The endocytic receptor megalin binds the iron transporting
neutrophil-gelatinase-associated lipocalin with high affinity and
mediates its cellular uptake
Vibeke Hvidberga, Christian Jacobsena, Roland K. Strongb, Jack B. Cowlandc,
Søren K. Moestrupa, Niels Borregaardc,*
aInstitute of Medical Biochemistry, University of Aarhus, Denmark
bDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, USA
cThe Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet 4042, 9 Blegdamsvej, DK-2100 Copenhagen Ø, Denmark
Received 17 November 2004; revised 1 December 2004; accepted 1 December 2004
Available online 24 December 2004
Edited by Lukas Huber
a prominent protein of specific granules of human neutrophils
also synthesized by epithelial cells during inflammation. NGAL
binds bacterial siderophores preventing bacteria from retrieving
iron from this source. Also, NGAL may be important in deliver-
ing iron to cells during formation of the tubular epithelial cells of
the primordial kidney. No cellular receptor for NGAL has been
We show here that megalin, a member of the low-density lipo-
protein receptor family expressed in polarized epithelia, binds
NGAL with high affinity, as shown by surface plasmon reso-
nance analysis. Furthermore, a rat yolk sac cell line known to
express high levels of megalin, endocytosed NGAL by a mecha-
nism completely blocked by an antibody against megalin.
? ? 2004 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Neutrophil-gelatinase-associated lipocalin (NGAL) is
Keywords: NGAL; Lipocalin; Megalin; Siderophore; Iron
Lipocalins constitute a family of more than 30 proteins,
found in species as separate as insects and man [1,2]. Lipoca-
lins share a common structural fold, often in the absence of
significant sequence homology, that forms a ligand-binding
site (calyx) that is typically lined with hydrophobic residues
[3,4]. Lipocalins function in general as transport proteins,
e.g., odorant-binding protein, bilin-binding protein, and reti-
nol-binding protein, but the physiological ligands have not
been identified for all lipocalins. Lipocalins are extracellular
proteins but some are involved in transport of substances that
must be delivered intracellularly to exert a function, e.g., reti-
nol. Very little is known about the receptors that mediate cel-
lular uptake of lipocalins and their ligands. It is not known
whether the ligands are presented by the lipocalins to ligand
receptors on cells or whether the lipocalins are taken up by
receptors and then deliver their cargo intracellularly . It is
assumed that most lipocalins are taken up by membrane recep-
tors but only two have been identified so far. One is a novel 51
kDa protein which binds tear-lipocalin (lipocalin-1 (Lcn-1))
and consequently is known as Lcn-1-Interacting Membrane
Protein [6,7]. The other receptor is megalin , a multi-ligand
endocytosis receptor that is expressed on a variety of epithelia,
primarily such that have a high absorptive capacity such as
tubular epithelial cells of kidneys, ileum, choroid plexus, and
yolk sac . Megalin belongs to the low density lipoprotein
receptor family . Megalin has been shown to bind the mouse
lipocalins retinol-binding protein, a1-microglobulin, mouse
major urinary protein, and odorant-binding protein [5,10].
We discovered a major human lipocalin, neutrophil-gelatin-
ase-associated lipocalin (NGAL) (also known as lipocalin-2
(lcn-2) or siderocalin), and have characterized its tissue expres-
sion . NGAL is constitutively expressed in human neutro-
phils but is also induced in epithelial cells when these are
engaged in inflammation [12–14]. This tissue expression indi-
cates that NGAL is involved in innate immunity. This notion
was supported when siderophores were identified as NGAL li-
gands  and followed by the recent demonstration of de-
creased survival of mice with a targeted disruption of the
NGAL (lcn-2) after intraperitoneal challenge with Escherichia
coli . Siderophores are extremely strong iron chelators se-
creted by microorganisms when iron is limiting . Sidero-
phores can extract iron out of iron-binding proteins such as
transferrin and lactoferrin for subsequent uptake by specific
siderophore receptors on the microorganisms. In this way,
microorganisms can secure a supply of this essential nutrient
at the expense of their host . It has been shown that apo-
NGAL is able to prevent siderophore producing E. coli from
dividing and that this restrain on bacterial growth is alleviated
by supplying iron in excess of the iron-siderophore binding
capacity of NGAL . However, such antimicrobial activity
can be expected to be temporary because several microorgan-
isms secrete proteases that may degrade NGAL even though
NGAL is known to be a very protease resistant molecule
. NGAL would therefore be expected to have a more sus-
tained antimicrobial effect, if NGAL is taken up by host cells.
Abbreviations: BN, Brown Norway; Lcn-1, lipocalin-1; Lcn-2, lipo-
calin-2; LRP, LDL-receptor related protein; NGAL, neutrophil-gela-
tinase-associated lipocalin; RAP, receptor associated protein; SPR,
surface plasmon resonance
*Corresponding author. Fax: +45 3545 4295.
E-mail address: email@example.com (N. Borregaard).
0014-5793/$30.00 ? 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS 29182 FEBS Letters 579 (2005) 773–777
This has been further indicated by the recent observation that
the mouse ortholog of NGAL, 24p3, is expressed in the fetal
kidney and delivers iron to cells at the stage where differentia-
tion of the budding kidney epithelia takes place during organ-
ogenesis of the kidneys [19,20]. The rapid clearance of NGAL
from the circulation indicates a mechanism of cellular uptake
. In addition, NGAL has been inferred as a factor inducing
apoptosis through a receptor mediated mechanism in IL-3
dependent cells such as bone marrow cells in the mouse ,
but this has not been confirmed .
Since none has yet been identified, we decided to test
whether megalin could also function as a receptor for NGAL.
2. Materials and methods
Recombinant human NGAL was generated either as apo-NGAL,
i.e., without bacterial siderophore, in Sf9 insect cells using the Baculo-
virus technique as previously described , or as NGAL with E. coli
siderophore as detailed in [15,25], with the modification that 50 ml
overnight culture of E. coli was added to 500 ml LB medium and incu-
bated for 90 min. Then, 0.1 mM IPTG and 0.1 mg/ml desferoxamine
(Desferal, Roche) were added to induce bacterial growth (IPTG) and
secure iron deprivation and enterobactin production by the bacteria.
After 3 h, 0.1 mg/ml FeCl3was added and the bacteria incubated for
another 90 min. The bacteria were then pelleted by centrifugation
and formed a bright red pellet. The bacteria were lysed and rhNGAL
was isolated as described . The isolated NGAL was scanned as de-
scribed from 250 to 800 nm, and showed the characteristic peaks at 330
and 500 nm indicative of enterobactin–Fe3+complex in the lipocalin
pocket . These were absent in the apo-NGAL from Sf9 cells (Fig.
1). It was ascertained that desferoxamine did not bind to apo-NGAL
by incubating apo-NGAL with desferoxamine and FeCl3.
Mouse monoclonal anti-NGAL antibody was used for Western blot-
ting . NGAL was quantitated as described by Kjeldsen et al. .
Megalin was purified by receptor-associated protein (RAP) affinity
chromatography from human kidney cortex according to standard
procedures . Human LDL-receptor related protein (LRP) was puri-
fied by a2macroglobulin-affinity chromatography as described .
Purified sheep polyclonal antibodies against rat megalin, previously de-
scribed , were used to block uptake of NGAL by megalin. Purified
sheep non-immune IgG served as negative control.
3. Surface plasmon resonance analysis
The binding to megalin and LRP was studied by surface
plasmon resonance (SPR) analysis on a Biacore 2000 instru-
ment (Biacore, Sweden). The procedure was as follows: Bia-
core sensor chips type CM5 were activated with a 1:1
mixture of 0.2 M N-ethyl-N0-(3-dimethylaminopropyl)carbo-
diimide and 0.05 M N-hydroxysuccimide in water according
to the manufacturer?s instructions. Megalin and LRP were
immobilized at a concentration of 10 lg/ml in 10 mM sodium
acetate, pH 4.5, and the remaining binding sites were blocked
with 1 M ethanolamine, pH 8.5. A control flow cell was made
by performing the activation and blocking procedures only.
The resulting receptor densities were 25–30 fmol receptor/
mm2. Samples were dissolved in 10 mM HEPES, 150 mM
NaCl, 1.5 mM CaCl2, 1.0 mM EGTA, and 0.005% Tween
20, pH 7.4, or in 10 mM HEPES, 150 mM NaCl, 20 mM
EGTA, and 0.005% Tween 20, pH 7.4. Sample and running
buffer were identical. Regeneration of the sensor chip after
each analysis cycle was performed with 1.6 M glycine–HCl
buffer, pH 3.0. The Biacore response is expressed in relative re-
sponse units (RU), i.e., the difference in response between pro-
tein and control flow channel (an activated but uncoupled flow
cell). Kinetic parameters were determined by BIAevaluation
4.1 software using a Langmuir 1:1 binding model and simulta-
neous fitting of all curves in the concentration range consid-
ered (global fitting).
4. Cellular uptake of NGAL
Megalin-expressing Brown Norway (BN) rat yolk sac epithe-
lial cells transformed with mouse sarcoma virus (BN cells) 
were grown on two-chamber Permanox slides (Nunc, Ros-
kilde, Denmark) and washed in PBS, pH 7.4. Cells were then
incubated for 1 h at 37 ?C in HyQ-CCM5 serum-free medium
(Hyclone, Logan, Utah) containing 1% BSA (w/v) and ?60 lg/
ml rhNGAL labeled with Alexa-488 (Molecular Probes, Lei-
den, The Netherlands). To some cells was also added sheep
polyclonal anti-rat megalin IgG antibody (200 lg/ml) or sheep
non-immune IgG antibody (200 lg/ml). After incubation, cells
were washed in PBS, pH 7.4, and fixed in 4% formaldehyde for
1 h at 4 ?C. Subsequently, cells were washed in PBS, pH 7.4,
containing 0.05% Triton X-100 and incubated with rabbit
anti-rat cubilin antibody  (10 lg/ml) in this buffer for 1 h
at room temperature in order to visualize the megalin-express-
ing structures of the cells. After washing in PBS, 0.05% Triton
X-100, cells were incubated for 1 h at room temperature with
Alexa-594-conjugated secondary anti-rabbit IgG (Molecular
Probes) diluted 1:200 in the same buffer. Slides were washed
in PBS, pH 7.4, mounted in Dako fluorescent mounting med-
ium (DakoCytomation, Glostrup, Denmark) and analyzed
Fig. 1. Absorption spectrum of NGAL. NGAL (1.2 mg/ml) with siderophore (A) and without siderophore (B) in PBS was scanned against PBS.
V. Hvidberg et al. / FEBS Letters 579 (2005) 773–777
using a Zeiss LSM-510 confocal microscope (Zeiss, Jena,
To test the hypothesis that NGAL might bind to megalin,
studies were conducted using SPR technique with matrix
bound megalin. Fig. 2 shows that binding (Kd= ?60 nM) of
apo-NGAL to megalin occurs with high affinity. Similar affin-
ity was measured with siderophore-bound NGAL (not shown).
The NGAL binding was prevented by EDTA, indicating that
the LDL-receptor type-A repeats of megalin are involved
in the binding of NGAL. Since this motif is also found in
the LRP , we tested the binding of NGAL to LRP. How-
ever, only a very weak signal was recorded (not shown) indi-
cating that this receptor has no major role in NGAL clearance.
The role of megalin in cellular uptake of NGAL was further
investigated in a rat yolk sac cell line that is known to express
high amounts of megalin . Fig. 3 shows that endocytosis of
fluorescently labeled NGAL by this cell line was extensive as
seen by the appearance of intracellular NGAL (Alexa-488,
green flourescence). A sheep polyclonal anti-megalin antibody
completely prevented cellular uptake of NGAL by these cells
(Fig. 3B), thus indicating that megalin is involved in mediating
the cellular uptake of NGAL by the cells. Cubilin, a receptor
colocalizing with megalin and recycling between coated pits
and endosomes, was visualized with Alexa-594 coupled anti-
body (red flourescense). The different localization of NGAL
and cubilin indicates that internalized NGAL is not recycled
but segregated from the receptor and targeted to endosomes
Neutrophil-gelatinase-associated lipocalin appears to be a
protein of importance in iron metabolism both during organ-
ogenesis [19,20] and in host defense [15,16], and possibly also
in tumorigenesis [33,34]. The ability of NGAL to sequester
iron and thus prevent its utilization by growing microorgan-
isms has been demonstrated in vitro , and NGAL/24p3
has been shown to mediate uptake of iron by the developing
kidney . Although not a priori apparent, it is highly likely
that the ability of NGAL to prevent iron utilization by micro-
organisms as demonstrated in vitro is ultimately dependent on
cellular uptake of iron, since NGAL might be degraded by
(microbial) proteases and the siderophore-bound iron re-
trieved by microorganisms if NGAL is not taken up by the
host epithelial cells. Certainly, the essential role of NGAL in
inducing epithelial cell differentiation in the developing kidney
is dependent on cellular uptake and delivery of NGAL bound
iron to the epithelial cells . It is similarly likely – but en-
tirely speculative – that the expression of NGAL by a variety
of epithelial tumors endows these with an iron retrieving mech-
anism that adds to the growth potential of the tumors [33–35],
and thus that for NGAL to exert its function, be it in host de-
fense or during cellular growth and differentiation, a cellular
receptor is needed which mediates uptake of NGAL with high
affinity. We demonstrate here that megalin acts as such a cel-
lular receptor for NGAL. Megalin is also known to bind an-
other iron-binding protein expressed and secreted from
human neutrophils, lactoferrin [36,37]. Although the ligands
for megalin are very diverse, positively charged amino acids
have been shown to be critical for binding of several ligands
to this receptor . The pI of NGAL of 8.4 distinguishes
NGAL from most other lipocalins . A number of positive
charges can by identified on NGAL from analysis of its crys-
talline structure . The affinity of megalin for NGAL as
determined by plasmon resonance is much higher than that ob-
served for other lipocalins and may likely relate to the unique
positive charge of NGAL among lipocalins.
The tissue expression of megalin fits well with the induction
of NGAL expression during inflammation. NGAL is highly
expressed by type 2 pneumocytes during inflammation .
These also express megalin . NGAL is also highly expressed
by epithelial cells of the intestines during inflammation .
These also express megalin .
It is noteworthy that we were not able to show any selectivity
of megalin for siderophore NGAL versus apo-NGAL. It may
be argued that in this way apoNGAL may decrease the
Fig. 2. Surface plasmon resonance analysis of the binding of NGAL to
purified megalin. Binding of 500 nM and 1 lM NGAL to megalin. No
binding of NGAL was seen in the presence of calcium-complexing
Fig. 3. Confocal fluorescence microscopy analysis of uptake of Alexa-488-labeled NGAL in BN cells. BN cells were incubated for 1 h with rhNGAL
(green) in the absence (A) or in the presence (B) of anti-megalin IgG or non-immune IgG (C). The red color represents Alexa-594 staining for cubilin,
a receptor colocalizing with megalin .
V. Hvidberg et al. / FEBS Letters 579 (2005) 773–777
efficiency by which megalin takes up siderophore-NGAL – this
would however only be a problem if the ability of megalin to
bind and endocytose NGAL is a limiting factor. The studies
of uptake indicate that the capacity of megalin to endocytose
NGAL is very high.
Our studies do not exclude the possibility that other recep-
tors other than megalin may be involved in cellular uptake
Acknowledgments: This study was supported by grants from the Dan-
ish Medical Research Council. The expert technical support of Char-
lotte Horn is greatly appreciated.
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