Role of LCAT in HDL remodeling: investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (−77% and −13%), and LCAT activities (−99% and −39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated preβ-1 and α-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with α-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, preβ-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with preβ-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of preβ-1 into α-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles. To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (−77% and −13%), and LCAT activities (−99% and −39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated preβ-1 and α-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with α-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, preβ-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with preβ-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of preβ-1 into α-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles. LCAT is a 416 amino acid protein that binds to lipoproteins or is present in lipid-free form in plasma and is secreted by the liver in humans (1McLean J. Fielding C. Drayna D. Dieplinger H. Baer B. Kohr W. Henzel W. Lawn R. Cloning and expression of human lecithin-cholesterol acyltransferase cDNA. Proc. Natl. Acad. Sci. USA. 1986; 83: 2335-2339Crossref PubMed Scopus (144) Google Scholar). LCAT synthesizes the majority of cholesteryl esters in plasma by transferring a fatty acid from lecithin (phosphatidyl choline) to the 3-hydroxyl group of cholesterol. It is generally believed that LCAT maintains the unesterified cholesterol gradient between peripheral cells and HDL. Efflux of free cholesterol (FC) from cells occurs by a passive diffusion of FC between cellular membranes and acceptors and by mechanisms facilitated by scavenger receptor type B-I (SR-BI) and ABCs. In the presence of LCAT, the bi-directional movement of cholesterol between cells and HDL results in net cholesterol efflux (2Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar, 3Czarnecka H. Yokoyama S. Regulation of cellular cholesterol efflux by lecithin:cholesterol acyltransferase reaction through nonspecific lipid exchange. J. Biol. Chem. 1996; 266: 2023-2028Google Scholar). Therefore, LCAT plays a central role in the initial steps of reverse cholesterol transport. LCAT is activated primarily by apolipoprotein A-I (apoA-I), but can also be activated by apoA-IV, apoC-I, and apoE (4Jonas A. von Eckardstein A. Kezdy K.E. Steinmetz A. Assmann G. Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants. J. Lipid Res. 1991; 32: 97-106Google Scholar, 5Steinmetz A. Kaffarnik H. Utermann G. Activation of phosphatidylcholine-sterol acyltransferase by human apolipoprotein E isoforms. Eur. J. Biochem. 1985; 152: 747-751Google Scholar). Both the binding and activation of LCAT on the surface of HDL are essential for esterification of FC and accumulation of cholesteryl esters in the core of HDL. Familial LCAT deficiency (FLD) is characterized by the absence of LCAT activity and reduced HDL cholesterol (HDL-C) level in plasma. In affected individuals, LCAT is either absent or present but inactive in plasma (6Kuivenhoven J.A. Pritchard H. Hill J. Frohlich J. Assmann G. Kastelein J. The molecular pathology of lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes. J. Lipid Res. 1997; 38: 191-205Google Scholar). LCAT has two distinct substrates: HDL and LDL. LCAT activity on HDL is called α-activity, and LCAT activity on LDL is called β-activity (7Santamarina-Fojo S. Hoeg J.M. Assmann G Brewer Jr., H.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Lecithin cholesterol acyltransferase deficiency and fish eye disease. In The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York.2001: 2817-2834Google Scholar, 8Gordon D.J. Rifkind B.M. High-density lipoprotein—the clinical implications of recent studies. N. Engl. J. Med. 1989; 321: 1311-1316Crossref PubMed Scopus (1407) Google Scholar). Lack of α-LCAT activity causes fish eye disease (FED). Homozygous subjects with FLD have corneal opacification, anemia, proteinuria, hematuria, and ultimately, renal failure, often requiring kidney transplantation (9Funke H. von Eckardstein A. Pritchard P.H. Albers J.J. Kastelein J.J. Droste C. Assmann G. A molecular defect causing fish eye disease: an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of alpha-LCAT activity. Proc. Natl. Acad. Sci. USA. 1991; 88: 4855-4859Google Scholar). FED subjects have no clinical manifestation other than an age-dependent corneal opacification. Although it is not clear whether LCAT deficiency is directly linked to premature coronary artery disease (CAD), increased risk for CAD has been reported in some patients (9Funke H. von Eckardstein A. Pritchard P.H. Albers J.J. Kastelein J.J. Droste C. Assmann G. A molecular defect causing fish eye disease: an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of alpha-LCAT activity. Proc. Natl. Acad. Sci. USA. 1991; 88: 4855-4859Google Scholar). Data obtained from cholesterol-fed human-LCAT transgenic rabbits indicated that HDL-C increased due to decreased catabolism of larger HDL particles, suggesting that the size of HDL may modulate the selective HDL-C uptake by the liver (10Brousseau M.E. Santamarina-Fojo S. Vaisman B.L. Applebaum-Bowden D. Berard A.M. Talley G.D. Brewer Jr., H.B. Hoeg J.M. Overexpression of human lecithin:cholesterol acyltransferase in cholesterol-fed rabbits: LDL metabolism and HDL metabolism are affected in a gene dose-dependent manner. J. Lipid Res. 1997; 38: 2537-2547Google Scholar). In human-LCAT transgenic mice, the liver uptake of HDL was reduced by 41%, resulting in a substantial increase of large HDL particles that might be atherogenic (11Berard A.M. Foger B. Remaley A. Shamburek R. Vaisman B.L. Talley G. Paigen B. Hoyt Jr., R.F. Marcovina S. Brewer Jr., H.B. High plasma HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin-cholesteryl acyltransferase. Nat. Med. 1997; 3 (et al.): 744-749Google Scholar) due to the fact that mice lack cholesteryl ester transfer protein (CETP) and that continued increase of cholesteryl ester in HDL by high levels of LCAT changes both the size and lipid composition of HDL. When CETP was coexpressed in LCAT transgenic mice, HDL size and composition changed and the animals were protected from atherosclerosis (12Foger B. Chase M. Amar M.J. Vaisman B.L. Shamburek R.D. Paigen B. Fruchart-Najib J. Paiz J.A. Koch C.A. Hoyt R.F. Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice. J. Biol. Chem. 1999; 274 (et al.): 36912-36920Google Scholar). These data suggest that under normal conditions in which CETP is present as in humans, increased LCAT activity is likely to increase HDL cholesterol and size and might reduce the risk for atherosclerosis. Our previous data suggest that the two largest, spherical, cholesteryl ester-rich HDL particles, α-1 and α-2, are good substrates for SR-BI in a human hepatoma cell line (13Asztalos B.F. de la Llera-Moya M. Dallal G.E. Horvath K.V. Schaefer E.J. Rothblat G.H. Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux. J. Lipid Res. 2005; 46: 2246-2253Google Scholar). Our aim was to gain insight into the role that LCAT plays in HDL metabolism as well as to better understand LCAT deficiency states. We have examined apoA-I-, -A-II-, -A-IV-, -C-I-, -C-III-, and -E-containing HDL subpopulation profiles in LCAT-deficient homozygotes and heterozygotes and in control subjects. The data we present indicate that LCAT plays a very significant role in HDL particle metabolism, composition, and remodeling. We examined plasma obtained from 11 homozygous LCAT-deficient subjects of Italian (n = 7), Japanese (n = 3), and US (n = 1) origin, as well as from 11 heterozygous LCAT-deficient subjects from Italy. Plasma obtained from gender-matched control subjects from the US (n = 15), Italy (n = 4), and Japan (n = 3) was used in this comparison. Homozygous and heterozygous subjects from Italy have been described previously (14Calabresi L. Pisciotta L. Costantin A. Frigerio I. Eberini I. Alessandrini P. Arca M. Bon G.B. Boscutti G. Busnach G. The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families. Arterioscler. Thromb. Vasc. Biol. 2005; 25 (et al.): 1972-1978Google Scholar). All homozygous subjects had primary hypoalphalipoproteinemia as defined by a plasma HDL-C level below the 5th percentile for the age- and gender-matched general populations of the specific countries. One homozygous subject from Japan had FED; however, none of the measured parameters of this subject were different by more than 1 SD from those of the other 10 homozygotes. Blood was collected from all subjects after an overnight fast and immediately placed on ice. Plasma was separated by low-speed centrifugation at 4°C and was stored at −°80 C until use. Samples were sent to the Lipid Metabolism Laboratory at Tufts University on dry ice and were thawed in a 37°C water bath for 1–2 min and then placed on ice just before use. Plasma total cholesterol, HDL-C, and triglyceride (TG) levels were determined using standard enzymatic techniques. Plasma concentrations of apoA-I, -A-II, and -B were determined by immunoturbidimetry. Plasma concentrations of apoA-IV, -C-I, -C-III, and -E were estimated by dot-blot analyses and expressed as arbitrary units. LCAT gene analyses, activity, and mass measurements were performed as described previously (14Calabresi L. Pisciotta L. Costantin A. Frigerio I. Eberini I. Alessandrini P. Arca M. Bon G.B. Boscutti G. Busnach G. The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families. Arterioscler. Thromb. Vasc. Biol. 2005; 25 (et al.): 1972-1978Google Scholar). HDL subpopulations were determined by nondenaturing two-dimensional PAGE, immunoblotting, and image analysis as described previously (15Asztalos B.F. Lefevre M. Foster T.A. Tulley R. Windhauser M. Wong L. Roheim P.S. Normolipidemic subjects with low HDL cholesterol levels have altered HDL subpopulations. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1885-1893Google Scholar). Four microliters of plasma was applied and electrophoresed on a vertical-slab agarose gel (0.7%) in the first dimension at 250 V until the α-mobility front moved 3.5 cm from the origin. The agarose gel was sliced, and the strips were applied onto 3–35% nondenaturing concave gradient polyacrylamide gels. In the second dimension, gels were electrophoresed to completion at 250 V for 24 h at 10°C, followed by electrotransfer to nitrocellulose membranes at 30 V for 24 h at 10°C. The specific apolipoproteins were immuno-localized on the membrane with mono-specific goat anti-human primary and 125I-labeled secondary antibodies immunopurified rabbit F(ab')2 fraction against goat IgG. The bound 125I-labeled secondary antibody was quantified in a FluoroImager (Molecular Dynamics). Each membrane was first probed for the apolipoprotein of primary interest and than reprobed for apoA-I for reference. Means and standard deviations were calculated for all study groups. Data obtained from homozygotes and heterozygotes were compared with data from controls using ANOVA analyses. A two-tailed P < 0.05 was considered as significant. 1 data on LCAT mass and activity as well as on and apolipoproteins in controls (n = heterozygotes (n = and homozygotes (n = 11) for LCAT Heterozygotes had of the LCAT activity and of the LCAT mass of had lower apoA-I apoA-II HDL-C and compared with had of the LCAT activity and of the LCAT mass of had lower HDL-C apoA-I apoA-II and and than of study (n = (n = (n = mass different < from activity different < from different < from cholesterol different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from apolipoprotein HDL-C, HDL Data are different < from in a apolipoprotein HDL-C, HDL Data are 1 and data on apoA-I-containing HDL subpopulations in controls and in heterozygous and homozygous LCAT-deficient subjects. Heterozygotes had particle in the preβ-1 however, all of other apoA-I-containing HDL subpopulations were to controls in and in heterozygotes was the HDL was a increase in preβ-1 a increase in and a increase in levels compared with were significant in the concentrations of all the other HDL particles, the of an was similar to that of In homozygotes, the majority of apoA-I was detected in small, lipid-poor, HDL particles and the low plasma concentrations of apoA-I in homozygous the apoA-I concentrations of particles were to those of We have also larger apoA-I-containing HDL particles with α-mobility in of the homozygotes. the of on apoA-I-containing and homozygous LCAT-deficient subjects. In control and HDL apoA-I and In some apoA-II was detected in the preβ-1 but the majority of apoA-II was in the and with a slight the particles, compared with In to controls, homozygotes had a very low level of which was detected in a small, with the preβ-1 HDL particles. or LCAT deficiency had no significant effect on the of apoA-IV or the of HDL particles 3 were no significant between heterozygotes and controls in apoC-I and In homozygotes had lower apoC-I and apoC-I was found on the of the gel with that apoC-I was present in particles, not in α-mobility HDL particles, as in controls and The concentrations and of apoC-III were different between LCAT-deficient subjects and controls In controls, the majority of apoC-III with apoA-I in α-1 and HDL, and some was also found in the and α-4 size with no with In all of the apoC-III was detected in small, HDL particles in homozygotes and ApoE-containing particles with in the size between and with a of in controls and no with apoA-I-containing HDL particles. In apoE was also found in large particles, with no with the size of apoE-containing particles was increased in heterozygotes compared with had apoE than concentrations in the larger particles and apoE-containing particles in the plasma of of HDL subpopulations as determined by apoA-I (n = (n = (n = different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from different < from are different < from in a HDL subpopulations of and homozygous LCAT-deficient subjects on the image of apoA-I-containing subpopulations. LCAT-deficient subjects have apoA-II in small, HDL particles. The the human the α-mobility image HDL subpopulations of and homozygous LCAT-deficient subjects on the image of apoA-I-containing subpopulations. or LCAT deficiency has no significant effect on the of HDL particles. The the human the α-mobility image HDL subpopulations of and homozygous LCAT-deficient subjects on the image of apoA-I-containing subpopulations. In homozygotes, apoC-I has only been detected on the of the gel with in to controls and The the human the α-mobility image HDL subpopulations of and homozygous LCAT-deficient subjects on the image of apoA-I-containing subpopulations. In controls, the majority of apoC-III with apoA-I in α-1 and HDL, and some has also been found in the and α-4 size with no with In homozygotes and all apoC-III has been detected in small, HDL particles. The the human the α-mobility image HDL subpopulations of and homozygous LCAT-deficient subjects on the image of apoA-I-containing subpopulations. is no of and apoA-I-containing particles. The the human the α-mobility image Data are The of this study was to gain insight into the role that LCAT plays in HDL metabolism as well as to better understand LCAT deficiency states. HDL particles in patients with of HDL metabolism has been in better HDL particle metabolism and reverse cholesterol transport. We have that disease patients 1) apoA-I only in the preβ-1 HDL particles, 2) no HDL, and 3) decreased size of apoE HDL. was not by the lack of cellular cholesterol efflux B.F. M.E. Horvath K.V. Roheim P.S. Schaefer E.J. of high density lipoproteins in homozygous and heterozygous disease. Scholar). We have reported that HDL subpopulations in homozygotes were very large, HDL particles B.F. Horvath K.V. C. M. Schaefer E.J. A. H. composition of HDL in cholesteryl ester transfer protein J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, CETP activity is essential for the formation of HDL particles in the normal size of HDL. CETP activity is essential for the formation of and HDL particles. In the present we the role of LCAT in HDL metabolism and in plasma. The first is that LCAT activity is not for the of preβ-1 HDL into α-mobility HDL. binds to and and unesterified cholesterol from cells (13Asztalos B.F. de la Llera-Moya M. Dallal G.E. Horvath K.V. Schaefer E.J. Rothblat G.H. Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux. J. Lipid Res. 2005; 46: 2246-2253Google Scholar). this are changes in apoA-I and We that α-4 HDL two of apoA-I, as is the for preβ-1 HDL. α-mobility HDL particles have also been in of the homozygotes. The of particles suggest that are poorly discoidal HDL We have no data whether LCAT can on large, or can only the small α-4 HDL as a The apoA-I-containing HDL subpopulation of heterozygotes that of low HDL-C CAD as apoA-I is the particles. is reduced in homozygous of fast catabolism D.J. N. H. Pritchard H. Frohlich J. M. L. catabolism of apolipoprotein and high density lipoproteins apoA-II in lecithin:cholesterol acyltransferase deficiency and disease. J. (et al.): it with preβ-1 which only apoA-II also with preβ-1 HDL in however, we not whether apoA-I and apoA-II are in the particles. a of the presence of cholesteryl ester in the core of HDL particles, apoA-II binds to and HDL particles very as indicated in heterozygotes are increased in particles. Our data also suggest that LCAT is not a in the formation of particles. the basis of and other findings B.F. M.E. Horvath K.V. Roheim P.S. Schaefer E.J. of high density lipoproteins in homozygous and heterozygous disease. Scholar, B.F. Horvath K.V. C. M. Schaefer E.J. A. H. composition of HDL in cholesteryl ester transfer protein J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google we that the metabolism of particles is of cellular cholesterol as well as of CETP and LCAT activities in The majority of apoC-I with apoA-I-containing α-1 HDL in of apoC-I in controls and of apoC-I in heterozygotes have α-mobility with larger than α-1 have apoC-I only in the that the lipid core is essential for the of apoC-I into HDL. has a in of apoC-III with with α-2, is found in and the is in the HDL size but not with apoA-I-containing particles. in both affected apoC-III has been detected in small, form that apoC-III is to the lipid and apolipoprotein composition of HDL. The large of free apoC-III in affected subjects also that the of this apolipoprotein is not increased with decreased particle which is not the for apoA-I and We that apoE-containing particles not with apoA-I-containing particles either in controls or in LCAT-deficient subjects in this have only apoE with apoA-I in homozygous subjects HDL size the size of LDL and the particles were with of cholesteryl ester B.F. Horvath K.V. C. M. Schaefer E.J. A. H. composition of HDL in cholesteryl ester transfer protein J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Although apoA-I concentrations were lower in the large particles in heterozygous LCAT-deficient apoE concentrations were increased in the large apoE HDL particles in subjects. We have no for this We not the composition of particles. In homozygous LCAT-deficient we only more apoE in particles. Therefore, LCAT activity not to be a in for apoE-containing HDL. to be for the formation of the core of apoE HDL in homozygotes. this is the as to this apoE HDL is and role in lipoprotein metabolism and CAD risk Our of HDL in in humans, in is on data in associated with in HDL metabolism LCAT, and CETP We are in the of HDL subpopulations in other as well apoE lipoprotein and the basis of we the steps in HDL is in the liver and small and two of apoA-I form a of to form discoidal preβ-1 HDL in the or plasma particles FC and more from cells the and are into small, lipid-poor, discoidal α-4 HDL particles. LCAT FC on the surface of HDL into cholesteryl which into the core with an increase in HDL particle in lipoprotein resulting in surface and and for HDL particle size The of LCAT, and increase HDL particle size exchange of core cholesteryl esters for between large HDL particles and and HDL particles apoA-I with or apoA-I or apoE CETP can also exchange cholesteryl esters for HDL particles as a a substantial of preβ-1 and and on HDL, resulting in size of large α-1 into HDL or of all larger HDL particles into α-4 and preβ-1 or preβ-1 and free apoA-I Cholesteryl esters on and α-1 HDL particles are from HDL particles to the liver SR-BI for of cholesterol into the resulting in of and FC from larger HDL particles to small α-4 HDL. The of and HDL particle size of small, apoA-I the in HDL particle metabolism is the uptake of HDL particles by the liver and of free apoA-I and preβ-1 HDL in the on the data are consistent with the that LCAT plays a role in the of HDL particles study was by from the of and Blood and from the Italian of University The are to M. S. G. G. G. G. L. G. I. G. and A. for the of the Italian LCAT-deficient families. apolipoprotein A-I coronary artery disease cholesteryl ester transfer protein free cholesterol fish eye disease LCAT deficiency HDL cholesterol scavenger receptor type B-I triglyceride