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Molecular Endocrinology 20(1):212–218
Copyright © 2006 by The Endocrine Society
doi: 10.1210/me.2005-0209
Megalin Is a Receptor for Apolipoprotein M, and
Kidney-Specific Megalin-Deficiency Confers Urinary
Excretion of Apolipoprotein M
Kirsten Faber, Vibeke Hvidberg, Søren K. Moestrup, Björn Dahlbäck, and Lars Bo Nielsen
Division of Clinical Chemistry (K.F., B.D.), Department of Laboratory Medicine, University of Lund,
University Hospital, S-20502 Malmö, Sweden; Department of Medical Biochemistry (V.H., S.K.M.),
University of Aarhus, DK-8000 Aarhus C, Denmark; and Department of Clinical Biochemistry (L.B.N.),
Rigshospital, University of Copenhagen, DK-2100 Copenhagen, Denmark
Apolipoprotein (apo) M is a novel apolipoprotein
belonging to the lipocalin protein superfamily, i.e.
proteins binding small lipophilic compounds. Like
other apolipoproteins, it is expressed in hepatocytes and secreted into plasma where it associates
with high-density lipoprotein particles. In addition,
apoM is expressed at high levels in the kidney
tubule cells. In this study, we show that the multiligand receptor megalin, which is expressed in kidney proximal tubule cells, is a receptor for apoM
and mediates its uptake in the kidney. To examine
apoM binding to megalin, a recombinant apoM was
expressed in Escherichia coli and used in surface
plasmon resonance and cell culture studies. The
results showed apoM binding to immobilized
megalin [dissociation constant (Kd) ⬃ 0.3–1 ␮M]
and that the apoM was endocytosed by cultured
rat yolk sac cells in a megalin-dependent manner. To examine the importance of apoM binding
by megalin in vivo, we analyzed mice with a tissue-specific deficiency of megalin in the kidney.
Megalin deficiency was associated with pronounced urinary excretion of apoM, whereas
apoM was not detected in normal mouse, human,
or rat urine. Gel filtration analysis showed that
the urinary apoM-containing particles were small
and devoid of apoA-I. The results suggest that
apoM binds to megalin and that megalin-mediated endocytosis in kidney proximal tubules prevents apoM excretion in the urine. (Molecular
Endocrinology 20: 212–218, 2006)
A
mouse and man (4). Structural analysis and molecular
modeling have suggested that apoM is a member of
the lipocalin protein family being composed of an
eight-stranded antiparallel ␤-barrel surrounding a hydrophobic ligand-binding interior (5). Among many diverse proteins, the lipocalin protein superfamily comprises the retinal-binding protein and apoD (6). Thus,
lipocalins can bind compounds of essential importance in human endocrinology. However, for most lipocalins, including apoM, the physiological ligand is
unknown.
Megalin (glycoprotein 330) is a 600-kDa endocytosis-mediating membrane receptor that is highly expressed in the proximal tubule of the kidney (7–9) and
in many other absorptive epithelia, e.g. in yolk sac,
brain, lung, retina, and the inner ear (10, 11). Megalin
binds a variety of substances, including albumin, basic
drugs, and apolipoproteins A-I, B, H, and J (12). Megalin deficiency in knockout mice results in abnormal
brain development and is associated with 98% mortality before adulthood (13). Nevertheless, Leheste et
al. (14) succeeded in studying the surviving mice and
showed that they had low molecular weight proteinuria. The predominant urinary proteins in megalin-deficient mice included albumin, vitamin D-binding protein, major urinary protein-6, ␣1-microglobulin, and
retinol-binding protein. The latter three proteins are
lipocalins and all bind megalin (14). Thus, it is conceiv-
POLIPOPROTEIN (apo) M is a novel apolipoprotein. Remarkably, apoM is secreted without prior
cleavage of its signal peptide, and the hydrophobic
signal peptide is suspected to anchor apoM in the
lipoprotein particles (1). Thus, in plasma, apoM is associated with lipoproteins, i.e. primarily apoA-I containing high-density lipoprotein (HDL) (1). Recent observations suggest that plasma apoM affects the
pre-␤ HDL formation and that overexpression of apoM
in the liver of hypercholesterolemic mice has a marked
antiatherosclerotic effect (2). A fraction of plasma
apoM also associates with the larger apoB-containing
lipoproteins in normolipidemic individuals (1), and
apoM is actually mainly recovered in low-density lipoprotein and very low-density lipoprotein in severely
hyperlipidemic low-density lipoprotein receptor-deficient and apoE-deficient mice (3).
Similar to other apolipoprotein genes, the apoM
gene is expressed in lipoprotein-producing hepatocytes (3, 4). However, the apoM gene is also expressed at a high level in kidney tubular cells, both in
First Published Online August 11, 2005
Abbreviations: apo, Apolipoprotein; HDL, high-density lipoprotein.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
212
Faber et al. • Megalin is a receptor for apoM
able that megalin might also be a receptor for other
members of the lipocalin family such as apoM.
In this study, we expressed a recombinant apoM in
Escherichia coli and examined whether it binds to
megalin and whether it is endocytosed by megalin in
cultured cells. We also studied mice with Cre/loxgene-targeted megalin deficiency (15) in the kidney to
investigate whether megalin is involved in tubular reabsorption of apoM in vivo.
RESULTS
Mol Endocrinol, January 2006, 20(1):212–218 213
confocal scanning microscopy revealed that green fluorescence-labeled apoM was taken up by these cells
(Fig. 2A). The uptake of apoM was completely blocked
by addition of a megalin-blocking antibody but not a
control IgG (Fig. 2, B and C). Cubilin colocalizes with
megalin in the endocytic recycling pathway (16). ApoM
uptake was seen in vesicular structures different from
the cellular structures (membrane and early endosomes)
staining for cubilin (Fig. 2, red fluorescence). This suggests that that apoM is transferred to the endocytic
degradation pathway (probably lysosomes) after segregation from megalin in the early endosomes.
Megalin Binds apoM and Mediates Its
Endocytosis
Megalin Deficiency Confers Urinary Excretion of
apoM in Megalinlox/lox; apoECre Mice
To examine whether megalin binds apoM, we expressed recombinant mouse apoM in E. coli and used
it in surface plasmon resonance studies. The results
suggested specific binding of apoM to immobilized
megalin with a dissociation constant (Kd) of 0.3–1
␮mol/liter (Fig. 1) as estimated by analyzing binding of
apoM at concentrations from 0.05–2.0 ␮mol/liter.
There was no binding of apoM to immobilized cubilin
(Fig. 1) or low-density lipoprotein receptor-related protein (data not shown). To examine whether megalin
binding of apoM confers apoM endocytosis in cells,
fluorescence-labeled recombinant apoM was added
to megalin-expressing yolk sac epithelial cells. Laser
The apoM gene is highly expressed in the kidney tubule cells (3, 4). However, apoM was not detected in
the urine of normal rats, mice, or humans (Fig. 3A). To
examine whether megalin prevents urinary excretion
of apoM, we studied megalinlox/lox; apoECre mice with
kidney-specific ablation of the megalin gene. These
mice display deficient megalin expression in the kidney proximal tubules, whereas megalin expression is
normal in other tissues (15). ApoM Western blot analysis of urine from megalinlox/lox; apoECre mice and
megalinlox/lox control mice showed that megalin defi-
Fig. 1. Characterization of apoM Binding to Megalin Using
Surface-Plasmon Resonance on a BIAcore 200 Instrument
Recombinant mouse apoM (2 ␮M) was injected over immobilized megalin (upper panel) or cubilin (lower panel). Binding of apoM to megalin, but not to cubilin, was observed. A Kd
value of 0.3–1 ␮M was calculated from the kinetic parameters.
s, Seconds.
Fig. 2. Megalin-Dependent Internalization of Recombinant
Mouse apoM by Cultured Rat Yolk Sac Epithelial Cells
A, Uptake of Alexa 488-conjugated apoM (green fluorescence) after addition to the cell medium. Cubilin (red fluorescence) was visualized by immunohistochemical staining after
fixation of the cells. B and C, Uptake of Alexa 488-conjugated
apoM (green fluorescence) after incubation with antimegalin
antibodies (100 ␮g/ml) (B) or sheep nonimmune IgG (100
␮g/ml) (C).
214 Mol Endocrinol, January 2006, 20(1):212–218
Fig. 3. Demonstration of apoM in Urine of Megalin-Deficient
Mice
For detection of apoM in normal rat, mouse, and human
urine as well as in urine from megalinlox/lox; apoECre and
megalinlox/lox mice, urine samples (10 ␮l) were separated on
12% SDS-PAGE under reduced conditions and subjected to
Western blotting using antimouse apoM. A (lane 1), Rat urine
(50⫻ concentrated); lane 2, mouse urine (50⫻ concentrated);
lane 3, human urine (50⫻ concentrated); and lane 4, 1␮l
human plasma (positive control; the polyclonal mouse apoM
antibodies cross-react with both human and rat plasma
apoM). B (lane 1), Megalinlox/lox; apoECre female; lane 2,
megalinlox/lox; apoECre female; lane 3, megalinlox/lox; apoECre
male; lane 4, pool of urine samples shown in lanes 1–3; lane
5, megalinlox/lox female; lane 6, megalinlox/lox female; lane 7,
megalinlox/lox male; lane 8, pool of urine samples shown in
lanes 5–7. C, Comparison of apoM in plasma and urine of
megalinlox/lox; apoECre mice. Lane 1, 1 ␮l plasma; lanes 2 and
3, two samples of urine from megalinlox/lox; apoECre mice (10
␮l each lane). Mr, Relative molecular mass.
ciency in the kidney leads to urinary excretion of apoM
(Fig. 3B). The size of urinary apoM in megalinlox/lox; apoECre mice corresponded to the size of plasma apoM and
there were no detectable lower molecular weight bands
that stained with the polyclonal apoM antibody (Fig. 3C).
This suggests that urinary apoM is excreted mainly in its
intact form. The urinary secretion of apoM was not due to
an up-regulation of apoM gene expression because
kidney and liver apoM mRNA concentrations were
similar in megalinlox/lox; apoECre mice and megalinlox/lox
control mice (Fig. 4A). Also, the plasma apoM concen-
Faber et al. • Megalin is a receptor for apoM
Fig. 4. ApoM Expression in Liver, Kidney, and Plasma of
Megalinlox/lox; apoECre and Megalinlox/lox Mice
A, apoM mRNA was quantified in liver and kidney with
real-time PCR. Individual values were normalized to the content of ␤-actin mRNA in the same sample. B, Western blot of
plasma from megalinlox/lox; apoECre and megalinlox/lox mice. C,
Plasma apoM concentrations in megalinlox/lox and megalinlox/lox;
apoECre were estimated from duplicate determinations of the
chemiluminescence intensity of individual bands after immunoblotting. Values are mean ⫾ SEM; n ⫽ 3.
tration was similar in megalinlox/lox; apoECre and
megalinlox/lox mice as judged by quantitative Western
blotting (Fig. 4, B and C).
Megalinlox/lox; apoECre mice have normal function of
the kidney glomeruli (15). Due to its preserved signal
peptide sequences, apoM is highly hydrophobic and is
attached to HDL in plasma of wild-type mice (3). Gel
filtration studies showed that plasma apoM in
megalinlox/lox; apoECre mice (as in control megalinlox/lox
mice) exclusively eluted with HDL-sized particles (Fig.
5, A and B). In contrast, urinary apoM particles were
considerably smaller than plasma HDL and eluted
slightly after albumin (Fig. 5C). Of note, ApoA-I was not
excreted into the urine of the megalinlox/lox; apoECre
mice (Fig. 5D).
Faber et al. • Megalin is a receptor for apoM
Mol Endocrinol, January 2006, 20(1):212–218 215
DISCUSSION
Fig. 5. Size Distribution of apoM in Plasma and Urine of
Megalin-Deficient Mice and apoA-I Western Blotting of
Mouse Urines
Pooled plasma samples from megalinlox/lox (A), littermate
megalinlox/lox; apoECre mice (B) and urine from megalinlox/lox;
apoECre male mice (C) were subjected to fast-phase liquid
chromatography by loading 500 ␮l on a Superose 6 HR 10/30
column. Subsequent to gel filtration, each fraction was analyzed by Western blotting with antibodies against mouse
apoM and apoA-I; wild-type mouse plasma was used as a
positive control. Absorbance at 280 nm was used to identify
the albumin peak for plasma and for urine. Note that the lower
intensity of the apoM bands in panel B compared with panel
A is paralleled by a lower absorbance at 280 nm. This reflects
that plasma from megalinlox/lox; apoECre mice was prediluted
with buffer before analysis. D, Urine samples (10 ␮l) were
subjected to Western blotting of apoA-1 after separation on
12% SDS-PAGE run under reducing conditions. Lane 1,
Megalinlox/lox; apoECre female; lane 2, megalinlox/lox; apoECre
female; lane 3, megalinlox/lox; apoECre male; lane 4, pool of
urine samples from megalinlox/lox; apoECre mice; lane 5,
megalinlox/lox female; lane 6, megalinlox/lox female; lane 7,
megalinlox/lox male; lane 8, pool of urine samples from
megalinlox/lox mice; lane 9, 1 ␮l human plasma (positive control). Mr, Relative molecular mass.
This study demonstrates that megalin binds apoM and
mediates its uptake in mouse kidney proximal tubules.
The Kd for apoM’s binding to megalin was smaller than
that reported for another lipocalin, i.e. retinol binding
protein (Kd ⬃ 2 ␮mol/liter) and larger than that of the
vitamin D-binding protein (Kd ⬃ 0.1 ␮mol/liter) (14). We
used a recombinant mouse apoM expressed in E. coli
for the plasmon surface resonance studies. Of note,
mouse apoM does not contain any glycosylation sites
(5). Thus, the results were not affected by the lack of
protein glycosylation in E. coli. Studies of the uptake of
fluorescence-labeled apoM in megalin-expressing
cells confirmed the binding studies and showed that
apoM is endocytosed by yolk sac endothelial cells in a
megalin-dependent fashion. Thus, the present results
add apoM to a list of megalin ligands that includes
nearly 40 proteins (12).
Kidney expression of megalin is reduced by more than
90% of normal in the megalinlox/lox; apoECre mice (15).
The present results showed that the megalin deficiency,
even though not absolute, resulted in urinary excretion of
apoM. In the plasma of wild-type mice, apoM is mainly
bound in HDL particles. Previous studies have shown
that apoA-I can be taken up by the kidney proximal
tubular epithelium (17). The apoA-I uptake is caused by
the binding of apoA-I to cubilin functioning in concert
with megalin (18). Recently, cubilin has been shown to
form a functional receptor complex with the protein amnionless (19), and both cubilin and megalin antibodies
attenuate HDL internalization in cubilin/megalin-expressing yolk sac cell (17). However, there was no excretion of
apoA-I in the urine of the megalinlox/lox; apoECre mice.
This is in accord with studies of megalin knockout mice
where apoA-I is also not detectable in the urine (Willnow,
T. E., and S. K. Moestrup, unpublished data) but is in
contrast to the finding of apoA-I loss in the urine of dogs
and humans with defective cubilin function (17). This
suggest that the cubilin-amnionless system is sufficiently
effective for apoA-I clearance in mice, or that mouse
apoA-I compared with human apoA-1 has a low glomerular clearance, e.g. because of a firmer binding to HDL
particles. If apoM is in complex exclusively with large
apoA-I-containing HDL particles in plasma, it is likely that
only limited amounts of apoM are filtered in glomeruli.
The present data showing renal tubule catabolism of
apoM may therefore be explained by clearance of apoM
derived from local synthesis in the kidney.
On Western blotting, urinary apoM had a size similar
to that of plasma apoM, indicating that apoM is secreted into the urine with its intact signal peptide (3).
Because of the strong hydrophobicity of the signal
peptide apoM is insoluble in water. Gel permeation
chromatography showed that urinary apoM was part
of particles that are larger than apoM itself (⬃22 kDa)
but smaller than albumin. Thus, we suspect that urinary apoM is secreted in complex with phospholipids
to make it soluble in the urine. Of note, we did not
216 Mol Endocrinol, January 2006, 20(1):212–218
detect low-molecular weight apoM degradation products in the urine. Presumably, degradation of apoM
would abolish the lipocalin structure-associated binding capacity of apoM.
The megalin-mediated reabsorption of low-molecular weight plasma proteins in the kidney is probably of minor importance in the whole-body protein
metabolism as a mechanism for preserving amino
acids (20). However, it can be essential in vitamin
metabolism. Kidney-specific megalin deficiency results in vitamin D deficiency and bone malformation
(due to loss of vitamin D-binding protein in the urine)
(15, 21). Megalin is also essential for kidney uptake
of retinal-binding protein and transcobalamin-vitamin-B12 complexes (22). Interestingly, urine from
megalin knockout mice contains both sterols and
lipophilic vitamins (14). Individual lipocalins tend to
be able to bind several and quite different lipophilic
molecules (6). ApoD, for instance, binds cholesterol,
bilirubin, and arachidonic acid in vitro (23–26). It is
conceivable that the biological role of apoM, being a
lipocalin, in the kidney may involve the binding of
one or more small lipophilic substances in the tubule
lumen. Subsequent uptake of apoM, together with
its putative ligand, by megalin could help preserve
the lipophilic substance in the body, which would
make the present finding highly relevant to human
endocrinology. Recent results suggest that plasma
apoM plays an important role in plasma HDL metabolism and formation of pre-␤ HDL particles (2).
Also, short-term adenovirus-mediated apoM overexpression in hyperlipidemic mice resulted in markedly reduced atherosclerosis (2). The latter observation may be due to apoM-induced alterations in
plasma HDL metabolism but could also reflect a
unique antiatherosclerotic property of the apoM particle itself, e.g. due to the binding of a small lipophilic ligand (e.g. an antioxidant) in the lipocalinbinding pocket of apoM. It should be kept in mind,
however, that so far the ligand-binding properties of
apoM only have been deduced from bioinformatics
(5) and that the endogenous ligand(s) is unknown.
In conclusion, the results are compatible with the
idea that megalin is a receptor for apoM and that
megalin mediates binding and reuptake of apoM in the
kidney proximal tubule.
MATERIALS AND METHODS
Animals
Lox P sites were introduced into the murine megalin gene
locus to generate megalinlox/lox mice (with normal megalin
expression). Mice with kidney expression of Cre recombinase
(Cre) were generated by introducing a Cre transgene that is
driven by a fragment of the human apoE promoter. The two
lines were crossed to obtain mice with kidney-specific
deficiency of megalin (megalinlox/lox; apoECre mice) (15). Megalinlox/lox mice were used as controls.
Faber et al. • Megalin is a receptor for apoM
Surface Plasmon Resonance Analysis
Recombinant mouse apoM was generated by expression of
amino acid residues 22–190 of mouse apoM (the signal peptide sequences were excluded) in E. coli [strain BL21 (DE3);
Stratagene, La Jolla, CA (3)]. The expressed truncated apoM
was purified from inclusion bodies and refolded before binding and uptake studies (3). Binding of mouse apoM to immobilized rabbit megalin was studied with surface plasmon resonance analysis in a BIAcore 2000 instrument (Biacore,
Uppsala, Sweden). The binding kinetics were analyzed by
using Biaevaluation software version 3.1 (Biacore) as described elsewhere (27). Megalin and cubilin were purified
from human kidney (28, 29) and immobilized to the chip
surface at a density of 45 and 40 fmol/mm2. ApoM (0.05–2
␮M) was diluted in 10 mM HEPES, 150 mM NaCl, 1.5 mM
CaCl2, 1 mM EGTA, 0.005% Tween 20 (pH 7.4), and the same
buffer was used as running buffer in surface plasmon resonance studies.
Cellular Uptake of apoM
Brown Norway rat yolk sac sarcoma epithelial cells (30, 31)
were grown in serum-free HyQ medium on four chamber
glass slides (Nunc, Roskilde, Denmark). The cells were
washed three times with PBS (37 C) before incubation with
Alexa 488 (Invitrogen, Taastrup, Denmark)-conjugated
mouse apoM (1.8 ␮mol/liter) (green fluorescence) at 37 C. For
inhibition controls, sheep antirat megalin IgG and sheep nonimmune IgG (16) (100 ␮g/ml) were added simultaneously with
the Alexa 488-apoM conjugate. After 60 min, the cells were
washed five times with PBS and fixed in 4% paraformaldehyde for 60 min at 4 C in darkness. After washing with PBS
with 0.05% Triton X-100 the cells were incubated at 22–24 C
with a polyclonal rabbit antirat cubilin antibody (32) for 1 h
followed by incubation with an goat antirabbit IgG Alexa
594-conjugated antibody (red fluorescence) for 1 h at room
temperature. Finally, the slides were washed with PBS with
0.005% Triton X-100 and mounted in DAKO fluorescence
mounting medium (DAKO A/S, Glostrup, Denmark) and examined with a Zeiss LSM-510 confocal microscope (Carl
Zeiss, Jena, Germany).
Western Blotting
Urine and gel filtration fractions were analyzed by analytical
Western blotting as described elsewhere (3). In brief, proteins
were separated on 12% SDS-PAGE gels and transferred to
polyvinylidine difluoride membranes (Gelman, Lund, Sweden)
using semidry electroblotting. After quenching for 1 h in
buffer [50 mM Tris-HCl, 150 mM NaCl (pH 8.0), containing
0.5% (wt/vol) Tween 20, and 3% fish gelatin (Norland Products, Inc., Cranbury, NJ)], the membranes were incubated for
1 h at 22–24 C with polyclonal rabbit antimouse apoM antibodies (30 ␮g/ml) (3) or polyclonal rabbit antimouse apoA-I
antibodies (1:2560 dilution) (BioSite, Täby, Sweden) in the
same buffer. Antibody binding was visualized with horseradish peroxidase-coupled swine antirabbit IgG antibodies (1:
10.000 dilution) (DAKO A/S, Copenhagen, Denmark) and
5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt and
p-nitroblue tetrazolium (Sigma-Aldrich, Stockholm, Sweden).
Plasma apoM was quantified with a slightly different Western
blotting protocol (3) using a dilution series of wild-type mouse
plasma to generate a standard curve and a chemiluminescence reader (Fujifilm LAS-1000 Intelligent Dark Box II; Fujifilms, Trorod, Denmark) to measure intensities of the apoM
bands. Chemiluminescence readings were corrected for
background before calculations using the Image Reader
LAS-1000 Pro version 2.5 ImageGauge 4.0 program
(Fujifilms).
Faber et al. • Megalin is a receptor for apoM
Mol Endocrinol, January 2006, 20(1):212–218 217
Gel Filtration of Plasma and Urine
ApoM-containing particles in mouse plasma and urine were
separated by gel permeation chromatography on a Superose 6
HR 10/30 fast pressure liquid chromatography column (Pharmacia Biotech, Uppsala, Sweden). Samples were pooled from
three megalinlox/lox; apoECre or three megalinlox/lox mice and
passed through 0.22-␮m filters before loading 0.5 ml on the
column. The column was run at room temperature with PBS, 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 analysis.
5.
6.
7.
8.
mRNA Purification and cDNA Amplification
Total RNA was isolated from kidney and liver of megalinlox/lox;
apoECre and megalinlox/lox mice with Trizol (Life Technologies,
Taastrup, Denmark). First-strand cDNA was synthesized from
1 ␮g total RNA with Moloney murine leukemia virus reverse
transcriptase (40 U, Roche A/S, Avedore, Denmark) and random hexamer primers in 10-␮l reactions. The cDNA was used
for real-time PCR quantification of apoM mRNA with the
LightCycler (Roche, Copenhagen, Denmark) and primers for
mouse apoM and ␤-actin amplification, as described in Refs.
3 and 33, respectively. All quantifications were performed in
duplicate in separate runs.
9.
10.
11.
Acknowledgments
12.
We thank Karen Rasmussen for technical assistance. We
also thank Christian Jacobsen and Anne-Marie Bundsgaard
for helping with the BIAcore experiments. Dr. Thomas Willnow, Max-Delbrueck-Center for Molecular Medicine, Berlin,
Germany, generously provided the tissue, plasma, and urine
from megalinlox/lox; apoECre and megalinlox/lox mice.
13.
14.
Received May 25, 2005. Accepted August 5, 2005.
Address all correspondence and requests for reprints to:
Lars B. Nielsen, Department of Clinical Biochemistry KB3011,
Rigshospitalet, University of Copenhagen DK-2100, Denmark. E-mail: [email protected]; or Björn Dahlbäck, Division of
Clinical Chemistry, Department of Laboratory Medicine, Lund
University, University Hospital, Malmö S-205 02, Sweden,
E-mail: [email protected].
This work was supported by grants from the Network for
Cardiovascular Research (to K.F. and B.D.) funded by the
Swedish Foundation for Strategic Research, the Swedish Science Foundation (Grant 07143), the Heart and Lung Foundation,
Söderberg’s and Österlund’s Foundations, and research funds
from the University Hospital Malmö (all to B.D.) and Grant No.
02-1-2-24-22980 (to L.B.N.) from the Danish Heart Foundation,
Rigshospitalet, and the Boserup Foundation.
REFERENCES
1. Xu N, Dahlback B 1999 A novel human apolipoprotein
(apoM). J Biol Chem 274:31286–31290
2. Wolfrum C, Poy MN, Stoffel M 2005 Apolipoprotein M is
required for pre␤-HDL formation and cholesterol efflux to
HDL and protects against atherosclerosis. Nat Med 11:
418–422
3. Faber K, Axler O, Dahlback B, Nielsen LB 2004 Characterization of apoM in normal and genetically modified
mice. J Lipid Res 45:1272–1278
4. Zhang XY, Dong X, Zheng L, Luo GH, Liu YH, Ekstrom U,
Nilsson-Ehle P, Ye Q, Xu N 2003 Specific tissue expression and cellular localization of human apolipoprotein M
15.
16.
17.
18.
19.
20.
21.
as determined by in situ hybridization. Acta Histochem
105:67–72
Duan J, Dahlback B, Villoutreix BO 2001 Proposed lipocalin fold for apolipoprotein M based on bioinformatics
and site-directed mutagenesis. FEBS Lett 499:127–132
Akerstrom B, Flower DR, Salier JP 2000 Lipocalins: unity
in diversity. Biochim Biophys Acta 1482:1–8
Kerjaschki D, Farquhar MG 1982 The pathogenic antigen
of Heymann nephritis is a membrane glycoprotein of the
renal proximal tubule brush border. Proc Natl Acad Sci
USA 79:5557–5561
Chatelet F, Brianti E, Ronco P, Roland J, Verroust P 1986
Ultrastructural localization by monoclonal antibodies of
brush border antigens expressed by glomeruli. I. Renal
distribution. Am J Pathol 122:500–511
Christensen EI, Gliemann J, Moestrup SK 1992 Renal
tubule gp330 is a calcium binding receptor for endocytic
uptake of protein. J Histochem Cytochem 40:1481–1490
Kounnas MZ, Haudenschild CC, Strickland DK, Argraves
WS 1994 Immunological localization of glycoprotein 330,
low density lipoprotein receptor related protein and 39
kDa receptor associated protein in embryonic mouse
tissues. In Vivo 8:343–351
Zheng G, Bachinsky DR, Stamenkovic I, Strickland DK,
Brown D, Andres G, McCluskey RT 1994 Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/␣ 2MR, and
the receptor-associated protein (RAP). J Histochem Cytochem 42:531–542
Barth JL, Argraves WS 2001 Cubilin and megalin: partners in lipoprotein and vitamin metabolism. Trends Cardiovasc Med 11:26–31
Willnow TE, Hilpert J, Armstrong SA, Rohlmann A, Hammer RE, Burns DK, Herz J 1996 Defective forebrain development in mice lacking gp330/megalin. Proc Natl
Acad Sci USA 93:8460–8464
Leheste JR, Rolinski B, Vorum H, Hilpert J, Nykjaer A,
Jacobsen C, Aucouturier P, Moskaug JO, Otto A, Christensen EI, Willnow TE 1999 Megalin knockout mice as an
animal model of low molecular weight proteinuria. Am J
Pathol 155:1361–1370
Leheste JR, Melsen F, Wellner M, Jansen P, Schlichting
U, Renner-Muller I, Andreassen TT, Wolf E, Bachmann S,
Nykjaer A, Willnow TE 2003 Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect.
FASEB J 17:247–249
Moestrup SK, Kozyraki R, Kristiansen M, Kaysen JH,
Rasmussen HH, Brault D, Pontillon F, Goda FO, Christensen EI, Hammond TG, Verroust PJ 1998 The intrinsic
factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins. J Biol
Chem 273:5235–5242
Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen C,
Cui S, Christensen EI, Aminoff M, de la Chapelle A, Krahe
R, Verroust PJ, Moestrup SK 1999 The intrinsic factorvitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density
lipoprotein. Nat Med 5:656–661
Hammad SM, Barth JL, Knaak C, Argraves WS 2000
Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins. J Biol Chem 275:
12003–12008
Fyfe JC, Madsen M, Hojrup P, Christensen EI, Tanner
SM, de la Chapelle A, He Q, Moestrup SK 2004 The
functional cobalamin (vitamin B12)-intrinsic factor receptor is a novel complex of cubilin and amnionless. Blood
103:1573–1579
Christensen EI, Willnow TE 1999 Essential role of megalin
in renal proximal tubule for vitamin homeostasis. J Am
Soc Nephrol 10:2224–2236
Nykjaer A, Dragun D, Walther D, Vorum H, Jacobsen C,
Herz J, Melsen F, Christensen EI, Willnow TE 1999 An
218 Mol Endocrinol, January 2006, 20(1):212–218
22.
23.
24.
25.
26.
27.
28.
endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 96:507–515
Moestrup SK, Verroust PJ 2001 Megalin- and cubilinmediated endocytosis of protein-bound vitamins, lipids,
and hormones in polarized epithelia. Annu Rev Nutr 21:
407–428
Vogt M, Skerra A 2001 Bacterially produced apolipoprotein D binds progesterone and arachidonic acid, but not
bilirubin or E-3M2H. J Mol Recognit 14:79–86
Morais Cabral JH, Atkins GL, Sanchez LM, Lopez-Boado
YS, Lopez-Otin C, Sawyer L 1995 Arachidonic acid binds
to apolipoprotein D: implications for the protein’s function. FEBS Lett 366:53–56
Patel RC, Lange D, McConathy WJ, Patel YC, Patel SC
1997 Probing the structure of the ligand binding cavity of
lipocalins by fluorescence spectroscopy. Protein Eng 10:
621–625
Rassart E, Bedirian A, Do Carmo S, Guinard O, Sirois J,
Terrisse L, Milne R 2000 Apolipoprotein D. Biochim Biophys Acta 1482:185–198
Moestrup SK, Schousboe I, Jacobsen C, Leheste JR,
Christensen EI, Willnow TE 1998 ␤2-Glycoprotein-I (apolipoprotein H) and ␤2-glycoprotein-I-phospholipid complex harbor a recognition site for the endocytic receptor
megalin. J Clin Invest 102:902–909
Kozyraki R, Kristiansen M, Silahtaroglu A, Hansen C,
Jacobsen C, Tommerup N, Verroust PJ, Moestrup SK
1998 The human intrinsic factor-vitamin B12 receptor,
Faber et al. • Megalin is a receptor for apoM
29.
30.
31.
32.
33.
cubilin: molecular characterization and chromosomal
mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region. Blood 91:
3593–3600
Moestrup SK, Nielsen S, Andreasen P, Jorgensen KE,
Nykjaer A, Roigaard H, Gliemann J, Christensen EI 1993
Epithelial glycoprotein-330 mediates endocytosis of
plasminogen activator-plasminogen activator inhibitor
type-1 complexes. J Biol Chem 268:16564–16570
Le Panse S, Galceran M, Pontillon F, Lelongt B, van de
Putte M, Ronco PM, Verroust PJ 1995 Immunofunctional
properties of a yolk sac epithelial cell line expressing two
proteins gp280 and gp330 of the intermicrovillar area of
proximal tubule cells: inhibition of endocytosis by the
specific antibodies. Eur J Cell Biol 67:120–129
Le Panse S, Ayani E, Nielsen S, Ronco P, Verroust P,
Christensen EI 1997 Internalization and recycling of glycoprotein 280 in epithelial cells of yolk sac. Eur J Cell Biol
72:257–267
Sahali D, Mulliez N, Chatelet F, Laurent-Winter C, Citadelle D, Roux C, Ronco P, Verroust P 1992 Coexpression
in humans by kidney and fetal envelopes of a 280 kDacoated pit-restricted protein. Similarity with the murine
target of teratogenic antibodies. Am J Pathol 140:33–44
Bartels ED, Lauritsen M, Nielsen LB 2002 Hepatic expression of microsomal triglyceride transfer protein and
in vivo secretion of triglyceride-rich lipoproteins are increased in obese diabetic mice. Diabetes 51:1233–1239
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