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* Division of Clinical Chemistry, Department of Laboratory Medicine, University of Lund, University Hospital Malmö, S-20502 Malmö, Sweden
Department of Clinical Biochemistry, Rigshospital, University of Copenhagen, Copenhagen DK-2100, Denmark
Published, JLR Papers in Press, April 21, 2004. DOI 10.1194/jlr.M300451-JLR200
1 To whom correspondence should be addressed. e-mail: larsbo{at}rh.dk (L.B.N.); bjorn.dahlback{at}klkemi.mas.lu.se (B.D.)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These data suggest that apoM mainly associates with HDL in normal mice but also with the pathologically increased lipoprotein fraction in genetically modified mice. The substantially decreased apoM levels in apoA-I-deficient mice suggest a connection between apoM and apoA-I metabolism.
Supplementary key words apolipoprotein A-I • apolipoprotein M • high density lipoprotein • low density lipoprotein • very low density lipoprotein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ApoM is a novel human apolipoprotein of 188 amino acids that is mainly associated with HDL (10). Structural analysis predicts that human apoM belongs to the lipocalin protein family (11). Lipocalins share a common tertiary structure with an eight stranded antiparallel ß-barrel surrounding a hydrophobic ligand binding interior (12–14). Lipocalins are extracellular proteins; some interact with specific cell surface receptors and/or are involved in the formation of macromolecular complexes (14). Several lipocalins are antioxidants or modulators of the immune system (12, 14, 15). However, for most lipocalins, including apoM, the biological role has not been determined (12).
Human apoM is secreted with its N-terminal signal peptide being retained in the mature protein (10). This is explained by the lack of a signal peptidase cleavage site after the signal peptide sequence. ApoM shares this unusual feature with two other HDL apolipoproteins, PON-1 and haptoglobin-related protein (16, 17). In the case of PON-1, the hydrophobic signal peptide has been shown to provide an anchor for PON-1 in the HDL particle (11, 16). PON-1 and haptoglobin-related protein are exclusively found in HDL, and HDL deficiency is accompanied by very low PON-1 plasma concentrations in both humans and mice (16, 18). However, although apoM is predominantly found in HDL in human plasma, apoM was initially isolated from chylomicrons, and the apparent content of apoM in chylomicrons was increased after ingestion of a fat-rich meal (10).
In this study, we demonstrate the presence of an apoM in murine plasma. We have characterized the expression of apoM mRNA during development and in adult mice and the apoM distribution among plasma lipoproteins in wild-type mice and mice with genetically modified plasma lipoprotein distribution.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4,000 g at 4°C. Plasma was stored at –20°C or –80°C until analysis. The experimental protocols were approved by the local ethics committee in Copenhagen, Denmark, or Lund, Sweden. Plasma from LDL receptor-deficient mice was a gift from Novo Nordisk A/S (Bagsvaerd, Denmark). Plasma from hepatic nuclear factor (HNF)-1
-deficent mice and littermate controls was provided my Dr. F. J. Gonzales (National Institutes of Health, Bethesda, MD). Pools of embryonic RNA were from BALB/C mice and were provided by Dr. F. C. Nielsen (Rigshospitalet, Copenhagen, Denmark) (19).
mRNA purification and cDNA amplification
Frozen biopsies of organs from wild-type and apoA-I-deficient mice were homogenized with a Polytron PT 1200CL (Buch and Holm, Herlev, Denmark) in TriZol reagent (Life Technologies, Taastrup, Denmark). Total RNA was isolated according to the manufacturer's manual and suspended in RNase-free water. The RNA concentration was calculated from the absorbance at 260 nm. The RNA integrity was ensured by 1% agarose gel electrophoresis or with an Agilent 2100 Bioanalyzer (Agilent Technologies Denmark A/S, Naerum, Denmark) using a RNA 6000 Nano Assay Kit (Agilent Technologies). First-strand cDNA was synthesized from 1 µg of total RNA with Moloney murine leukemia virus reverse transcriptase (40 units; Roche A/S, Hvidovre, Denmark) and random hexamer primers in 10 µl reactions. Sense and antisense primers for amplification of mouse apoM mRNA were 5'-CCT GGG CCT GTG GTA CTT TA-3' and 5'-CCA TGT TTC CTT TCC CTT CA-3' (Sigma-Genosys, Pampisford, UK), the sequences being derived from the available cDNA sequence (accession number AF207820). Primers for ß-actin amplification have been reported elsewhere (20). DNA sequencing and agarose gel electrophoresis of apoM and ß-actin PCR products confirmed the specificity of the RT-PCR.
mRNA quantification with a real-time PCR assay
Quantitative real-time PCR analysis of apoM mRNA expression was performed with a LightCycler (software version 3.39) and the DNA master SYBR GREEN kit (Roche A/S). Each PCR sample (20 µl) contained 2 µl of SYBR GREEN I mix, 2–2.5 mM MgCl2, 10 pmol of each primer, cDNA synthesized from 20 ng of total RNA, and PCR-grade water. The amount of mRNA transcript in each tissue sample was determined from the time point of the log-linear increase in amplified DNA during the PCR using the fit point option of the LightCycler. To determine the relation between the time point of the log-linear increase of the fluorescence signal and the relative concentration of a mRNA transcript in a sample, a dilution series of a pool of cDNA from mouse liver was analyzed in each run.
Anti-mouse apoM antibodies
A mouse apoM cDNA (National Center for Biotechnology Information GenBank accession number AF207820) encoding the full-length 190 amino acid residue mouse apoM protein was used for the cloning of a truncated cDNA into an expression vector (pET-30 Xa/LIC; Novagen, Sigma-Aldrich, Stockholm, Sweden) and expressed in Escherichia coli [strain BL21 (DE3); Stratagene]. The expressed truncated apoM included a His tag, a S tag, and amino acid residues 22–190 of mouse apoM (signal peptide was excluded) and was purified from inclusion bodies and refolded (K. Faber and B. Dahlbäck, unpublished data). The purified truncated recombinant apoM was mixed with complete Freund's adjuvant and used to immunize two female rabbits. The Ig fraction of the apoM antiserum was purified on a protein G column (5 ml; Hi-trap, Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's protocol. Affinity-purified apoM antibodies were further affinity purified on a column coupled with 8.5 mg of recombinant mouse apoM (1 ml; Hi-trap, Amersham Pharmacia Biotech). The apoM column was equilibrated with 50 mM Tris-HCl, 0.15 M NaCl, pH 7.5, and the bound antibodies were eluted with 0.1 M glycine HCl, pH 2.7, and collected in tubes containing one-tenth volume of 1 M Tris-HCl, pH 9.0, to neutralize the pH. The concentration of purified antibodies was determined from the absorbance at 280 nm using an extinction coefficient (1%, 1 cm) of 12.5. The antibodies were stored at 4°C after the addition of 0.02% NaN3. A similar truncated human apoM was constructed in the pET-30 Xa/LIC vector, and the protein was expressed in E. coli, isolated, and refolded as described for mouse apoM. This apoM was used to immunize mice, and three monoclonal antibodies were created with standard techniques. None of these monoclonal antibodies reacted with mouse apoM.
Western blotting
Plasma, gel filtration fractions, or PBS homogenates of liver or kidney were separated on 12% SDS-PAGE gels, and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Gelman, Lund, Sweden) using semidry electroblotting. The membrane was quenched for 1 h in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, containing 0.5% (w/v) Tween 20 and 3% fish gelatin (Norland Products, Inc., Cranbury, NY) followed by incubation for 1 h at room temperature with anti-mouse apoM antibodies (30 µg/ml), polyclonal rabbit anti-mouse apoA-I antibodies (1:2,560; BioSite, Täby, Sweden), monoclonal mouse anti-mouse apoB-100 antibodies (1:500; kindly provided by Dr. S. Young, Gladstone Institute of Cardiovascular Disease, San Francisco, CA), or rabbit anti-mouse apoA-II antibodies (Biodesign International) in the same buffer. The membranes were then washed and incubated with swine anti-rabbit or rabbit anti-mouse antibodies coupled with horseradish peroxidase (1:10,000; DAKO, Copenhagen, Denmark) (21). After another round of washes, the membranes were developed with 5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt and p-nitroblue tetrazolium (Sigma-Aldrich, Stockholm, Sweden). All incubations were done at room temperature.
Endoglycosidase digestion
ApoM-containing HDL was purified from human plasma using a monoclonal antibody against apoM that we recently developed (our unpublished data). This material (10.5 µl with an optical density at 280 nm of 0.07) was mixed with 60 µl of 0.1% sodium dodecyl sulfate containing 0.1 M 2-mercaptoethanol and denatured for 5 min at 90°C. After cooling, 18 µl of 0.5 M sodium citrate, pH 5.5, 1.5 µl of water, and peptide:N-glycanase [15 µl of 0.12 mg/ml, kindly provided by Dr. J. Stenflo, Department of Laboratory Medicine, University Hospital (Malmö, Sweden)] were added. After incubation at 37°C overnight, the samples were analyzed by Western blotting using a 12% SDS-PAGE run under reducing conditions. For immunochemical detection, a mixture of polyclonal anti-mouse and anti-human apoM antibody was used to visualize both human and mouse apoM.
Quantification of plasma apoM
A dilution series of a pool of wild-type mouse plasma (corresponding to 0, 0.1, 0.25, 0.5, and 1 µl of plasma) and plasma samples from individual mice (0.5 µl) were analyzed on the same gel by 12% SDS-PAGE. After transfer to a Hybond-P 0.45 µm PVDF membrane (Amersham Biosciences, Copenhagen, Denmark), the blots were incubated at 4°C with anti-apoM antibodies for 14–18 h and a horseradish peroxidase-conjugated goat anti-rabbit antibody for 1 h. Antibody binding was detected with a chemiluminescence reader (Fujifilm LAS-1000 Intelligent Dark Box II, Fujifilms, Trorod, Denmark) after incubation of the blot with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemicals, Copenhagen, Denmark). Chemiluminescence readings were corrected for background before calculations using the Image Reader LAS-1000 Pro V2.5 ImageGauge 4.0 program (Fujifilms).
Gel filtration of plasma
Lipoproteins in mouse plasma were separated by gel permeation chromatography on a Superose 6 HR 10/30 fast-protein liquid chromatography column (Amersham Pharmacia Biotech). Samples were pooled from 4–11 mice and passed through 0.22 µm filters before loading 0.5 ml on the column. The column was run at room temperature with phosphate-buffered saline, pH 7.4, at a flow rate of 0.1 ml/min. Fractions of 0.5 ml were collected and stored at –20°C until protein and lipid analyses.
Quantification of plasma lipids
Cholesterol concentrations were determined enzymatically as described (20).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma lipid and apolipoprotein profiles
Mean plasma cholesterol concentrations were 1 mmol/l in wild-type mice, 0.6 mM in apoA-I-deficient mice, 6.5 mmol/l in LDL receptor-deficient mice, 14 mmol/l in chow-fed apoE-deficient mice, and 41 mmol/l in apoE-deficient mice fed a high-fat and high-cholesterol diet. To analyze the distribution of apoM among plasma lipoproteins, we used gel filtration chromatography and Western blot analysis. As expected, in wild-type mouse plasma, cholesterol was predominantly found in HDL and apoM was recovered in the same fractions as HDL, suggesting that murine apoM, like its human counterpart, is associated with the HDL-sized particles (Fig. 3A)
. There was no difference in the size distributions of apoM- and apoA-I-containing particles according to Western blotting. In LDL receptor-deficient mice, the HDL cholesterol level was similar to that in wild-type mice, but the major fraction of the plasma cholesterol in these mice was in LDL (Fig. 3B). Remarkably, apoM was abundant in LDL-sized particles of the LDL receptor-deficient mice, whereas apoA-I was exclusively found in HDL particles (Fig. 3B). ApoE-deficient mice had increased VLDL cholesterol concentrations when fed a normal mouse chow (Fig. 3C). This phenotype was accentuated when the diet was changed to a high-fat/high-cholesterol-enriched diet (Fig. 3D). In chow-fed apoE-deficient mice, apoM was mainly present in the HDL fractions but was also clearly detectable in LDL- and VLDL-sized particles (Fig. 3C). In high-fat- and high-cholesterol-fed apoE-deficient mice, apoM was most prominent in the VLDL fraction and only traces of apoM were found in HDL-sized particles (Fig. 3D). Additional immunoblotting with an antibody against mouse apoB-100 confirmed the presence of apoB in the cholesterol containing VLDL and LDL particles of apoE- and LDL receptor-deficient mice (data not shown).
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Plasma concentrations of apoM
The plasma concentrations of apoM in the different types of mice were determined with a Western blotting technique using a chemiluminescence detection system. Plasma apoM was similar in wild-type mice and high-fat- and high-cholesterol-fed apoE-deficient mice (Fig. 4A)
, despite the pronounced difference in the plasma cholesterol concentrations. Normal plasma apoM concentrations were also found in chow-fed apoE-deficient mice and in LDL receptor-deficient mice. In contrast, plasma from apoA-I-deficient mice had significantly lower apoM concentrations than did wild-type mice (Fig. 4A). The apoM concentration in apoA-I-deficient mice was on average 33% of that in wild-type mice (Fig. 4C). A recent study suggested that deficiency of HNF-1 causes low plasma apoM as a result of decreased mRNA expression (23). Western blotting of plasma from HNF-1-deficient mice confirmed this result, as apoM was virtually absent in HNF-1-deficent mice (Fig. 4D). To determine whether the lower apoM plasma concentration in apoA-I-deficient mice could be attributable to decreased mRNA expression, we analyzed liver and kidney apoM mRNA. There was no statistically significant difference in apoM mRNA expression between wild-type and apoA-I-deficient mice (Fig. 5)
, although there was a trend for liver apoM mRNA to be slightly increased in apoA-I-deficient compared with wild-type mice. We could not detect apoM on Western blots of liver or kidney protein extracts or urine from apoA-I-deficient mice (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue mRNA studies suggested that mouse apoM is produced primarily in liver and kidney. This expression pattern is similar to what was previously observed in a multiple-tissue Northern blot with human mRNA (10). The kidney expresses other apolipoprotein genes, including apoB and apoA-I. However, in rodents and mammals, the kidney expression of apoB and apoA-I is much smaller than the liver expression (24). In contrast, kidney apoM expression was almost 20% of that in the liver. ApoM is structurally similar to mouse major urinary protein-1, which is secreted into the urine (10). However, we could not detect apoM in either mouse or rat urine, even after concentration of the urine samples (our unpublished data). Thus, the role of kidney-derived apoM remains to be determined.
Like apoM, PON-1 and haptoglobin-related protein circulate in plasma with uncleaved signal peptides that anchor the proteins onto lipoproteins (16). Because PON-1 and haptoglobin-related protein are not associated with lipoproteins other than HDL, we investigated whether apoM would be associated primarily with HDL even in the setting of increased LDL and VLDL concentrations. We compared the lipoprotein distribution and total plasma levels of apoM in wild-type and genetically modified mice. The total concentration of apoM in plasma was similar in wild-type mice and mice with genetically induced hyperlipoproteinemia, despite a striking 40-fold difference in the total plasma cholesterol concentration between wild-type and high-fat/high-cholesterol-fed apoE-deficient mice. Although apoM was predominantly found in HDL in wild-type mice (as in humans), it tended to associate with the most prominent plasma lipoprotein fraction (i.e., LDL in LDL receptor-deficient mice and VLDL in apoE-deficient mice) (10). Notably, apoM was found in apoB-containing LDL-sized particles totally devoid of apoA-I in the plasma of LDL receptor-deficient mice.
Interestingly, in high fat/high cholesterol-fed apoE-deficient mice, apoM was predominantly recovered in intestinally derived VLDL even though apoM mRNA was undetectable in the intestine. This suggests that secretion of apoM from the liver is independent of apoB-containing lipoproteins and that apoM is transferred to these particles in plasma. In contrast, apoA-I-deficient mice had dramatically decreased apoM levels (33% of those of wild-type mice), suggesting a connection between apoM and apoA-I metabolism in the normal mouse. Although decreased apoM gene expression causes low apoM plasma levels in HNF-1-deficient mice (23), apoM mRNA expression levels were similar in wild-type and apoA-I-deficient mice. Thus, the lower apoM levels presumably reflect increased clearance. Interestingly, gel filtration studies suggested that apoM in apoA-I-deficient mice is mainly associated with apoA-I-free and apoA-II- and cholesterol-containing HDL-sized particles and not with apoB-containing lipoproteins. Altogether, the present results might indicate that the plasma lipoprotein association of apoM is dependent on lipid interactions that occur after its secretion. Thus, it is conceivable that the decrease of apoM in apoA-I knockout mice simply reflects the presence of fewer HDL particles to interact with. However, it should be kept in mind that normally the plasma concentration of apoM is much lower (20 mg/l) compared with that of apoA-I (1 g/l) and that only a subfraction of the HDL particles contain apoM. Although our studies to date have elucidated some basic aspects of apoM biology, the physiological role of apoM remains enigmatic. Like several other apolipoproteins, apoM appeared to be expressed during murine embryonic life. In the mouse embryos, apoM expression was activated from approximately day 10 and throughout embryonic life. DNA sequencing of the apoM gene in more than 200 human subjects (A. Hillarp and B. Dahlbäck, unpublished data) revealed no mutations that would result in the production of a truncated or defect apoM. This may indicate an important physiological role of apoM either before or after birth. The present demonstration of apoM expression in the mouse has paved the way for generating new mouse models with altered apoM expression. Such models should be helpful in providing insights into the physiological role of apoM.
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ACKNOWLEDGMENTS |
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-deficient mice, respectively, Dr. Finn C. Nielsen (Rigshospitalet) for embryonic mRNA, and Dr. Johan Stenflo (University Hospital Malmö) for peptide: N-glycanase. These studies were supported by grants from the Network for Cardiovascular Research, funded by the Swedish Foundation for Strategic Research, the Swedish Science Foundation (07143), Söderberg's, Påhlsson's, and Österlund's Foundations, and research funds from University Hospital Malmö, the Danish Heart Foundation, Rigshospitalet, and the Boserup Foundation. Manuscript received October 30, 2003
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REFERENCES |
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