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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1746-1754.)
© 1995 American Heart Association, Inc.

Articles

Generation of Pre-ß₁-HDL and Conversion Into -HDL

Evidence for Disturbed HDL Conversion in Tangier Disease

Yadong Huang; Arnold von Eckardstein; Shili Wu; Claus Langer; Gerd Assmann

From the Institut für Arterioskleroseforschung an der Universität Münster (Y.H., S.W., G.A.) and the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster (A. von E., C.L., G.A.), Federal Republic of Germany.

Correspondence to Arnold von Eckardstein, Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster, Albert-Schweitzer-Str 33, D-48129 Münster, Federal Republic of Germany.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract HDL encompasses several apoA-I–containing particles that differ by size and show pre-ß- or -mobility on agarose gel electrophoresis: pre-ß₁-LpA-I, pre-ß₂-LpA-I, pre-ß₃-LpA-I, -LpA-I₂, and -LpA-I₃. The quantitatively minor subclass pre-ß₁-LpA-I serves as an initial acceptor of cell-derived cholesterol. In this study, we generated a pre-ß₁-LpA-I–like particle in vitro by the incubation of biotinylated apoA-I with cholesterol-loaded macrophages. Both native pre-ß₁-LpA-I and in vitro–generated pre-ß₁-LpA-I were indistinguishable from lipid-free apoA-I by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis but exhibited a different size upon gel filtration. In vitro–generated biotin–pre-ß₁-LpA-I took up twofold to threefold more [³H]cholesterol from labeled fibroblasts during a 1-minute pulse incubation than lipid-free apoA-I. The in vitro conversion of biotin–pre-ß₁-LpA-I was investigated in the presence of plasmas of healthy probands and patients with Tangier disease, with apoA-I deficiency, and with lecithin-cholesterol acyltransferase (LCAT) deficiency. Incubation of biotin–pre-ß₁-LpA-I with plasmas either from normoalphalipoproteinemic probands or from a patient with apoA-I deficiency generated a biotinylated particle with the size and electrophoretic mobility of -LpA-I₂. This conversion was sensitive to heating at 56°C but not to the removal of calcium. Inhibition of LCAT by dithiobisnitrobenzoic acid led to the formation of -LpA-I₃ instead of -LpA-I₂. Incubation of biotin–pre-ß₁-LpA-I with the plasma of an LCAT-deficient patient also led to the generation of biotin–-LpA-I₃ instead of -LpA-I_2. By contrast, incubation of biotin–pre-ß₁-LpA-I with plasma of patients with Tangier disease did not cause the disappearance of biotin–pre-ß₁-LpA-I and the formation of biotin–-LpA-I. However, co-incubation of Tangier disease plasma or of pre-ß₁-LpA-I isolated from Tangier disease plasma with apoA-I–deficient plasma generated -LpA-I₂. In conclusion, our data indicate that (1) pre-ß₁-LpA-I can be formed in vitro by the interaction of free apoA-I with cholesterol-loaded macrophages, (2) both normal and apoA-I–deficient plasmas contain a factor that converts pre-ß₁-LpA-I into -LpA-I, and (3) this factor is absent in the plasma of patients with Tangier disease.

Key Words: HDL subclasses • reverse cholesterol transport • apoA-I deficiency • familial LCAT deficiency • cholesterol efflux

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The HDLs encompass several particles that can be classified according to a variety of properties, including hydrated density,¹ apolipoprotein composition,² and charge.^{3 4} The putative ability of HDL to protect the vessel wall from atherosclerosis has usually been explained by the reverse cholesterol transport model, in which HDL mediates the flux of excess cholesterol from peripheral cells to the liver (reviewed in References 5 through 7^{5 6 7} ). In the extracellular compartment, this process is initiated by the uptake of cholesterol from cell membranes by two quantitatively minor subgroups of HDL that can be differentiated from the bulk of HDL on the basis of their apolipoprotein composition and electrophoretic mobility^{8 9 10 11} : Pre-ß₁-LpA-I contains apoA-I as the only apolipoprotein and exhibits pre-ß-mobility upon electrophoresis; -LpE contains apo E as the only apolipoprotein and exhibits -mobility.^{8 9 10} After uptake of cellular cholesterol by pre-ß₁-LpA-I, cell-derived cholesterol is rapidly transferred via other pre-ß-migrating particles (pre-ß₂-LpA-I and pre-ß₃-LpA-I) to the bulk of HDL, which has electrophoretic -mobility and is termed -LpA-I.^{9 11} -LpA-I can be differentiated into smaller -LpA-I₃ with a Stokes diameter of 7.2 to 8.8 nm and larger -LpA-I₂ with a Stokes diameter of 8.9 to 11.4 nm.

It is not fully understood how the various HDL subfractions are formed and interconverted. It has been reported that free apoA-I incubated with cholesterol-loaded macrophages gives rise to "HDL-like lipoprotein particles" that release cholesterol from cells.^{12 13} These particles resemble particles in lymph that are rich in phospholipids and unesterified cholesterol.^{14 15 16} Both "HDL-like lipoproteins" generated in vitro and HDL of the lymph have electrophoretic pre-ß-mobility.^{12 13 16} Pre-ß₁-LpA-I is also produced from HDL₂ by the action of HTGL and CETP^{17 18} as well as during lipolysis of triglyceride-rich lipoproteins.¹⁹ Moreover, it has been proposed that LCAT is involved in the interconversion of pre-ß₁-LpA-I into -LpA-I.^{19 20 21 22} The origin of the other HDL subfractions, ie, pre-ß₂-LpA-I, pre-ß₃-LpA-I, and -LpE, is unknown.

In this study, we used biotinylated apoA-I to investigate the effects of cells and plasmas on the generation and conversion of HDL subfractions. We also took advantage of the selective absence of distinct HDL subfractions from plasmas of patients with the various familial HDL-deficiency syndromes to demonstrate the specificity of the conversion processes. The plasma of apoA-I–deficient patients completely lacks apoA-I–containing lipoproteins but contains all the other apolipoproteins, enzymes, and lipids that are involved in HDL metabolism.^{11 23} Familial LCAT deficiency is accompanied by a lack of pre-ß₃-LpA-I and -LpA-I₂.¹¹ Plasmas of patients with Tangier disease lack all HDL subfractions except pre-ß₁-LpA-I.¹¹ Our data demonstrate that pre-ß₁-LpA-I can be formed in the extracellular compartment by the interaction of free apoA-I with cell membranes and also provide evidence for the presence in normal human plasma of a factor that converts pre-ß₁-LpA-I and lipid-free apoA-I into -LpA-I. This factor is absent in the plasma of patients with Tangier disease.

	Methods

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Subjects and Plasma Samples
Three normolipidemic probands, one patient with apoA-I deficiency, one patient with familial LCAT deficiency, and three patients with Tangier disease participated in this study. Characteristics of their lipid metabolism have been described previously.¹³ The 32-year-old Italian woman with apoA-I deficiency is homozygous for a nonsense mutation in codon 32 of the apoA-I gene.^{11 23} The patient with familial LCAT deficiency is homozygous for a ThrLeu substitution at residue 321 of LCAT.²⁴ The three patients with Tangier disease have been described previously.^{25 26} Blood samples were collected after overnight fasting and were cooled immediately on ice. Streptokinase was used as the anticoagulant at a final concentration of 150 U/mL blood. Plasmas were obtained by centrifugation at 4°C (2000g, 15 minutes), divided into aliquots, and stored at -70°C. In previous studies, we found that storage at -70°C affects neither the electrophoretic appearance of apoA-I–containing lipoproteins upon 2D-PAGGE nor the ability of normal plasma to take up and esterify cell-derived cholesterol.

Preparation of Lipoproteins and Apolipoproteins
HDLs (d=1.063 to 1.21 g/mL) were isolated from fresh normal human plasma by sequential isopycnic ultracentrifugation.¹ Ultracentrifugation was also used to isolate ß-VLDL from plasmas of hypercholesterolemic rabbits that were fed a high-fat diet for 4 weeks.²⁷ Human apoA-I was isolated as described previously.²⁸ Briefly, after delipidation with ethanol/ether (3:1), HDL apolipoproteins were dissolved in 50 mmol/L PBS (pH 7.4) containing 6 mol/L urea and then dialyzed against 50 mmol/L PBS (pH 7.4). ApoA-I was isolated from HDL by reversed-phase high-performance liquid chromatography.²⁸ For some experiments, apoA-I–containing lipoproteins were isolated from plasma of normoalphalipoproteinemic subjects and two individuals with Tangier disease by anti–apoA-I immunoaffinity chromatography.²⁹ The bound fraction was eluted and characterized by 2D-PAGGE (see below) or gel filtration (see below).

Nondenaturing Two-Dimensional Electrophoresis
The distribution of apoA-I–containing lipoproteins in cell culture medium and in plasma samples from healthy or HDL-deficient probands was analyzed by 2D-PAGGE in which agarose gel electrophoresis was followed by PAGGE.^{8 9} Briefly, in the first dimension, 30-µL plasma samples were separated by electrophoresis at 4°C in a 0.75% agarose gel using a 50 mmol/L merbital buffer (pH 8.7, Serva). Bromphenol blue was added to a standard sample to visualize albumin in the native gel. The electrophoresis was stopped when the albumin/bromphenol blue marker had migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins were then transferred to a 2% to 20% polyacrylamide gradient gel. Separation in the second dimension was performed at 40 mA for 4 to 5 hours at 10°C. During this time, the endogenous plasma albumin, which because of bromphenol blue added to the cathodic buffer (300 µL/L buffer) was visible in the native gel as a faint blue band, had migrated 10 cm. The proteins separated in the PAGGE gel were electroblotted onto a nitrocellulose membrane. ApoA-I–containing lipoproteins were detected by the use of sheep antibodies against human apoA-I (Boehringer Mannheim) that had been biotinylated according to the manufacturer's recommendations (Sigma) and streptavidin–biotinylated horseradish peroxidase complex (Amersham). In experiments with biotinylated apoA-I, streptavidin-biotinylated horseradish peroxidase complex was used directly instead of anti–apoA-I antibodies to detect lipoproteins that contained biotin–apoA-I. The lower detection limit of this visualization method was established by SDS-PAGE³⁰ and subsequent anti–apoA-I immunoblotting of a dilution series of plasma with known apoA-I concentration. It was <0.15 ng apoA-I. The apparent size of the HDL subclasses was determined by the comparison of their mobility with the mobility of protein standards with known Stokes diameter (Pharmacia).

Gel Filtration
Gel filtration was also used to separate by size lipid-free apoA-I, apoA-I–containing lipoproteins in cell culture medium, and plasma fractions obtained by anti–apoA-I immunoaffinity chromatography. A Hiload HR16/60 column filled with Sephacryl S100HR was run with the fast protein liquid chromatography system of Pharmacia. Lipid-free apoA-I (200 µg), 2 mL J774 macrophage medium with 200 µg apoA-I, or anti–apoA-I–immunoreactive plasma fractions containing 1350 µg protein were separated in 10 mmol/L phosphate buffer containing 150 mmol/L sodium chloride and 0.05% (wt/vol) sodium azide (pH 7.4). The flow was 0.1 mL/min. Fractions (2 mL) were collected. The apparent size of the fractions was determined by comparison of their elution times with those of protein standards with known Stokes diameters (Pharmacia). The fractions were then concentrated by factor of four through ultrafiltration using Centriprep-10 concentrators from Amicon and were subsequently analyzed by 2D-PAGGE.

Cell Culture
J774 macrophages and normal human skin fibroblasts were cultured in DMEM containing 10% fetal calf serum in dishes 3.5 cm in diameter as described previously.³¹ For the generation of pre-ß₁-LpA-I, J774 macrophages were preloaded with cholesterol by incubation with 100 µg rabbit ß-VLDL for 24 hours at 37°C. These conditions changed them microscopically into foam cells. For pulse-chase experiments, at the state of near confluence, fibroblasts of some dishes were labeled for 72 hours at 37°C with 0.5 mCi [1,2-³H]cholesterol (New England Nuclear, 51.7 Ci/mmol) that had been complexed with fetal calf serum. The final specific radioactivity in the labeled cells amounted to 1.5±0.6x10⁷ cpm/µg cell unesterified cholesterol (mean±SD).

In Vitro Generation of Pre-ß₁-LpA-I
To investigate the formation of pre-ß₁-LpA-I in vitro, several aliquots of 0.5 mL DMEM supplemented with 50 µg human apoA-I were incubated for 12 hours at 37°C with ß-VLDL cholesterol–loaded J774 macrophages. The apoA-I–conditioned media were centrifuged at 3000 rpm for 20 minutes at 4°C to remove cell debris. For control experiments, we used the medium of ß-VLDL cholesterol–loaded J774 macrophages, which were depleted of cell debris by centrifugation, for cell-free incubation under the same conditions. The apoA-I–containing media were used for the following experiments: First, 30 µL of the medium containing biotinylated apoA-I was separated by 2D-PAGGE (see above). Second, we applied 2 mL medium with 200 µg apoA-I to gel filtration (see above). Third, to determine the ability of in vitro–generated pre-ß₁-LpA-I to promote cholesterol efflux, 0.5 mL of the macrophage-conditioned apoA-I medium or 0.5 mL unconditioned medium with 50 µg human apoA-I was pulse-incubated with [³H]cholesterol–labeled fibroblasts for 1 minute as described previously.¹¹ Thereafter, the medium was separated by agarose gel electrophoresis under the conditions described above. After separation, each lane of the gel was cut into segments 0.5 cm long. Lipids were extracted from these slices by incubation three times with chloroform/methanol (2:1, vol/vol). Radioactivity was counted by scintillation spectrometry.

Generation of Biotin–Pre-ß₁-LpA-I
To follow the metabolic fate of pre-ß₁-LpA-I, we needed a labeled particle. Pre-ß₁-LpA-I was labeled by biotinylation of apoA-I before it was incubated with J774 macrophages. After determination of protein concentration by Lowry's method,³² some of the purified apoA-I was biotinylated by use of a biotinylation kit (Sigma) according to the manufacturer's recommendations. Both biotinylated and native apoA-I were characterized by SDS-PAGE.³⁰ To compare their ability to promote cholesterol efflux from cells, 50 µg/mL of either native or biotinylated apoA-I was incubated with [³H]cholesterol-labeled fibroblasts for various time intervals. After incubation, the radioactive cholesterol released into the medium was measured by liquid scintillation spectrometry. Biotin–pre-ß₁-LpA-I was generated in vitro by the same conditions as described above for the unbiotinylated particle. Electrophoretic properties and [³H]cholesterol efflux promotion by biotin–apoA-I and biotin–pre-ß₁-LpA-I were also analyzed as described above.

Conversion of Biotin–Pre-ß₁-LpA-I
To investigate the conversion of in vitro–generated pre-ß₁-LpA-I, 100 µL macrophage medium conditioned with biotin–apoA-I was incubated with 100 µL plasma of healthy probands for a further 12 hours at 37°C. Incubations were performed in the absence or presence of 2 mmol/L DTNB. After these incubations, the distribution of apoA-I–containing lipoproteins was analyzed by 2D-PAGGE, blotting, and detection with streptavidin–biotinylated horseradish peroxidase complex. Alternatively, 100 µL macrophage medium conditioned with unbiotinylated apoA-I was incubated with 100 µL plasma of the apoA-I–deficient patient for a further 12 hours at 37°C and separated by 2D-PAGGE. In this case, apoA-I–containing lipoproteins were visualized by the use of biotinylated anti–apoA-I antibodies and strepatividin–horseradish peroxidase.

Conversion of HDL Subfractions in HDL Deficiency
Conversion of pre-ß₁-LpA-I by HDL-deficient plasmas was analyzed by two methods: (1) Macrophage culture medium containing biotinylated pre-ß₁-LpA-I was incubated with plasmas of patients with apoA-I deficiency, LCAT deficiency, or Tangier disease and analyzed by 2D-PAGGE under the same conditions as described before for normal plasma; or (2) in other experiments, plasmas of patients with Tangier disease or LCAT deficiency as a source of pre-ß₁-LpA-I were incubated with the plasma of the apoA-I–deficient patient. In this case, anti–apoA-I immunoblotting was used for the detection of apoA-I–containing liproproteins.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Generation of Pre-ß₁-LpA-I In Vitro
Fig 1a presents the distribution of apoA-I–containing lipoproteins in normal plasma after separation by 2D-PAGGE. As described previously,^{8 9} apoA-I is immunodetected in a major HDL subfraction with -mobility (-LpA-I) as well as in three minor subfractions with pre-ß-mobility that differ from one another by their size (pre-ß₁-LpA-I, pre-ß₂-LpA-I, and pre-ß₃-LpA-I). Incubation of human apoA-I with cholesterol-loaded J774 macrophages for 12 hours at 37°C generated an apoA-I–containing particle that upon 2D-PAGGE had electrophoretic pre-ß-mobility and a diameter of 6.8 to 7.6 nm (Fig 1b). This particle, however, was electrophoretically indistinguishable from lipid-free apoA-I (Fig 1c).

View larger version (66K): [in this window] [in a new window]   Figure 1. Electrophoretic comparison of native and in vitro–generated apoA-I–containing lipoproteins and lipid-free apoA-I. ApoA-I–containing lipoproteins after separation by 2D-PAGGE in the sequence agarose gel electrophoresis, nondenaturing polyacrylamide gradient gel electrophoresis, and subsequent anti–apoA-I immunoblotting. a, Distribution of apoA-I–containing lipoproteins in normal human plasma; b, appearance of apoA-I–containing lipoproteins after 12 hours' incubation of 50 µg/mL free human apoA-I with cholesterol-loaded J774 macrophages; and c, 2D electropherogram of unconditioned, lipid-free apoA-I. Note that the in vitro–generated pre-ß1-LpA-I in b is electrophoretically indistinguishable from native pre-ß1-LpA-I in normal plasma (a) and lipid-free apoA-I (c).

Gel filtration of anti–apoA-I–immunoreactive plasma lipoproteins separated two fractions (Fig 2B). The quantitatively major fraction, with an apparent Stokes diameter of 7.2 to 9.2 nm, exhibited electrophoretic -mobility upon 2D-PAGGE, and the quantitatively minor fraction, with an apparent Stokes diameter of 6.0 to 7.0 nm, exhibited the mobility of pre-ß₁-LpA-I. J774 macrophage–conditioned apoA-I was eluted in a single fraction that exhibited an apparent Stokes diameter of 5.9 to 6.9 nm (Fig 2C). Lipid-free, unconditioned apoA-I was eluted in a fraction with an apparent molecular Stokes diameter of 4.4 to 5.3 nm (Fig 2D). After a 12-hour-long cell-free incubation of apoA-I with the medium of cholesterol-loaded macrophages, gel filtration revealed the presence of a protein with the size of lipid-free apoA-I (Fig 2D). If the medium was not depleted of cell debris by centrifugation, 50% of the apoA-I was converted into a particle with an apparent Stokes diameter of 5.9 to 6.9 nm (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2. Graphs showing gel filtration of apoA-I–containing plasma lipoproteins, in vitro–generated pre-ß1-LpA-I, and lipid-free apoA-I. Sephacryl S100HR was used for separation. A, Separation of albumin (molecular mass, 67 kD; Stokes diameter, 7.1 nm), ovalbumin (43 kD, 6.1 nm), chymotrypsinogen A (25 kD, 4.2 nm), and ribonuclease A (13.7 kD, 3.3 nm). The "void" fraction contains dextran blue (2000 kD). B, Separation of 1350 µg anti–apoA-I–immunoreactive lipoproteins that were previously isolated from plasma by anti–apoA-I–immunoaffinity chromatography. C, Separation of 200 µg apoA-I that was previously incubated with J774 macrophages for 12 hours. D, Elution profile of unconditioned, lipid-free apoA-I. Essentially the same chromatogram depicted in D was obtained if apoA-I was separated that had been preincubated for 12 hours with the cell-free medium of cholesterol-loaded macrophages. Note the identical sizes of native (fraction II in B) and in vitro–generated pre-ß1-LpA-I (fraction II in C) but different size of lipid-free apoA-I (fraction III in D).

Next we used lipid-free apoA-I, apoA-I preincubated with cholesterol-loaded J774 macrophages, or apoA-I preincubated with J774 macrophage medium for pulse incubations with [³H]cholesterol-labeled fibroblasts. After 1 minute of incubation with radiolabeled cells, the media were separated by agarose gel electrophoresis. In all cases, radioactivity was found in a particle with pre-ß-mobility (Fig 3). However, apoA-I preincubated with cholesterol-loaded macrophages took up twofold to threefold more radioactivity (Fig 3a) than unconditioned apoA-I (Fig 3b) or apoA-I that was preincubated with the medium of macrophages but in the absence of cells (not shown). Taking together the data on the electrophoretic, chromatographic, and cholesterol efflux–stimulating properties, incubation of apoA-I with J774 macrophages thus appears to generate in vitro a pre-ß₁-LpA-I–like particle.

View larger version (20K): [in this window] [in a new window]   Figure 3. Graphs showing uptake of cell-derived [3H]cholesterol by in vitro–generated pre-ß1-LpA-I and lipid-free apoA-I. DMEM (0.5 mL) containing 50 µg apoA-I that was either preincubated for 12 hours with cholesterol-loaded J774 macrophages (a) or remained unconditioned (b) was incubated with [3H]cholesterol-labeled fibroblasts for 1 minute. After incubation, the samples were separated by agarose gel electrophoresis. Every lane of the gel was cut into segments 0.5 cm long. Lipids in these slices were extracted by chloroform/methanol (2:1, vol/vol), and the radioactivity was determined by liquid scintillation spectrometry. Note that conditioning of apoA-I with macrophages (a) increases its ability to take up [3H]cholesterol from labeled fibroblasts severalfold compared with unconditioned apoA-I (b).

To study the metabolic fate of pre-ß₁-LpA-I, apoA-I was biotinylated and incubated with cholesterol-loaded J774 macrophages. Fig 4 shows, for comparison, electrophoretic and cholesterol efflux–stimulating properties of biotinylated and native apoA-I. After biotinylation, the labeled apoA-I was indistinguishable from native apoA-I on the basis of its mobility on SDS-PAGE (Fig 4a) and its ability to promote cholesterol efflux from [³H]cholesterol-labeled fibroblasts (Fig 4b). Upon 2D-PAGGE, both biotin–apoA-I incubated for 12 hours with cholesterol-loaded J774 macrophages and unconditioned biotin–apoA-I were indistinguishable from native pre-ß₁-LpA-I, native apoA-I, and in vitro–generated pre-ß₁-LpA-I (Fig 5a and 5b). Like macrophage-conditioned native apoA-I, conditioned biotin–apoA-I was twofold to threefold more active than unconditioned biotin–apoA-I in taking up [³H]cholesterol from labeled fibroblasts during a 1-minute incubation (Fig 6). Thus, preincubation of biotin–apoA-I with J774 macrophages also appears to form a particle that accepts cell-derived cholesterol and that we call biotin–pre-ß₁-LpA-I.

View larger version (28K): [in this window] [in a new window]   Figure 4. Comparison of biotinylated apoA-I and native apoA-I. a, SDS-PAGE of biotinylated apoA-I (1), native apoA-I (2), and low-molecular-weight standard (3). b, Time-dependent efflux of [3H]cholesterol from fibroblasts into medium containing 50 mg/mL of either native () or biotinylated () apoA-I.

View larger version (90K): [in this window] [in a new window]   Figure 5. 2D-PAGGE of biotin–pre-ß1-LpA-I and biotin–apoA-I. Biotinylated human apoA-I was incubated with cholesterol-loaded J774 macrophages for 12 hours at 37°C at a concentration of 100 µg/mL medium. After incubation, 30 µL medium was separated by 2D-PAGGE (a). As a control, 30 µL unconditioned DMEM with 3 µg biotin–apoA-I was also separated by 2D-PAGGE (b). After electroblotting onto nitrocellulose membranes, biotin–apoA-I–containing lipoproteins were detected by streptavidin–biotinylated horseradish peroxidase complex (a). Note that both conditioned (ie, biotin–pre-ß1-LpA-I) and unconditioned biotin–apoA-I exhibit electrophoretic properties indistinguishable from those of pre-ß1-LpA-I in native plasma, in vitro–generated pre-ß1-LpA-I, and lipid-free apoA-I (see Fig 1).

View larger version (21K): [in this window] [in a new window]   Figure 6. Graphs showing uptake of cell-derived [3H]cholesterol by in vitro–generated biotin–pre-ß1-LpA-I and lipid-free biotin–apoA-I. DMEM (0.5 mL) containing 50 µg biotinylated apoA-I that either was preincubated for 12 hours with cholesterol-loaded J774 macrophages (a) or remained unconditioned (b) was incubated with [3H]cholesterol-labeled fibroblasts for 1 minute. After incubation, the samples were separated by agarose gel electrophoresis. Every lane of the gel was cut into segments 0.5 cm long. Lipids in these slices were extracted by chloroform/methanol (2:1, vol/vol), and the radioactivity was determined by liquid scintillation spectrometry. Note that conditioning of biotin–apoA-I with macrophages (a) increases its ability to take up [3H]cholesterol from labeled fibroblasts severalfold compared with unconditioned apoA-I (b) (see also Fig 2).

Conversion of Pre-ß₁-LpA-I Into -LpA-I
To analyze whether pre-ß₁-LpA-I can be converted into other apoA-I–containing particles, we incubated the biotin–pre-ß₁-LpA-I–containing macrophage medium with normal plasma for 12 hours at 37°C. Subsequent 2D-PAGGE and Western blotting identified a biotinylated particle with -mobility and a diameter of 8.8 to 10.9 nm that resembled native -LpA-I₂ (Fig 7a). Heating of the plasma at 56°C for 1 hour prior to its incubation with biotin–pre-ß₁-LpA-I completely prevented the formation of biotin–-LpA-I (Fig 7b), whereas heating of normal plasma did not affect the electrophoretic appearance of its endogenous apoA-I–containing lipoproteins (Fig 7c). The conversion of biotin–pre-ß₁-LpA-I into biotin–-LpA-I was not sensitive to the removal of calcium by the addition of 5 mmol/L EDTA (data not shown). Twelve hours of incubation of unconditioned biotin–apoA-I with normal plasma also caused the heat-sensitive formation of biotin–-LpA-I (data not shown).

View larger version (61K): [in this window] [in a new window]   Figure 7. Effects of normal plasma on the conversion of biotin–pre-ß1-LpA-I. Medium containing biotin–pre-ß1-LpA-I was incubated with native (a) or heated (b) (56°C, 1 hour) human plasma (1:1, vol/vol) for 12 hours at 37°C. After 2D-PAGGE and subsequent electroblotting onto a nitrocellulose membrane, biotin–apoA-I–containing lipoproteins were detected by streptavidin–biotinylated horseradish peroxidase complex. c, Anti–apoA-I immunoblot of heated human plasma separated by 2D-PAGGE. Note that normal plasma converts biotin–pre-ß1-LpA-I into biotin–-LpA-I2 (a). Heating of normal plasma prevents the conversion of biotin–pre-ß1-LpA-I into biotin–-LpA-I (b). Heating, however, does not affect the electrophoretic appearance of endogenous apoA-I–containing lipoproteins of plasma (c).

To investigate whether biotinylated apoA-I exchanged between pre-ß₁-LpA-I generated in vitro and endogenous -LpA-I, we incubated biotin–pre-ß₁-LpA-I–containing macrophage medium with apoA-I–deficient plasma. ApoA-I–containing lipoproteins were undetectable in this sample (Fig 8a). However, apoA-I–deficient plasma converted biotin–pre-ß₁-LpA-I into biotin–-LpA-I₂ (Fig 8b). Conversion was abolished by heating (Fig 8c) but not by removal of calcium (data not shown). These results strongly suggest the presence of a heat-sensitive factor present in normal and apoA-I–deficient plasma that converts pre-ß₁-LpA-I and apoA-I into -LpA-I.

View larger version (61K): [in this window] [in a new window]   Figure 8. Effects of apoA-I–deficient plasma on the conversion of biotin–pre-ß1-LpA-I. a, Anti–apoA-I immunoblot after 2D-PAGGE of apoA-I–deficient plasma. Medium containing biotin–pre-ß1-LpA-I (see Fig 4b) was incubated with native (b) or heated (c) apoA-I–deficient plasma (1:1, vol/vol) for 12 hours at 37°C. The mixtures were then separated by 2D-PAGGE, and the proteins were electroblotted onto nitrocellulose membranes. Biotin–apoA-I–containing lipoproteins were detected by streptavidin–biotinylated horseradish peroxidase.

Effects of LCAT on the Conversion of Pre-ß₁-LpA-I
To test whether LCAT is involved in the conversion of pre-ß₁-LpA-I into -LpA-I, biotin–pre-ß₁-LpA-I–containing macrophage medium was incubated with plasma in the presence of the LCAT inhibitor DTNB. 2D-PAGGE and Western blotting led to the detection of a biotinylated particle with -mobility and a diameter of 7.5 to 8.6 nm (Fig 9a) that resembles -LpA-I₃. Moreover, under this condition, a higher proportion of biotin–pre-ß₁-LpA-I remained unconverted compared with the experiment without LCAT inhibitor (see Fig 7a). Similarly, incubation of biotin–pre-ß₁-LpA-I–containing macrophage medium with LCAT-deficient plasma also led to the formation of biotin–-LpA-I₃ instead of biotin–-LpA-I₂ (Fig 9b). Since pre-ß₁-LpA-I and -LpA-I₃ were present, the distribution of apoA-I–containing lipoproteins resembled that of LCAT-deficient plasma (Fig 9c). Incubation of LCAT-deficient plasma with apoA-I–deficient plasma (as a source of LCAT) generated -LpA-I₂ (Fig 9d). These results suggest that the conversion of pre-ß₁-LpA-I into -LpA-I₃ is at least partially independent of LCAT. LCAT, however, is mainly responsible for converting -LpA-I₃ into -LpA-I₂.

View larger version (46K): [in this window] [in a new window]   Figure 9. Effects of LCAT on the conversion of pre-ß1-LpA-I. Medium containing biotin–pre-ß1-LpA-I was incubated with apoA-I–deficient plasma for 12 hours at 37°C in the presence of 2 mmol/L DTNB (a). This led to the formation of a smaller biotin–-LpA-I3 particle compared with the biotin–-LpA-I2 particle generated by apoA-I deficient plasma in the absence of DTNB (see Fig 8b). Moreover, a great proportion of biotin–pre-ß1-LpA-I was not converted. By the occurrence of smaller -LpA-I3, the 2D-electrophoretic pattern resembles the distribution of LpA-I in plasma of a patient with familial LCAT deficiency (b). Incubation of biotin–pre-ß1-LpA-I with LCAT-deficient plasma leads to the formation of small amounts of biotin–-LpA-I3 (c). d, LCAT-deficient plasma and apoA-I–deficient plasma were mixed at a ratio of 1:1 and incubated for 12 hours at 37°C. The mixture was then separated by 2D-PAGGE. Proteins were electroblotted onto nitrocellulose membranes, and apoA-I–containing lipoproteins were immunodetected with a biotinylated anti–apoA-I antibody and streptavidin–biotinylated horseradish peroxidase complex. Note the formation of -LpA-I2.

Conversion of Pre-ß₁-LpA-I Into -LpA-I in Tangier Disease Plasma
Incubation of biotin–pre-ß₁-LpA-I–containing macrophage medium with Tangier disease plasma did not lead to a decrease of biotin–pre-ß₁-LpA-I with generation of biotin–-LpA-I (Fig 10). Since pre-ß₁-LpA-I was present but pre-ß₂-LpA-I, pre-ß₃-LpA-I, and -LpA-I were absent, apoA-I–conditioned macrophage medium resembled the plasma of patients with Tangier disease (Fig 11b and 11d). After 12 hours' incubation of Tangier disease plasma at 37°C, the electrophoretic appearance of pre-ß₁-LpA-I did not change, whereas incubation of normal plasma led to the disappearance of pre-ß₁-LpA-I (Fig 11a and 11c). These observations indicate that the conversion of pre-ß₁-LpA-I occurring in normal plasma is disturbed in the plasma of patients with Tangier disease. To confirm that deficiency of a converting activity was the basis of -LpA-I deficiency in Tangier disease, Tangier disease plasma was incubated with apoA-I–deficient plasma. ApoA-I–deficient plasma converted pre-ß₁-LpA-I of Tangier disease plasma into -LpA-I₂ (Fig 11e). Incubation of apoA-I–deficient plasma with pre-ß₁-LpA-I isolated from Tangier disease plasmas by anti–apoA-I immunoaffinity chromatography also formed -LpA-I₂ (Fig 12). As with the conversion of pre-ß₁-LpA-I generated in vitro, this process was sensitive to heating at 56°C but insensitive to the removal of calcium by EDTA (data not shown). Inhibition of LCAT by DTNB led to the formation of small amounts of -LpA-I₃ (Fig 11f). Under this condition, however, a large proportion of pre-ß₁-LpA-I remained unconverted. These data demonstrate that LCAT is necessary but not sufficient for the conversion of pre-ß₁-LpA-I into -LpA-I.

View larger version (82K): [in this window] [in a new window]   Figure 10. Effect of Tangier disease plasma on the conversion of biotin–pre-ß1-LpA-I. Medium containing biotin–pre-ß1-LpA-I was incubated with plasma of a patient with Tangier disease at a ratio of 1:1 (vol/vol) for 12 hours at 37°C. The mixture was then separated by 2D-PAGGE. After 2D-PAGGE and subsequent electroblotting onto a nitrocellulose membrane, biotin–apoA-I–containing lipoproteins were detected by streptavidin–biotinylated horseradish peroxidase complex. Note that Tangier disease plasma does not convert biotin–pre-ß1-LpA-I into biotin–-LpA-I as does normal or apoA-I–deficient plasma (see Figs 7a and 8b).

View larger version (49K): [in this window] [in a new window]   Figure 11. Distribution and generation of LpA-I in Tangier disease plasma. The figure shows the distribution of apoA-I–containing lipoproteins in various plasmas under different conditions as obtained by 2D-PAGGE and subsequent anti–apoA-I immunoblotting (see Fig 1a; for details, see "Methods"). a and b, 2D-electrophoretic distribution of LpA-I in the plasma of healthy control subjects and Tangier disease patients, respectively. Note the exclusive presence of pre-ß1-LpA-I in Tangier disease plasma. Twelve hours' incubation at 37°C leads to the disappearance of pre-ß1-LpA-I in normal plasma (c) but not in Tangier disease plasma (d). Coincubation of Tangier disease plasma with apoA-I–deficient plasma generates -LpA-I2 (arrow) if LCAT is not inhibited (e) but -LpA-I3 (arrow) if LCAT is inhibited by DTNB (f). In this case, less pre-ß1-LpA-I disappears.

View larger version (104K): [in this window] [in a new window]   Figure 12. Conversion of pre-ß1-LpA-I from Tangier disease. Pre-ß1-LpA-I was isolated from 2.5 mL Tangier disease plasma by anti–apoA-I immunoaffinity chromatography. ApoA-I–containing lipoproteins were eluted, dialyzed, and concentrated to 100 µL. Of this specimen, 50 µL was diluted with 50 µL PBS (a); another 50-µL aliquot was incubated with 50 µL plasma of an apoA-I–deficient patient for 12 hours' incubation at 37°C (b). Forty microliters of both mixtures was then separated by 2D-PAGGE. After 2D-PAGGE and subsequent electroblotting onto a nitrocellulose membrane, apoA-I–containing lipoproteins were detected with biotinylated IgG of an anti–apoA-I antiserum and streptavidin–biotinylated horseradish peroxidase complex. Note that before incubation with apoA-I–deficient plasma, anti–apoA-I immunoreactive lipoproteins in Tangier disease plasma exhibit electrophoretic pre-ß-mobility (a). Incubation with apoA-I–deficient plasma converts Tangier disease pre-ß1-LpA-I into -LpA-I (b).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
It is generally thought that mature HDLs are formed by HDL precursors in the plasma compartment. These include nascent HDL of hepatic and intestinal origin as well as particles that are generated (1) by the interaction of free apolipoproteins with phospholipids from cell membranes and (2) from the surface components of triglyceride-rich particles during lipolysis.^{2 12 13 14 15 16 33 34 35 36 37 38 39} Hara and Yokoyama^{12 13} found that incubation of lipid-free apoA-I with macrophages generates a particle with pre-ß-mobility that releases cholesterol from cells. In our studies, both lipid-free apoA-I and apoA-I preincubated with cholesterol-loaded macrophages exhibited electrophoretic properties that were indistinguishable from those of pre-ß₁-LpA-I (Figs 1 and 5). However, gel filtration differentiates lipid-free apoA-I and pre-ß₁-LpA-I (Fig 2b and 2d) and revealed that incubation of apoA-I with J774 macrophages generates a particle with the size of pre-ß₁-LpA-I (Fig 2c). Moreover, preincubation of apoA-I with J774 macrophages greatly increased the cholesterol efflux–stimulating properties of apoA-I (Figs 3 and 6). Both the increase in size and enhanced cholesterol efflux stimulation are presumably due to the binding of cellular phospholipids to apoA-I. Together with the finding that large proportions of HDL in the lymph also have pre-ß-mobility,¹⁶ it thus appears that pre-ß₁-LpA-I is an HDL precursor in plasma.

It is not known how HDL precursors are converted into mature HDL particles. In previous studies, culture media of hepatocytes or liver perfusates, which both contain HDL precursors of different shapes, sizes, and apolipoprotein compositions, were incubated with LCAT. This led to the formation of spherical HDL of a different size, so that LCAT has generally been considered to be responsible for the conversion and maturation of HDL.^{40 41 42} Several authors have also implicated LCAT as the factor that causes the disappearance of pre-ß₁-LpA-I during storage of plasma at 37°C.^{19 20 21 22} In this study, we demonstrate that pre-ß₁-LpA-I can be converted into an -migrating lipoprotein by heat-sensitive factors present in both normal and apoA-I–deficient plasma but missing in plasmas of patients with Tangier disease (Figs 7, 8, 10, and 11). Also, lipid-free apoA-I was converted into -LpA-I. Our data, however, do not rule out that association of lipid-free apoA-I with phospholipids from plasma lipoproteins first generates pre-ß₁-LpA-I, which is then converted into -LpA-I. The conversion of pre-ß₁-LpA-I and apoA-I into -LpA-I is not fully explained by LCAT, since inhibition of LCAT by DTNB prevents the formation of -LpA-I₂ but not of -LpA-I₃ and since both pre-ß₁-LpA-I and small -LpA-I (ie, -LpA-I₃) are present in the plasma of LCAT-deficient patients (Fig 9). Similarly, other investigators found that a subgroup of small and discoidal HDL precursors secreted by HepG2 cells can be maturated to larger, spherical particles independently of LCAT.³⁹

Previous work has shown that HTGL and CETP are not involved in the conversion of pre-ß₁-LpA-I, since these enzymes produce rather than remove pre-ß₁-LpA-I.^{17 43 44} It thus appears that an as yet unidentified plasma factor contributes to the conversion of pre-ß₁-LpA-I and apoA-I into -LpA-I. Heat sensitivity of this process may indicate that this factor is a protein but does not exclude a nonprotein factor, since, for example, some phospholipids also undergo gross structural changes when heated. Thus, Davidson and colleagues⁴⁵ reported that the charge difference between -LpA-I and pre-ß-LpA-I is determined by the negative charge of phosphatidylinositol molecules, which are present more on -LpA-I than on pre-ß₁-LpA-I. The effects of different phospholipids on the conversion of pre-ß₁-LpA-I are currently being investigated in our laboratory.

Since pre-ß₁-LpA-I not only accepts cell-derived cholesterol but also transfers it to other apoA-I–containing lipoproteins, the conversion of pre-ß₁-LpA-I provides a mechanism directing cholesterol away from cells. Previous pulse-chase experiments indicated that cell-derived cholesterol is not transferred directly to -LpA-I but rather moves via pre-ß₂-LpA-I and pre-ß₃-LpA-I.^{8 9} In our in vitro system, however, we did not detect these two intermediate particles. Since the plasma concentrations of pre-ß₂-LpA-I and pre-ß₃-LpA-I are considerably lower than that of pre-ß₁-LpA-I, their concentrations might have been below the limits of detection. Alternatively, despite the precursor-product relationships in the kinetics of [³H]cholesterol transfer between pre-ß₁-LpA-I, pre-ß₂-LpA-I, pre-ß₃-LpA-I, and -LpA-I, it cannot be excluded that pre-ß₂-LpA-I or pre-ß₃-LpA-I takes up cholesterol from sources other than pre-ß₁-LpA-I. Indirect evidence excluding at least pre-ß₃-LpA-I as an intermediate product in the pre-ß₁-LpA-I- LpA-I conversion is the lack of this particle in the plasma of patients with familial LCAT deficiency or fish-eye disease, although each plasma contains both pre-ß₁-LpA-I and -LpA-I₃ (Reference 11¹¹ and Fig 9b).

Pre-ß₁-LpA-I is the only apoA-I–containing HDL particle in the plasma of patients with Tangier disease. Like both pre-ß₁-LpA-I in normal plasma and in vitro–generated pre-ß₁-LpA-I, it serves as an initial acceptor of cell-derived cholesterol.¹¹ Unlike the pre-ß₁-LpA-I of normal plasma, pre-ß₁-LpA-I of Tangier disease plasma does not disappear during storage of Tangier plasma at 37°C. However, pre-ß₁-LpA-I in Tangier plasma is converted into -LpA-I when incubated with apoA-I–deficient plasma. This indicates that a precursor-product relationship exists between pre-ß₁-LpA-I and -LpA-I and that this pathway is interrupted in Tangier disease.

On the basis of previous studies, it was concluded that Tangier disease is due to defective intracellular trafficking of lipids, specifically in macrophages and Schwann cells, rather than to a defect in plasma lipid transfer.^{46 47} In particular, experiments with monocyte-derived macrophages of patients with Tangier disease suggested that HDLs are taken up and erroneously degraded in lysosomes²⁵ and that the synthesis and catabolism of lipids, especially phospholipids, are disturbed.^{26 48} The findings of this study suggest that models of the pathogenesis of Tangier disease must take into account a deficiency in plasma of a factor converting pre-ß₁-LpA-I to -LpA-I. This factor may act in both the intracellular and extracellular compartments or may be absent from the plasma compartment because of the dysregulated metabolism of Tangier disease cells.

In conclusion, our data provide the first evidence for the presence of a factor in human plasma that, together with LCAT, converts pre-ß₁-LpA-I and apoA-I into -LpA-I and that is inactive or absent in the plasma of patients with Tangier disease. This factor does not correspond to CETP or HTGL.

	Selected Abbreviations and Acronyms

 

2D-PAGGE = two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis apoA-I = apolipoprotein A-I CETP = cholesterol ester transfer protein DMEM = Dulbecco's modified Eagle's medium DTNB = dithiobisnitrobenzoic acid HTGL = hepatic lipase LCAT = lecithin-cholesterol acyltransferase LpA-I = lipoprotein A-I

	Acknowledgments

 
This project was supported by grants from the Deutsche Forschungsgemeinschaft (Ec 116, 2-1; Ec 116, 3-1) to Dr von Eckardstein. We are indebted to Prof G. Noseda, Mendrisiso, Switzerland, for providing us with plasma samples from the apoA-I–deficient patient and to Dr Paul Cullen for critically reading our manuscript.

Received February 12, 1995; accepted July 10, 1995.

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