PubMed Google W. Davidson W.S. proteolytic for between and Lipid Res. Full Text Full Text PDF PubMed Google Scholar). the that LpA-I/A-II cholesterol efflux we that apoA-II can of apoA-I that with apoA-I to in the presence of were on LpA-I and for isolated from and the HDL particles are in The apoA-I from is for the isolated from plasma and the particles in of the isolated from was not were for different only on two of the however, those are in and are with of apoA-I at a in particles containing This difference was suggesting that apoA-II apoA-I such that it is more on the of HDLs by sequential has been widely for composition, and was the effect that on the proteins in HDLs. proteins were identified in particles isolated by those by even for with the LpA-I and LpA-I/A-II differences found in plasma particles were isolation of HDLs by is that in plasma. with apoA-I be by the C. M. of proteins in lipoprotein using gel electrophoresis and PubMed Scopus Google Scholar), even particles The and of in human J. PubMed Scopus Google Scholar). However, our gel filtration revealed and in our the is and significantly with HDL (2.Davidson, W. S., 2017. Davidson Lab Webpage: Lipoprotein Proteome Watch. Accessed March 15, 2017, at www.DavidsonLab.com.Google W. M. in human HDL by Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar) and not be to new proteins to the particles have not been to significant of apoA-I M. S. Characterization of HDL particles and by ABCA1-mediated efflux of cellular to Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar) with a lack of protein in the of the gel filtration Thus, we that HDL high and have been F. of in plasma high density lipoproteins from and Biol. Full Text PDF PubMed Google for the of between lipoprotein in human plasma and for the of Lipid Res. Full Text PDF PubMed Google A. isolation of Biophys. Acta. PubMed Scopus Google effect of ultracentrifugation on A-I-containing lipoprotein Lipid Res. Full Text PDF PubMed Google M. R. F. C. heterogeneity of lipoprotein particles containing and with isolated from Lipid Res. Full Text PDF PubMed Google Scholar) that can the from HDL cholesterol and a it that to isolation to be in functional In and to human LpA-I and LpA-I/A-II Characterization of lipoprotein particles isolated by containing and and particles containing but no Biol. Full Text PDF PubMed Google and of and in A-I-containing Lipid Res. Full Text PDF PubMed Google M. M.A. Anantharamaiah G.M. Characterization of high density lipoprotein structural studies by ultracentrifugation and chromatography.J. Lipid Res. Full Text PDF PubMed Google size of A-I-containing lipoproteins in with coronary Lipid Res. Full Text PDF PubMed Google Scholar). the of MS our differences in the of these In the to be in in in most with Characterization of lipoprotein particles isolated by containing and and particles containing but no Biol. Full Text PDF PubMed Google M. R. F. C. heterogeneity of lipoprotein particles containing and with isolated from Lipid Res. Full Text PDF PubMed Google of human high density lipoproteins and isolation of lipoproteins and Biophys. Acta. PubMed Scopus Google Scholar). We that of and in This from and of and in A-I-containing Lipid Res. Full Text PDF PubMed Google Scholar), there have been Characterization of lipoprotein particles isolated by containing and and particles containing but no Biol. Full Text PDF PubMed Google C. S. on the of the protein in lipoprotein Lipid Res. Full Text PDF PubMed Google Scholar) studies and protein a our analysis that the LpA-I/A-II particles were best associated with lipid transport functions. can at three that apoA-II the of lipid transport apoA-II to accessory protein proteins with apoA-II through apoA-II apoA-I to affect its with apoA-II has been to affect apoA-I A. The effect of on the structure and of in a high density lipoprotein Biol. Full Text Full Text PDF PubMed Scopus Google K. J.L. S. R. of in high density Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and regulate its with A. Rye K.A. that high density lipoproteins the of Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). LpA-I particles the most of the more proteins in the HDL such and of the protease This is that the LpA-I only about of HDLs Characterization of lipoprotein particles isolated by containing and and particles containing but no Biol. Full Text PDF PubMed Google Scholar). LpA-I particles were best associated with response, hemostasis, immune response, metal ion binding, and protease functions. that most of these proteins be associated with LpA-I to be lost was the in ABCA1-mediated efflux from to LpA-I/A-II particles. was not both LpA-I and LpA-I/A-II particles cholesterol efflux and However, in the presence of the LpA-I/A-II particles were This of the subspecies were isolated directly from were in the of its in cholesterol the has been that is only with However, a L. J.R. HDL size is a of ABCA1-mediated cellular cholesterol Res. PubMed Scopus Google Scholar) has that HDL particles can with to cholesterol This effect with LpA-I and LpA-I/A-II were of similar size, in the size our is that the LpA-I/A-II preparations of apoA-I that it is to the that apoA-I be from the particles the cholesterol efflux we apoA-I was added to our particles in that be in our In the cholesterol efflux by apoA-I not for the differences with the particles our we that the best for our is that apoA-II can a change in apoA-I that it to with in with a HDL and A. of proteolytic of in and in high density Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) that apoA-I is by at the of the Thus, apoA-II the of apoA-I, which has been by numerous laboratories to be the in the S. Characterization and of particles by ABCA1-mediated cellular lipid efflux to Lipid Res. Full Text Full Text PDF PubMed Scopus Google Davidson W.S. The of in cholesterol efflux the Biol. Full Text Full Text PDF PubMed Scopus Google F. L. The of is in and cholesterol Biophys. Res. PubMed Scopus Google Scholar). it affect other This not when apoA-I apoA-II were in the suggesting that a lipid is for this In our are with the that apoA-I a scaffold that the of numerous HDL accessory proteins with diverse functions. The presence of apoA-II can the of of these proteins, but with a to the lipid transport functions. the HDL more on and is by the proteins, we that it is to the that mediate protein HDL particles. with lipoprotein cholesterol protein cholesterol chromatography lipoproteins with apoA-I and no apoA-II lipoproteins with apoA-I and apoA-II lipoproteins with apoA-I and no apoA-II lipoproteins with apoA-I and apoA-II cholesterol ultracentrifugation HDL isolated by ultracentrifugation