| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Lipid Research, Vol. 47, 1833-1843, August 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
* Department of Clinical Biochemistry, Rigshospital, University of Copenhagen, Copenhagen DK-2100, Denmark
Department of Laboratory Medicine, Division of Clinical Chemistry, University of Lund, University Hospital, SE-20502 Malmö, Sweden
Published, JLR Papers in Press, May 8, 2006.
1 To whom correspondence should be addressed. e-mail: larsbo{at}rh.dk (L.B.N.); bjorn.dahlback{at}med.lu.se (B.D.)
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDLapoM+) contained significantly more free cholesterol than HDL lacking apoM (HDLapoM–) (5.9 ± 0.7% vs. 3.2 ± 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDLapoM+ inhibited Cu2+-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDLapoM–. In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.
Abbreviations: apoM, apolipoprotein M; HRP, haptoglobin-related protein; PON1, paraoxonase 1
Supplementary key words cholesterol efflux • oxidation • apoM
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Efflux of cholesterol from macrophages protects the cells from lipid toxicities and attenuates foam cell formation. Cholesterol and phospholipid efflux can be mediated by lipid-free apolipoprotein A-I (apoA-I) or lipid poor preß-HDL particles in a process that involves the binding of apoA-I to the membrane transporter protein ABCA1 (5, 6). Efflux of cholesterol to spherical HDL particles involves ABCG1 and ABCG4 (7) but also may occur by free diffusion (8). In addition to apoA-I, several other HDL-associated proteins have been reported to affect cholesterol efflux: PON1 enhances HDL-induced cholesterol efflux by increasing the binding affinity of HDL to macrophages and by stimulating ABCA1-mediated cholesterol transport (9), and apoJ, which is associated with lipid-poor HDL, stimulates reverse cholesterol transport by an unknown mechanism (10).
Recently, a novel apolipoprotein, apoM, was described (11). ApoM is predominantly present in HDL and to lesser extent in chylomicrons, VLDL, and LDL. ApoM belongs to the lipocalin family, the members of which share a common tertiary structure of an eight stranded antiparallel ß-barrel that forms a hydrophobic ligand binding pocket (12). The lipocalin protein family comprises many proteins with very diverse functions, some of which are antioxidants (13). However, for many lipocalins, including apoM, the endogenous ligand(s) and biological function have not been identified.
Like PON1, apoM lacks a signal peptidase cleaving site (11). The retained signal peptide may serve as a lipid anchor for the protein necessary for its association with lipoprotein particles (11). In addition to apoM and PON1, the haptoglobin-related protein (HRP) shares this unusual lipid binding property (14). ApoM has been suggested to play an important role in reverse cholesterol transport (15). Wolfrum, Poy, and Stoffel (15) demonstrated that mice made deficient in apoM using small, interfering RNA had large HDL particles that do not stimulate cholesterol efflux from macrophages to the same extent as HDL from wild-type mice. Moreover, LDL receptor-deficient mice treated with a murine apoM adenovirus construct to increase the apoM expression level developed less extensive atherosclerotic plaques in aorta than LDL receptor-deficient mice treated with a control adenovirus, suggesting that apoM protects against atherosclerosis.
To increase the knowledge of normal human apoM particles, and to better understand the role of apoM in human cholesterol metabolism, we have established a method for purifying apoM-containing lipoproteins from human plasma and explored the protein and lipid composition, size, density, and charge of apoM-containing lipoproteins. Also, we have investigated whether the apoM-containing particles inhibit the oxidation of lipoproteins and stimulate cholesterol efflux from macrophage-derived foam cells.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
100 mM NaCl) and the second apoA-I/apoM positive peak eluting at 300 mM NaCl. The peaks were pooled separately and subjected to immunoaffinity chromatography using a monoclonal antibody against apoM (see below). The apoM-containing fractions were dialyzed against 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5, before immunoaffinity chromatography. HDL (1.063 < d < 1.21 g/l) and LDL (1.019 < d < 1.063 g/l) were isolated by ultracentrifugation of a pool of plasma from 15 normal individuals with a Beckman Ti 50.3 rotor and a Beckman Optima LE-80K ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA). The density was adjusted with NaBr. Ultracentrifugation was performed at 50,000 rpm at 4°C for 16 h (d = 1.019 and 1.063 g/l) or 24 h (d = 1.21 g/l). The purified LDL and HDL were dialyzed extensively against PBS with Na2EDTA (0.1 mg/ml) at 4°C and stored at –80°C.
Anti-apoM and anti-apoA-I immunoaffinity chromatography
Polyclonal and monoclonal antibodies were raised against truncated (residues 22–188) recombinant human apoM expressed in Escherichia coli using the pET-30 Xa/Lic vector (Novagen) as described previously (16) with standard techniques. A monoclonal antibody against human apoA-I (M12) was obtained after immunization of mice with proteins extracted from isolated chylomicrons. The specificity of the M12 antibody was ascertained with apoA-I Western blotting and from its usefulness for the purification of apoA-I-containing HDL from human plasma. Monoclonal antibodies against apoM (M23) or apoA-I (M12) were coupled to 5 ml HiTrap N-hydroxy-succinimide (NHS)-activated columns (Amersham Biosciences) at 10 mg/ml gel, according to the manufacturer's instructions.
The apoA-I/apoM-containing lipoproteins obtained from either the ion-exchange chromatography or the ultracentrifugation were applied first to the M23 anti-apoM column to isolate apoM-containing lipoproteins. The column was washed with TBS followed by TBS with 0.5 mol/l NaCl, and bound particles were eluted with glycine (0.1 mol/l, pH 2.2). One milliliter fractions were collected in tubes containing 50 µl of 1 M Tris (pH 9.0). The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Bie and Berntsen A-S, Rodovre, Denmark) using BSA as a standard. The HDL and LDL preparations were subjected to several rounds of M23 anti-apoM chromatography until all apoM particles had been removed from the respective lipoprotein. In some cases, apoM-containing lipoproteins were concentrated by centrifugation at 10°C for 4 h at 100,000 g in a S/N O4U 1660 TLA 100 rotor head with a Beckman OptimaTM MAX-E ultracentrifuge (Beckman Coulter). ApoM-free apoA-I-containing particles were isolated from apoM-free HDL with an M12 anti-apoA-I column and a protocol similar to that used for the M23 anti-apoM column.
Characterization of lipoprotein density, size, and charge
The density of apoM-containing lipoproteins was assessed by gradient density ultracentrifugation. A gradient was formed in Ultra-Clear tubes (Beckman Coulter) by layering from the bottom 2 ml of 50% sucrose, 2 ml of 25% sucrose, 5 ml of 12.5% sucrose with 50 µl of apoM-containing lipoproteins, and 3 ml of PBS (17). After centrifugation for 70–71 h at 35,000 rpm and 12°C in a Beckman SW41 Ti rotor using the Beckman Optima LE-80K ultracentrifuge, 12 fractions of 1 ml were collected by aspiration from the top and apoM concentration was measured with an apoM ELISA; the cross-contamination between fractions was <10% (17).
The size of the isolated lipoprotein particles was assessed by nondenaturing gel electrophoresis: lipoproteins and the native high-molecular-weight marker (Amersham Biosciences Europe GmbH, Horsholm, Denmark) were electrophoresed at room temperature for 3 h at 125 V on 4–20% or 3–8% Novex Tris-glycine gels (Invitrogen A/S, Taastrup, Denmark) and stained with SimpleBlue SafeStain (Invitrogen A/S).
Gel-permeation chromatography was performed at 20–24°C using PBS with EDTA on serially connected Superose 6 and Superose 12 10/300 GL fast-protein liquid chromatography columns (Amersham Biosciences Europe). ApoM-containing HDL (HDLapoM+) was analyzed with 1 mg/ml albumin in sample and buffer. The flow rate was 0.2 ml/min, and fractions of 250 µl were collected. Void volume (V0) and total column volume (Vt) were determined with intralipid and glycerol, respectively, and the column was calibrated with different molecular markers (high-molecular-weight and low-molecular-weight calibration standards; Amersham Biosciences).
Agarose (0.8% Litex agarose; Medinova Scientific A/S) gel electrophoresis was done in a barbital buffer (pH 8.5). Proteins on the agarose gels were stained with Coomassie Brilliant Blue. Lipid free apoA-I (a gift from Dr. M. H. Andersen, Borean Pharma, Aarhus, Denmark) and human LDL purified by ultracentrifugation were used to identify ß- and preß-migrating particles.
Human apoM ELISA
An ELISA for human apoM based on two monoclonal apoM antibodies (M58 and M42) was used to quantify apoM in fractions. The details of this ELISA will be presented elsewhere. In brief, 96-well Costar plates (Corning BV, Biotech Line A/S, Slangerup, Denmark) were coated with M58, and the plates were quenched and washed using standard techniques. Samples were diluted in TBS with 1% Triton X-100 (Sigma-Aldrich Denmark A/S, Vallensbaek Strand, Denmark) and 1% BSA (Sigma) and added to the wells. After incubation to allow binding of apoM to the immobilized M58, bound apoM was detected with biotinylated M42, streptavidin-avidin-horseradish peroxidase (DAKO A/S, Glostrup, Denmark), and o-phenylenediamine (OPD) (DAKO A/S) according to the manufacturer's instructions.
Protein identification by mass spectrometry
Reduced and nonreduced isolated apoM-containing lipoprotein fractions were separated on 12% SDS-PAGE gels and silver-stained. The protein was identified as described previously (18). In short, the relevant bands were excised from the polyacrylamide gel, reduced, alkylated using iodacetamiden, and digested by trypsin. The resulting fragments were extracted, purified using C18 ZipTip (Millipore), and measured by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry on a Biflex instrument (Bruker, Bremen, Germany). For identification, the mass spectral patterns of the fragments were used to search against several databases.
Western blotting
For Western blotting, proteins were separated on 12% or 16% polyacrylamide gels (Kem En Tec, Copenhagen, Denmark) and transferred to Hybond-P 0.45 µm polyvinylidene difluoride membranes (RPN303F; Amersham Biosciences Europe) using a semidry electroblotter (Kem En Tec). The membranes were quenched for 1 h in skim milk (100 mg/ml) followed by incubation for 1 h at room temperature or for 16 h at 4°C with primary antibodies [goat anti-human apoJ (1:1,000; Abcam, Cambridge, UK), rabbit anti-human haptoglobin (1:4,000; DAKO, Copenhagen, Denmark), mouse anti-human PON1 (1:2,000; a gift from Dr. Dragomir Draganov, Michigan Medical School), mouse anti-human apoB (1:6,000; a gift from Dr. Sally McCormick, Otago University, New Zealand), mouse anti-human apoA-I (1:500; Autogen Bioclear, Wiltshire, UK), or rabbit anti-human apoM IgG (2.5 µg/ml)]. The dilutions of antibodies were made in the quenching buffer. The membranes were then washed and incubated with horseradish peroxidase-coupled secondary antibodies (goat anti-rabbit or rabbit anti-goat from DAKO, anti-biotin from Trichem Aps, or goat anti-mouse from BD Biosciences). After another round of washes, binding of antibody was detected with a chemiluminiscence reader (Fujifilm LAS-1000 Intelligent Dark Box II; Fujifilms, Trorod, Denmark) after incubation of the membrane with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemicals, Copenhagen, Denmark).
Lipid analysis
TLC (19) was used to measure the lipid composition of isolated lipoproteins. Briefly, lipids were extracted with chloroform-methanol. The extracts were washed (20) and applied on 20 x 20 cm silica-coated plates (DC-fertigplatten SIL G-25; Macherey-Nagel, Duren, Germany). Lipid extracts and standards were separated in six different solvent systems to enable separation of triglycerides, free cholesterol, esterified cholesterol, and individual phospholipid classes and visualized by heating the plate to 200°C for 2 min before quantification with digital image analysis. Details of the TLC system and intra-assay and interassay variations have been reported previously (20). Cholesterol in gel filtration fractions was measured by an enzymatic method with the CHOD-PAP reagents (Roche A/S, Applied Science, Hvidovre, Denmark).
Lipoprotein oxidation
Isolated lipoproteins were dialyzed against PBS, pH 7.4, for 16–20 h at 4°C. Nine hundred fifty microliters was placed in a quartz cuvette (VWR International ApS, Albertslund, Denmark) and mixed with 50 µl of PBS (control) or 50 µl of CuSO4 (final concentration, 2.5 µmol/l). The formation of conjugated dienes at 37°C was measured at 234 nm every minute for 4 h with a Hewlett-Packard 8452A diode array spectrophotometer. The lag time was estimated by drawing a perpendicular line to the x axis from the intersection of the two straight lines drawn through the lag and propagation phases of the absorption curve (21). The propagation rate was calculated as the slope of the curve during the linear propagation phase.
Efflux of cholesterol from THP-1 foam cells
Human monocytic THP-1 cells (3.5 x 105) were grown on 24-well plates (Multidishes NunclonTM; VWR International) in 1 ml of medium [RPMI-1640 (–L-glutamine); Sigma-Aldrich Denmark] containing 4 mM L-glutamine (Sigma-Aldrich Denmark), 10% fetal calf serum, and 1% penicillin/streptomycin at 37°C in 5% CO2. Cells were grown in the presence of phorbol myristate acetate (Sigma-Aldrich Denmark) for 5 days to induce differentiation into macrophages. For the first 24 h, the concentration of phorbol myristate acetate was 50 ng/ml; for the next 4 days, it was 25 ng/ml. The medium was changed on days 1, 3, and 5. On day 5, the same medium as described above without calf serum and with 1.5% BSA, 200,000 dpm/ml [3H]cholesterol [1
,2(n)-3H-cholesterol; Amersham Bioscience], and 50 µg protein/ml acetylated LDL [prepared according to Basu et al. (22)] was added. This resulted in foam cell formation as judged by light microscopic inspection of Oil Red O-stained cells. After 48 h, cells were washed three times with serum-free medium and incubated with 0.7 ml of serum-free medium containing isolated lipoproteins (25 µg protein/ml) that had been dialyzed against PBS for 16–20 h at 4°C. Twenty-five microliters of medium was collected from each well after 4, 8, 16, 24, and 40 h. At each time point, the cells were inspected by microscopy to ensure that no morphological changes or accumulation of dead cells had occurred. After 40 h, cells were washed twice with PBS and lipids were extracted in 1 ml of hexane-isopropanol (3:2; 20–24°C) for 1 h followed by 0.5 ml of hexane-isopropanol for 15 min. The extracts were dried under N2 and resuspended in 100 µl of isopropanol with 1% Triton X-100 (Sigma). [3H]cholesterol in cell extracts and media was measured by scintillation counting using OptiPhase Hisafe 3 Scintillation Liquid (Perkin-Elmer Life Sciences, Hvidovre, Denmark) and a Wallac 1409 Liquid Scintillation Counter (Perkin-Elmer Life Sciences). Total cholesterol in cell extracts was measured enzymatically. Cell proteins were hydrolyzed in 0.5 ml of 0.5 M NaOH at 37°C for 24 h and measured with the Pierce BCA Protein Assay Kit (Bie and Berntsen). Efflux was calculated as the percentage of [3H]cholesterol in medium at a given time compared with the total [3H]cholesterol contents in medium and cells.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
100 mM NaCl (pool 1) and 300 mM NaCl (pool 2) (data not shown). The two pools were separately applied to an anti-apoM monoclonal antibody (M23) column to isolate the apoM-containing particles. The pools were reapplied to the M23 column multiple times until depleted of apoM. ApoM-free apoA-I-containing lipoproteins were then purified using an anti-apoA-I (M12) column. The apoM-containing particles obtained from pools 1 and 2 had densities of 1.07–1.21 g/l, which is comparable to HDL2 and HDL3 (Fig. 1A
). The size of the apoM-containing particles was estimated by nondenaturing polyacrylamide gel electrophoresis (Fig. 1B). Pool 1 demonstrated apoM particles of three different sizes. A minor fraction comigrated with LDL (23 nm), whereas the two major fractions migrated corresponding to 8.0 nm particles or 10–12 nm particles. The apoM-containing particles of pool 2 were all of HDL size (i.e., they migrated similarly to apoM-free apoA-I-containing particles and to total HDL; the migration corresponded to 12 nm particles). This indicated that the isolated plasma apoM was located mainly in HDL and to a minor extent in LDL. However, we cannot exclude the possibility that a minor fraction of the isolated apoM particles were larger than LDL, as VLDL did not enter the gel. Also, because the nondenaturing gels were not to equilibrium, the actual size of the apoM-containing particles should be judged with caution.
|
|
Basic characterization of apoM-containing HDL and LDL particles from plasma
As apoM was associated with both HDL and LDL, we purified apoM particles from HDL and LDL that had been isolated by ultracentrifugation. ApoM-containing HDL and LDL were compared with HDL and LDL lacking apoM. Purification of apoM-containing particles from both HDL and LDL was effectively performed using multiple rounds of purification on a HiTrap column with a monoclonal antibody (M23) against apoM (Fig. 2A
), resulting in preparations of apoM-containing HDLapoM+ and LDLapoM+ and apoM-free HDLapoM– and LDLapoM– particles (Fig. 2B, C). Analysis using several techniques including Western blotting against apoA-I, apoB, and apoM demonstrated good separation of lipoprotein fractions: LDL and HDL contained no detectable apoA-I and apoB, respectively (Fig. 2C). ApoM could not be detected in LDLapoM– and HDLapoM– (Fig. 2C). On analysis of HDL, HDLapoM–, and HDLapoM+ with nondenaturing gradient gels, the major portion of HDLapoM+ migrated similarly to HDL but a minor portion migrated corresponding to 7.2 nm particles (Fig. 3A
). Small apoM-containing particles were also seen on gel filtration analysis of HDLapoM+ (Fig. 3B). The band corresponding to the smaller particles in HDLapoM+ contained both apoA-I and apoM: apoA-I was seen on Western blots of nondenaturing gels with an apoA-I antibody, and apoM was detected in the smaller band when the portion of the nondenaturing gel with 7.2 nm particles was cut out and incubated with buffer followed by quantification of apoM in the eluate with ELISA (data not shown). Of note, the small apoM-containing particles in HDLapoM+ were not seen in plasma on gel filtration analyses (Fig. 3B). Thus, they probably arose during the ultracentrifugation or column purification procedures. LDLapoM+ tended to be slightly smaller than total LDL and LDLapoM– (Fig. 3A).
|
|
|
- and preß-migrating bands (Fig. 4B), whereas apoM was predominantly in -migrating particles (Fig. 4C). Compared with total HDL, HDLapoM+ contained similar amounts of phosphatidylcholine, triglyceride, and cholesteryl esters but slightly more free cholesterol (Fig. 4C).
Antioxidative effect of apoM-containing HDL and LDL
To measure the antioxidant effect of HDLapoM+ and LDLapoM+, we monitored Cu2+-induced development of dienes at 234 nm (Fig. 5
, Table 2
). The oxidation curve obtained for HDLapoM+ demonstrated increased lag time (see Methods for a definition) after the addition of Cu2+ compared with corresponding curves obtained with total HDL and HDLapoM–. This suggests that the apoM-containing HDL particles were more resistant to oxidation than the other HDL particles (Fig. 5A). Moreover, HDLapoM+ was able to prolong the lag time and reduce the propagation rate when added to LDL (Fig. 5B, Table 2) or HDL (Fig. 5C) at a level identical to albumin, thus appearing to protect both LDL and HDL against Cu2+-induced oxidation. Finally, LDLapoM+ demonstrated a slightly shorter lag time than did total LDL or LDLapoM–, but the maximal amount of Cu2+-induced diene formation was decreased (Fig. 5D, Table 2). To ensure that these results were not attributable to the purification of HDLapoM+ by immunoaffinity chromatography, HDLapoM– was further purified by chromatography with immobilized antibody against apoA-I (M12) using the same protocol. However, the results obtained with HDLapoM– with or without immunoaffinity chromatography purification were essentially identical (data not shown).
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
5% of HDL particles and in <2% of LDL particles. The apoM-containing particles contained both HDL-related (apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, and apoJ) and LDL-related (apoB) apolipoproteins. ApoJ-containing HDL particles are known to be heterogeneous in size and composition, and apoJ has been found in both HDL2 and HDL3 (23). However, apoJ could not be identified in the HDLapoM+ that was isolated by the combination of ultracentrifugation and immunoaffinity chromatography. Indeed, control experiments showed that apoJ was lost from HDL during ultracentrifugation of plasma for 24 h at d = 1.21 g/l (data not shown). A similar phenomenon has been reported for other lipoproteins, such as LCAT (24).
ApoM contains no signal peptidase cleaving site, and the signal peptide is retained in the mature apoM in plasma (11, 12). Although both PON1 and HRP also contain retained signal peptides, they were present only in small amounts in the isolated preparations of apoM-containing particles. This suggests that although the hydrophobic signal peptides probably serve to anchor the respective apolipoproteins in the lipid moiety of the lipoprotein particle, the signal peptide per se does not confer any specificity for association with a certain subpopulation of HDL.
Based on experiments performed in mice, it was recently proposed that apoM is a constitutive component of preß HDL and is required for preß HDL formation (15). In this study, the size and charge patterns of the isolated HDLapoM+ particles suggest them to be as extensively heterogeneous as regular HDL. The main part of HDLapoM+ particles were -migrating. Interestingly, our analyses suggest that the content of free cholesterol relative to other lipids is increased in HDLapoM+ compared with total HDL particles.
We explored the function of the HDL particles containing apoM and found that HDLapoM+ could protect itself, as well as other HDL and LDL particles, from Cu2+-induced oxidation. The HDL-associated proteins PON1 and apoJ have previously been reported to have antioxidative effects (25–27). Although these proteins were present in some apoM-containing particles, it is important to stress that the antioxidative effects of HDLapoM+ were not attributable to increased levels of PON1 or apoJ. Indeed, LDLapoM+ also seemed more resistant to oxidation than LDLapoM–. The mechanism for the antioxidative effect of apoM is still unknown, and it is interesting that several other lipocalins have been described to have similar antioxidant properties (13).
The HDLapoM+ particles were more efficient than total HDL or HDLapoM– in stimulating cholesterol efflux from THP-1 foam cells and reducing total levels of intracellular cholesterol. The results obtained with the THP-1 cells and apoM-containing particles are in agreement with observations reported by Wolfrum, Poy, and Stoffel (15) suggesting that apoM can stimulate cholesterol efflux from murine Raw cells. It is possible that the increased efflux capacity of the apoM-containing HDL simply is a reflection of them being enriched in small preß-migrating HDL. Thus, whether apoM contributes to apoA-I-mediated cholesterol efflux or uses other unknown pathways remains to be investigated. However, the data suggest unique properties of apoM in relation to cholesterol accumulation in foam cells. Thus, the protein composition of LDLapoM+ and LDLapoM– was mainly distinguished by the presence of apoM, and LDLapoM+ mediated more [3H]cholesterol net efflux and induced less cholesterol accumulation than LDLapoM–.
Although these results suggest unique properties of apoM-containing HDL and LDL, it is important to note that these results suggest that apoM is present in only 5% of HDL particles and <2% of LDL particles. Hence, the bulk cholesterol efflux and antioxidative capacity of HDL resides with apoM-free HDL. Accordingly, there was no difference between total HDL and HDLapoM– in the oxidation and cholesterol efflux evaluations. These investigations of the biological effects of purified HDLapoM+ should be interpreted with caution, because purification by ultracentrifugation leads to a loss of apolipoproteins that may have physiologically important effects (e.g., apoJ). Also, the purification of HDLapoM+ resulted in the formation of small apoA-I- and apoM-containing particles that could not be seen in plasma and that may have effects in the oxidation and cholesterol efflux assays. Nevertheless, these particles constituted <10% of the total HDLapoM+. Moreover, multiple passages of apoM-free HDL over the anti-apoM column did not change its protein composition (data not shown), and the effects of HDLapoM– (which had passed over the affinity column multiple times) and total HDL were similar both in the oxidation and cholesterol efflux assays. Finally, there was no effect of immunoaffinity purification of apoM-free apoA-I-containing HDL on its ability to protect LDL from oxidation. These results suggest that the immunoaffinity purification procedures did not confer major changes in the biological properties of the HDL subclasses.
In conclusion, human apoM is associated with plasma lipoproteins that are heterogeneous in size and charge as well as in protein and lipid composition. Nevertheless, as judged from the in vitro investigations of lipoprotein oxidation and cholesterol efflux, apoM appears to designate a small subpopulation of HDL (and LDL) that potentially may be relatively more antiatherogenic than the HDL population as a whole.
|
ACKNOWLEDGMENTS |
|---|
Manuscript received February 1, 2006 and in revised form April 19, 2006 and in re-revised form May 8, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Ahnstrom, O. Axler, M. Jauhiainen, V. Salomaa, A. S. Havulinna, C. Ehnholm, R. Frikke-Schmidt, A. Tybjaerg-Hansen, and B. Dahlback Levels of apolipoprotein M are not associated with the risk of coronary heart disease in two independent case-control studies J. Lipid Res., September 1, 2008; 49(9): 1912 - 1917. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Christoffersen, J. Ahnstrom, O. Axler, E. I. Christensen, B. Dahlback, and L. B. Nielsen The Signal Peptide Anchors Apolipoprotein M in Plasma Lipoproteins and Prevents Rapid Clearance of Apolipoprotein M from Plasma J. Biol. Chem., July 4, 2008; 283(27): 18765 - 18772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wolfrum, J. J. Howell, E. Ndungo, and M. Stoffel Foxa2 Activity Increases Plasma High Density Lipoprotein Levels by Regulating Apolipoprotein M J. Biol. Chem., June 13, 2008; 283(24): 16940 - 16949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Venteclef, A. Haroniti, J.-J. Tousaint, I. Talianidis, and P. Delerive Regulation of Anti-atherogenic Apolipoprotein M Gene Expression by the Orphan Nuclear Receptor LRH-1 J. Biol. Chem., February 15, 2008; 283(7): 3694 - 3701. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Christoffersen, M. Jauhiainen, M. Moser, B. Porse, C. Ehnholm, M. Boesl, B. Dahlback, and L. B. Nielsen Effect of Apolipoprotein M on High Density Lipoprotein Metabolism and Atherosclerosis in Low Density Lipoprotein Receptor Knock-out Mice J. Biol. Chem., January 25, 2008; 283(4): 1839 - 1847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ahnstrom, K. Faber, O. Axler, and B. Dahlback Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M J. Lipid Res., August 1, 2007; 48(8): 1754 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Axler, J. Ahnstrom, and B. Dahlback An ELISA for apolipoprotein M reveals a strong correlation to total cholesterol in human plasma J. Lipid Res., August 1, 2007; 48(8): 1772 - 1780. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||
