Báo cáo khóa học: Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells potx

Surface nucleolin participates in both the binding and endocytosis
of lactoferrin in target cells
Dominique Legrand
1
, Keveen Vigie
´
1
, Elias A. Said
2
, Elisabeth Elass
1
, Maryse Masson
1
,
Marie-Christine Slomianny
1
, Mathieu Carpentier
1
, Jean-Paul Briand
3
, Joe¨ l Mazurier
1
and Ara G. Hovanessian
2
1
Unite
´
de Glycobiologie Structurale et Fonctionnelle et Unite
´
Mixte de Recherche n°8576 du CNRS, Institut Fe
´

de
´
ratif de Recherche
n°118, Universite
´
des Sciences et Technologies de Lille, Villeneuve d’Ascq, France;
2
Unite
´
de Virologie et Immunologie Cellulaire,
URA 1930 CNRS, Paris, France;
3
Institut de Biologie Mole
´
culaire et Cellulaire, UPR 9021 CNRS, Strasbourg, France
Lactoferrin (Lf), a multifunctional molecule present in
mammalian secretions and blood, plays important roles in
host defense and cancer. Indeed, Lf has been reported to
inhibit the proliferation of cancerous mammary gland epi-
thelial cells and manifest a potent antiviral activity against
human immunodeficiency virus and human cytomegalo-
virus. The Lf-binding sites on the cell surface appear to be
proteoglycans and other as yet undefined protein(s). Here,
we isolated a Lf-binding 105 kDa molecular mass protein
from cell extracts and identified it as human nucleolin.
Medium–affinity interactions ( 240 n
M
) between Lf and
purified nucleolin were further illustrated by surface plas-
mon resonance assays. The interaction of Lf with the cell

surface-expressed nucleolin was then demonstrated through
competitive binding studies between Lf and the anti-human
immunodeficiency virus pseudopeptide, HB-19, which binds
specifically surface-expressed nucleolin independently of
proteoglycans. Interestingly, binding competition studies
between HB-19 and various Lf derivatives in proteoglycan-
deficient hamster cells suggested that the nucleolin-binding
site is located in both the N- and C-terminal lobes of Lf,
whereas the basic N-terminal region is dispensable. On intact
cells, Lf co-localizes with surface nucleolin and together they
become internalized through vesicles of the recycling/deg-
radation pathway by an active process. Morever, a small
proportion of Lf appears to translocate in the nucleus of
cells. Finally, the observations that endocytosis of Lf is
inhibited by the HB-19 pseudopeptide, and the lack of Lf
endocytosis in proteoglycan-deficient cells despite Lf bind-
ing, point out that both nucleolin and proteoglycans are
implicated in the mechanism of Lf endocytosis.
Keywords: lactoferrin; surface nucleolin; receptor binding;
HIV; cancer.
Lactoferrin (Lf) is an 80 kDa iron-binding glycoprotein
found in external secretions (mainly milk) and in the
secondary granules of leukocytes. It has important func-
tions, such as modulation of the inflammatory response and
inhibition of cancer cell proliferation [1,2]. Lf has also been
reported to have potent antiviral activity against human
immunodeficiency virus (HIV)-1 and human cytomegalo-
virus infection in in vitro cell cultures [3–5]. In the case of its
anti-HIV activity, Lf appears to inhibit virus binding and/or
entry into permissive cells [5]. Although most Lf-binding

sites on cells are reported to be proteoglycans [6,7],
additional sites have also been proposed on the surface of
lymphocytes, platelets, mammary gland cells and entero-
cytes [8–11]. Accordingly, a partially characterized protein
of 105 kDa molecular mass [8], the lipoprotein receptor-
related protein (LRP) [11], and an enterocytic protein of
136 kDa molecular mass [10], have been proposed as
complementary receptors for Lf.
The events that follow the binding of Lf to cells have not
been clearly established. In lymphocytes, Lf was shown to
differentiate cells by activating pathways mediated by the
mitogen-activated protein kinase (MAPK) [12], most
probably through Lf–receptor interactions on the surface
of cells. Furthermore, it was proposed that Lf acts as a
gene trans-activator through MAPK signaling [13]. On the
other hand, the antiproliferative activities of Lf on
cancerous cells favour endocytosis and nuclear targeting
mechanisms. Indeed, Lf has been found in the nucleus of
human leukemia K562 cells and was shown to bind distinct
DNA sequences [14,15]. In our laboratory, Lf was shown
to induce the growth arrest of human breast carcinoma
cells, MDA-MB-231, at the G1 to S transition [16]. This
latter effect is associated with both inhibition of Cdk2 and
Cdk4 activities and increase of the Cdk inhibitor p21
expression. Although Lf binding to proteoglycans seems
essential for its activity on MDA-MB-231 cells, the
Correspondence to D. Legrand, Unite
´
de Glycobiologie Structurale
et Fonctionnelle, UMR CNRS 8576, Universite

´
des Sciences
et Technologies de Lille, 59655 Villeneuve d’Ascq cedex, France.
Fax: + 33 320436555, Tel.: + 33 320337238,
E-mail:
Abbreviations: AZT, azidothymidine; bLf, bovine Lf isolated from
milk; bLfc, bovine lactoferricin (residues 17–41 of bLf); CHO, Chinese
hamster ovary; FITC, fluorescein isothiocyanate; HB-19,
5[Kw(CH
2
N)PR]-TASP; HB-19-biotin, HB-19 labeled with biotin;
HB-19-fluo, HB-19 labeled with FITC; hLf, human Lf isolated from
milk; hLf-biotin, hLf labeled with biotin hydrazide; hTf, human
transferrin; Lf, lactoferrin; TRITC, tetrarhodamine isothiocyanate.
(Received 28 August 2003, revised 13 November 2003,
accepted 17 November 2003)
Eur. J. Biochem. 271, 303–317 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03929.x
involvement of higher affinity binding sites has been
hypothesized [7].
Nucleolin, a major ubiquitous 105 kDa nucleolar protein
of exponentially growing eukaryotic cells, has been des-
cribed as a cell surface receptor for several ligands, such as
matrix laminin-1, midkine, attachment factor J, apo-B and
apo-E lipoproteins [17–21]. This RNA-binding phospho-
protein was found primarily in the nucleus where it is
involved in the regulation of cell proliferation and growth,
cytokinesis, replication, embryogenesis and nucleogenesis
[22]. More recently, nucleolin has been described as a shuttle
between the cell surface and the nucleus [17,21,23] and it was
proposed as a mediator for the extracellular regulation of

nuclear events [22]. The transport of nucleolin to the cell
surface implies an alternative secretion pathway that is
independent of the classical pathway of secretion through
the endoplasmic reticulum (ER) and Golgi apparatus [23].
Furthermore, nucleolin is tightly associated with the intra-
cellular actin at the cell surface [23]. Finally, surface
nucleolin was reported as an attachment target for some
viruses, such as HIV [23–26].
Consistently, a 105 kDa protein (identified in the present
work as human nucleolin) was retained on an affinity matrix
containing purified Lf. In view of this and of a previous
observation pointing out that nucleolin serves as a binding
protein for various ligands, we investigated the implication
that surface nucleolin is a putative Lf receptor. Using
proteoglycans expressing mutant Chinese hamster ovary
cells (CHO), cancerous mammary gland cells MDA-MB-
231, and HB-19 (an anti-HIV pentameric pseudopeptide
that binds specifically to nucleolin) [23–26], we show that in
addition to proteoglycans, surface nucleolin is a major cell
surface Lf-binding site and participates in Lf endocytosis.
We also partially delineate the nucleolin-binding site in Lf.
Materials and methods
Cells
All cells were obtained from the American Type Culture
Collection (ATCC). They were maintained in a humidified
atmosphere of 95% air and 5% CO
2
at 37 °C and in cell
culture media containing 10% (v/v) heat-inactivated fetal
bovine serum. The human breast tumour cell line MDA-

MB-231 was grown in Eagle’s minimal essential medium, as
described previously [16]. Three CHO cell lines were used
and propagated in Ham’s F12 medium: wild-type cells
(CHO K1); mutant cells defective in heparan-sulfate
proteoglycan expression (CHO 677); or mutant cells
defective in heparan- and chondroitin-sulfate proteoglycan
expression (CHO 618) [27]. The human T lymphocyte cell
lines Jurkat and MT-4 were routinely grown in RPMI-1640,
and HeLa-CD4-LTR-lacZ cells (HeLa P4 cells) were
cultured in Dulbecco’s modified Eagle’s medium, as
described previously [8,19,25,26]. The HIV-1 LAI isolate
was propagated and purified as reported previously [25].
Proteins
Native human Lf (hLf) was purified from fresh human milk
(obtained from a single donor) by ion-exchange chroma-
tography, as described previously [28]. Bovine Lf (bLf) was
kindly provided by Biopole (Brussels, Belgium). Chicken egg
white lysozyme and human transferrin (hTf) were purchased
from Sigma. In order to avoid possible steric hindrance of
the interactions of the hLf polypeptide with nucleolin, hLf
used for microscopy studies was labeled with biotin
hydrazide (Pierce, Rockford, IL, USA) through its glycan
moiety after mild periodate oxidation of N-acetylneuraminic
acid residues, as described previously [9]. Radioiodination of
hLf was carried out as described previously [8]. The purity
of native Lf and Lf derivatives used in the experiments
was confirmed by the migration of single protein bands in
SDS/PAGE.
Antibodies
Antibodies to hLf and nonimmune rabbit polyclonal sera

were obtained from healthy hLf-injected and nonimmunized
rabbits, respectively. Mouse mAbs to nucleolin, clones
3G4B2 and D3, were purchased from Upstate biotechnology
(Lake Placid, NY, USA) and provided by Dr J. S. Deng [29],
respectively. Mouse mAb against either human EEA1 or hTf
receptor (CD71), rabbit polyclonal antibodies to human
caveolin-1, and goat fluorescein isothiocyanate (FITC)-
conjugated anti-rabbit Ig were from Becton-Dickinson
Biosciences. Rabbit polyclonal antibodies against human
lysosomal protein LAMP-1/CD107A were from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Goat FITC- or
tetrarhodamine isothiocyanate (TRITC)-conjugated anti-
rabbit IgG were obtained from Sigma. Rabbit Alexa Fluor
546-labeled anti-mouse IgG was from Molecular Probes
(Eugene, OR, USA). Donkey Texas Red dye (TR) conju-
gated anti-rabbit polyclonal Ig were from Jackson Immuno
Research Laboratories, Inc. (West Grove, PA, USA).
Polyclonal antibodies against the C-terminal fragment of
human nucleolin (residues 345–706) produced in Escheri-
chia coli, were raised in a rabbit. Briefly, 5 lgofthetotal
RNA preparation from Jurkat cells was reverse transcribed
into first-strand cDNA using oligo dT primers (Stratagene)
and 20 U of reverse transcriptase MMLV (Promega). The
truncated nucleolin was generated by PCR amplification
using total first-strand cDNA as template and the following
oligonucleotides: 5¢-TGGTATGACTAGGAAATTTGGT
TATGTG-3¢ and 5¢-GACAGAAGCTATTCAAACTTC
GTCTTC-3¢. The PCR product was subcloned in plasmid
pGEX-4T-2 (Amersham Pharmacia Biotech), in-frame to
glutathione S-transferase. The nucleolin-derived protein

was produced in E. coli BL21 cells transformed with the
expression plasmid and purified by passing the cell lysate
through a 1 mL glutathione Sepharose 4B column (Amer-
sham Pharmacia Biotech). After washing, the gel was
incubated with thrombin (Amersham Pharmacia Biotech)
(50 U in 1 mL of NaCl/P
i
) overnight at 20 °Cwithgentle
mixing. The 40 kDa protein released from the gel was
injected into rabbit.
Preparation of Lf derivatives
Mild enzymatic digestion of hLf gave the N-terminally
deleted proteins hLf
)2N
(residues 3–692), hLf
)3N
(residues
4–692) and hLf
)4N
(residues 5–692) [6], the 30 kDa hLf N-t
(residues 4–283), 50 kDa hLf C-t (residues 284–692) and
18 kDa hLf N2 (residues 91–255) fragments [30]. rhLf
EGS
,
304 D. Legrand et al. (Eur. J. Biochem. 271) Ó FEBS 2003
a recombinant hLf whose sequence 28RKVRGPP34 was
replaced with EGS (the 365–367 C-terminal counterpart of
sequence 28–34), was produced in a baculovirus expression
system, as reported previously [6]. The 30 kDa bLf N-t and
50 kDa bLf C-t fragments, which are homologous to their

human counterpart, were obtained from bLf, as previously
described [31]. An octadecapeptide, CFQWQRNMRKVR
GPPVSC, corresponding to residues 20–37 of hLf, was
chemically synthesized by Dr A. Tartar (Pasteur Institute of
Lille, Lille, France). Lactoferricin B (bLfc), a cationic
antimicrobial peptide isolated by pepsin digestion of bLf
(residues 17–41) was a gift from Morinaga Milk Industry
(Tokyo, Japan). The purity of proteins and peptides was
assessed by the presence of a single band on SDS/PAGE
stained with Coomassie blue. The absence of protease
activity in protein fractions was tested by incubating
aliquots of proteins with azocoll substrate (Sigma) in
NaCl/P
i
for 1–6 h at 37 °C and according to the manufac-
turer’s instructions.
Preparation and labeling of HB-19
The HB-19 pseudopeptide 5[Kw(CH
2
N)PR]-TASP
mimicks the gp120 V3 loop of HIV, binds specifically to
the C-terminal domain of nucleolin and is a potent inhibitor
of HIV entry into permissive cells [25,32,33]. The template
presents pentavalently the tripeptide Kw(CH
2
N)PR, where
w(CH
2
N) represents a reduced peptide bond between lysine
and proline residues. The synthesis of HB-19, and its

labeling with fluorescein (HB-19-fluo) or biotin (HB-19-
biotin), were as described previously [32].
Affinity chromatography studies
Purified hLf was immobilized on an Ultralink hydrazide gel
(Pierce), according to the manufacturer’s instructions, and
used to study the binding of proteins from MDA-MB-231
cell lysates. Two milligrams of protein was bound per mL of
Ultralink hydrazide gel. A total of 50 · 10
6
MDA-MB-231
cells were washed twice with NaCl/P
i
and lysed in NaCl/P
i
,
1% Triton-X-100 (w/v) containing 1 m
M
of the protease
inhibitor Pefabloc [4-(2-aminoethyl)-benzenesulfonyl fluor-
ide] (Roche Diagnostics, Mannheim, Germany) for 1 h at
4 °C. After centrifugation at 10 000 g for 30 min, the
supernatant was recovered, diluted 10-fold with NaCl/P
i
containing 1 m
M
Pefabloc and incubated overnight at 4 °C
with 150 lL of hLf-Ultralink gel (250 lg of protein). The
hLf-Ultralink gel was collected by centrifugation at 600 g
for 5 min and washed with 10 mL of NaCl/P
i

. The proteins
bound to the gel were sequentially eluted with two volumes
of 200 lLof0.5
M
NaCl in 20 m
M
sodium phosphate
buffer, pH 7.4, two volumes of 200 lLof1
M
NaCl in this
buffer, two volumes of 0.2
M
glycine/HCl, pH 2.3, contain-
ing 0.5% (v/v) Triton-X-100, and 300 lL of 10% (w/w)
SDS. Polypeptides in 100 lL of each fraction were separ-
ated by SDS/PAGE in 7.5% (w/v) acrylamide gels that were
then stained with Coomassie Brilliant Blue.
MS analysis
To identify the 105 kDa protein eluted from the hLf-
Ultralink affinity chromatography, the stained protein band
in the SDS/PAGE gel was cut from the gel and treated as
described previously [34]. MS measurements were made on
a Voyager DE-STR MALDI-TOF instrument (Applied
Biosystems, Foster City, CA) and proteins were identified
according to their tryptic peptide mass fingerprint after
database searching using
PROTEIN PROSPECTOR
(http://
prospector.ucsf.edu).
Purification of extranuclear nucleolin from Jurkat cells

Extranuclear nucleolin was prepared by lysis of 0.9–
1.2 · 10
9
NaCl/P
i
-washed Jurkat cells at 4 °Cfor1hin
25 mL of 20 m
M
Tris/HCl, pH 7.6, 150 m
M
NaCl, 5 m
M
MgCl
2
,5 m
M
b-mercaptoethanol, 0.5% (v/v) Triton X-100,
1m
M
Pefabloc and Complete (Roche Diagnostics), a
protease inhibitor cocktail. The nuclei were pelleted by
centrifugation at 1000 g for 5 min and the supernatant was
then centrifuged at 12 000 g for 10 min prior to storage at
)80 °C. A rapid two-step chromatography procedure was
used to purify nucleolin from nucleus-free extracts. All steps
were performed at 4 °C using ice-cold buffers and columns
in the presence of 1 m
M
Pefabloc and Complete protease
inhibitor cocktail. The cytoplasmic extract of Jurkat cells

(25 mL) was diluted 10-fold with 20 m
M
sodium phosphate,
pH 7.0, and passed through a 5 mL DEAE–Sepharose Fast
Flow column (Amersham Pharmacia Biotech). After wash-
ing the column with 150 mL of 20 m
M
sodium phosphate,
pH 7.0, elution of the adsorbed proteins was performed
with 10 mL of the same buffer containing 1
M
NaCl. The
eluant was diluted 10-fold with 50 m
M
Tris/HCl, pH 7.9,
5m
M
MgCl
2
,0.1m
M
EDTA, 1 m
M
b-mercaptoethanol
(buffer A) and loaded onto a 1 mL Heparin–Sepharose
column (Amersham Pharmacia Biotech) equilibrated with
the same buffer. The gel was washed with 20 mL of buffer A
containing 0.2
M
ammonium sulfate, and proteins were

eluted in 50 lL fractions with 2 mL of buffer A containing
0.6
M
ammonium sulfate. Five or six eluted fractions
contained a single 105 kDa protein band corresponding
to nucleolin, as confirmed by immunoblotting with anti-
nucleolin Ig. Nucleolin was pooled and dialyzed against
NaCl/P
i
containing 1 m
M
Pefabloc at 4 °Cfor2hbefore
storage at )80 °C. A further control on a 7.5% SDS
acrylamide gel, stained with Coomassie blue, confirmed the
presence of the purified nucleolin as a single 105 kDa
protein band. Two 70 and 50 kDa protein bands, corres-
ponding to partial degradation products of nucleolin
[23,32], were observed in amounts lower than 5% of the
total protein.
Analysis of Lf binding to nucleolin in a surface plasmon
resonance biosensor
All materials and chemicals were from BIAcore AB
(Uppsala, Sweden). Analyses were performed at 25 °Con
a BIAcore 3000 biosensor, and Hepes-buffered saline (HBS-
EP) was used as a running buffer and for the dilution of
ligands and analytes. Human nucleolin, purified from the
extranuclear fraction of Jurkat cells, was immobilized at a
concentration of 1.6 lgÆmL
)1
in 0.1

M
sodium acetate,
pH 5, at a flow rate of 10 lLÆmL
)1
,toaCM5sensor
chip that had been previously activated according to the
manufacturer’s instructions. Covalent binding resulted in a
Ó FEBS 2003 Nucleolin is a cell surface lactoferrin-binding site (Eur. J. Biochem. 271) 305
signal of 4200 resonance units (RU). An empty flow cell was
used as a control for nonspecific binding and bulk effects.
The ligand concentrations and a flow rate of 10 lLÆmL
)1
were found to avoid mass-transport limitations and rebind-
ing. Human Lf was injected at seven concentrations,
ranging from 40–2560 n
M
, in HBS-EP. Each sample was
injected for 1 min followed by dissociation buffer flow for
1 min. After the dissociation phase, the sensor chip was
regenerated by injection of 5 lLof10m
M
HCl at a flow
rate of 10 lLÆmL
)1
. After subtraction of the blank sensor-
gram, kinetic rate constants were calculated from an overlay
of the sensorgrams of all Lf concentrations using a method
based on the Langmuir’s 1 : 1 binding model (
BIAEVALUA-
TION

3.1 software).
Analysis of the inhibition of HB-19 binding
to CHO cells by Lf and Lf derivatives
The inhibition of HB-19 binding to CHO cells was
investigated by fluorescence flow cytometry on a FACScal-
ibur flow cytometer (Becton-Dickinson). Preconfluent cells,
propagated in six-well cell culture plates (Nalge Nunc,
Rochester, NY, USA), were removed from plastic using the
nonenzymatic cell dissociation solution (Sigma) and gentle
pipetting. Pooled cells were washed twice with NaCl/P
i
.
They were then resuspended in fresh RPMI containing 1%
heat-inactivated fetal bovine serum and distributed into
1.5 mL centrifuge tubes ( 500 000 cells per tube). The cells
were incubated at 15 °C for 45 min in 100 lL of cell culture
medium containing 1 l
M
HB-19-fluo and 0–8 l
M
hLf.
After seven washes with NaCl/P
i
, the cells were analyzed by
flow cytometry. Binding specificity and reversibility controls
were performed with 0–50 l
M
unlabeled HB-19. For studies
on the nucleolin-binding site of Lf, CHO 618 cells were
incubated at 15 °C for 45 min in 100 lL of cell culture

media containing 1 l
M
HB-19-fluo and one of the follow-
ing: unlabeled HB-19 (50 l
M
), hTf, bLf, hLf or hLf and bLf
protein variants and peptides (8 l
M
). To assess potential
interactions between hLf and HB-19, hLf (1 l
M
)was
incubated with HB-19-biotin (1 l
M
) in 1 mL of NaCl/P
i
for
1 h at room temperature and the mixture was then mixed
with 50 lL of streptavidin-agarose (Sigma) for 30 min.
After seven washes with NaCl/P
i
, the agarose was boiled
and submitted to SDS/PAGE. Coomassie blue staining was
used to detect hLf bound to HB-19.
125
I-labeled Lf-binding assays
The binding parameters of
125
I-labeled hLf to CHO lines
were investigated on cells grown to preconfluency in 12-well

culture plates in Ham’s F12 containing 10% fetal bovine
serum. In some experiments, cells were incubated with
Ham’s F12 containing 1% fetal bovine serum for 12 h prior
to performing the binding assays. Cells were then incubated
for 1 h at 4 °C with 250 lL of 0–3 l
M
125
I-labeled hLf in
Ham’s F12 containing 1% fetal bovine serum. Non-specific
binding was measured in the presence of a 100-fold molar
excess of unlabeled hLf. Cells were washed seven times with
fresh Ham’s F12 medium containing 1% fetal bovine
serum, and then lysed with 0.1
M
NaOH. The cell lysates
were recovered for gamma counting. For the binding
competition assays between hLf and HB-19, CHO cells were
incubated at 15 °Cfor45minwith1l
M
125
I-labeled hLf
and 0–100 l
M
HB-19. Washes were performed five times
with NaCl/P
i
containing 1% BSA and twice with NaCl/P
i
containing 0.3
M

NaCl, prior to cell lysis and counting.
Binding experiments to MDA-MB-231 cells
Preconfluent MDA-MB-231 cells, propagated in six-well
cell culture plates (Nalge Nunc) in Eagle’s medium
containing 10% fetal bovine serum, were removed from
plastic using a cell dissociation solution (Sigma) and gentle
pipetting. After two washes with NaCl/P
i
, the cells were
resuspended in fresh Eagle’s medium containing 1% fetal
bovine serum and incubated at 15 °Cfor45minwith1l
M
HB-19-fluo, 1 l
M
hLf-biotin or rabbit polyclonal antibodies
to nucleolin (1 : 200 dilution). A similar dilution of
antibodies from a nonimmunized rabbit was used as a
control. These ligands were presented to cells either alone or
in the presence of competitors, as described in the legend of
Fig. 6. Cells incubated with hLf-biotin and anti-nucleolin Ig
were washed five times with NaCl/P
i
and further incubated
with streptavidin-FITC (1 : 2000) and FITC-labeled anti-
rabbit IgG (1 : 4000), respectively. After five washes with
NaCl/P
i
and two washes with NaCl/P
i
containing 0.3

M
NaCl, the fluorescence intensity was measured by flow
cytometry.
Confocal microscopy
Indirect immunofluorescence staining and confocal micros-
copy were used to visualize the fate of hLf in MDA-MB-231
cells and its co-localization with nucleolin and endosome
markers. For these experiments, cells were grown on eight-
well glass slides (Laboratory-Tek Brand Products, Naper-
ville, IL, USA) coated with collagen. Cells in Eagle’s
medium containing 10% fetal bovine serum were incubated
at 15 or 37 °C for 1–14 h with 3 l
M
hLf-biotin, alone or in
the presence of either polyclonal anti-nucleolin rabbit Ig
(1 : 100) or mouse mAb to the hTf receptor (CD71)
(1 : 200). Thirty minutes before the end of incubation at
37 °C, the ligand-containing medium was replaced with
fresh 37 °C-warmed Eagle’s medium containing 10% fetal
bovine serum, to allow endocytosis of the cell bound ligand.
Cells were washed a further five times with NaCl/P
i
and
twice with NaCl/P
i
containing 0.3
M
NaCl, prior to fixation
with 4% paraformaldehyde in NaCl/P
i

(4 °C, 30 min). Cells
were then washed with NaCl/P
i
, permeabilized with 0.15%
Triton-X-100 in NaCl/P
i
(20 °C, 2 min), washed again,
blocked by 1% ethanolamine in NaCl/P
i
(4 °C, 20 min) and
extensively washed with NaCl/P
i
containing 1% BSA. The
treated cells were incubated (37 °C, 45 min) with FITC-
conjugated streptavidin (1 : 800) and TRITC-conjugated
goat anti-rabbit IgG (1 : 800) or Alexa Fluor 546-labeled
rabbit anti-mouse IgG (1 : 800). In some co-localization
experiments, cells, following endocytosis of hLf-biotin, were
fixed and incubated with antibodies against endosome
markers (37 °C, 45 min): rabbit polyclonal antibodies
against human lysosomal protein LAMP-1/CD107A
(1 : 50); mouse mAb against human EEA1 (1 : 100), a
protein specifically associated to early endosomes; and
rabbit polyclonal antibodies to human caveolin-1 (1 : 100),
which are specific for caveolae, nonclathrin membrane
306 D. Legrand et al. (Eur. J. Biochem. 271) Ó FEBS 2003
invaginations. Fluorescence staining was performed with
FITC-conjugated streptavidin, TRITC-conjugated goat
anti-rabbit IgG and/or Alexa Fluor 546-labeled rabbit
anti-mouse IgG, as reported above. After extensive washing

with NaCl/P
i
containing 1% BSA, cells were examined
using an LSM 510 confocal microscopic system (Carl Zeiss,
Esslingen, Germany). Procedures used to evidence capping
of surface nucleolin on MT-4 cells and endocytosis of hLf
into CHO cells [19,26], are briefly described in the legends
of Figs 5 and 9, respectively.
Results
The purified hLf is functional as an inhibitor of cell
proliferation and virus infection
Lf from fresh human milk was purified as described
previously [28]. This purified preparation inhibited the
proliferation of breast cancer MDA-MB-231 cells in a
dose-dependent manner, as reported previously [16]. In
[
3
H]thymidine incorporation experiments, the 50% inhibi-
tion of cell proliferation was observed at 50 lgÆmL
)1
(0.62 l
M
) Lf (data not shown). To study its antiviral
activity, we investigated the action of hLf on infection of
HeLa-CD4-LTR-lacZ cells (HeLa P4 cells) by the HIV-1
LAI isolate. HIV entry and replication in HeLa P4 cells
resulted in activation of the HIV long terminal repeat
(LTR), leading to expression of the lacZ gene. Conse-
quently, the b-galactosidase activity could be measured in
cell extracts to monitor HIV entry into cells [25]. The value

of the b-galactosidase activity obtained in the presence of
the HIV-replication inhibitor, azidothymidine (AZT), is
referred to as the background value in a given experiment.
Another control for the inhibition of HIV infection was
obtained by the nucleolin-binding anti-HIV pseudopeptide,
HB-19, that, by its capacity to bind the cell-surface
expressed nucleolin, blocks HIV attachment to cells and
thus inhibits HIV infection [25,35]. Consistent with previous
reports [3,4], hLf inhibited, in a dose-dependent manner,
HIV infection of HeLa P4 cells with a 50% inhibitory
concentration (IC
50
) value of 0.25 l
M
. A complete inhibi-
tion of HIV infection was observed at 2 l
M
Lf (Fig. 1A). As
a preliminary step to investigate the mechanism of the
inhibitory effect of hLf on HIV infection, we investigated
HIV attachment to HeLa P4 cells. AZT had no effect,
whereas the HB-19 pseudopeptide, as expected, completely
inhibited HIV attachment [25,35]. Interestingly, we found
that Lf is a very potent inhibitor of HIV attachment to cells
(Fig. 1B). These observations indicated that our purified
preparation of hLf was functionally active as far as its
antiproliferative (not shown) and antiviral (Fig. 1) activities
were concerned.
Nucleolin is an hLf-binding protein
To investigate major Lf-binding proteins in total extracts

of cancerous human mammary gland MDA-MB-231
cells, affinity chromatography was performed on immo-
bilized hLf. A complex pattern of protein bands was
retained on Ultralink-immobilized hLf and eluted by
increasing salt concentrations. One of the major proteins
that were preferentially and quantitatively retained on
immobilized hLf was a 105 kDa protein, which mostly
eluted at 0.5
M
NaCl (data not shown, see the Materials
and methods). This band was not observed in a control
experiment using Ultralink-immobilized hTf (data not
shown). Trypsin degradation of the 105 kDa protein
band generated peptides whose molecular ion masses
were used for identification by MALDI-TOF. As shown
in Table 1, the measured masses of seven out of nine
Fig. 1. Human lactoferrin (hLf) inhibition of HIV entry by blocking
virus particle attachment to cells. HIV entry (A) and attachment (B)
were assayed in HeLa P4 cells, as described previously, at 37 and
20 °C, respectively [25]. (A) Entry of the HIV-1 isolate, LAI, was
monitored in HeLa P4 cells by expression of the lacZ gene (corres-
ponding to b-galactosidase) under the control of the HIV-1 LTR. Cells
were infected in the presence of azidothymidine (AZT) (5 l
M
), HB-19
(1 l
M
), or hLf (0.25, 0.50, 1, 2 or 4 l
M
). The b-galactosidase activity

was measured at 48 h postinfection (at an absorbance of 570 nm). The
mean ± SD of triplicate assays of a representative experiment is
shown. (B) Assay of HIV-1 LAI attachment was performed in the
presence of AZT, HB-19, or hLf (as above). The concentration of the
HIV-1 core protein p24 was measured in cell extracts as an estimation
of the amount of HIV attached to cells. The mean ± SD of triplicate
assays of a representative experiment is shown.
Table 1. MALDI-TOF identification of the 105 kDa protein bound to Ultralink-immobilized lactoferrin (Lf) as human nucleolin.
Measured molecular ion masses
used for identification by MALDI-TOF Computed masses Human nucleolin residues
812.4 811.9
555
LELQGPR
561
1178.7 1178.3
411
EVFEDAAEIR
420
1561.8 1561.6
611
GFGFVDFNSEEDAK
624
1595.0 1594.8
524
GYAFIEFASFEDAK
537
1649.0 1648.8
349
FGYVDFESAEDLEK
362

2312.7 2312.6
298
VEGTEPTTAFNLFVGNLNFNK
318
2501.8 2501.8
487
TLVLSNLSYSATEETLQEVFEK
508
Ó FEBS 2003 Nucleolin is a cell surface lactoferrin-binding site (Eur. J. Biochem. 271) 307
major peptides between 812.39 and 2501.79 Da matched
with the computed masses of peptides between residues
298 and 624 of human nucleolin (Swiss-Prot accession
number P19338). Finally, the identity of the 105 kDa
band as nucleolin was further confirmed by immunoblot-
ting using a mAb specific for human nucleolin (3G4B2)
(data not shown).
Lf binds to human nucleolin through medium–affinity
interactions
Mainly characterized as a nucleolar protein, nucleolin is
continuously expressed on the surface of different types of
cells along with its intracellular pool within the nucleus and
cytoplasm. Surface and cytoplasmic nucleolin are similar
and can be differentiated from nucleolar nucleolin by their
distinct isoelectric points, occurring, most probably, as a
consequence of post-translational modifications [23]. To
assess hLf binding to surface and/or cytosolic nucleolin, we
isolated the protein from the extranuclear pool and
investigated the binding parameters and kinetics in a surface
plasmon resonance biosensor. Jurkat cells were used as a
source for human nucleolin because they express substantial

amounts of surface nucleolin [20] and can conveniently be
cultured at a preparative scale. Furthermore, some reports
have proposed the existence of a 105 kDa hLf receptor on
dividing Jurkat cells [6,8]. Because of a high susceptibility to
proteolysis, a new procedure was implemented to prepare
purified nucleolin. This procedure takes advantage of the
ability of nucleolin to bind both anionic heparin and
cationic groups owing to the presence of large acidic
stretches in its N-terminal domain [36]. Rapid chromato-
graphy steps, together with the use of potent protease
inhibitors, allowed the preparation of pure nucleolin, which
was then immediately immobilized via its amino groups
onto the sensor chip. The overlay surface plasmon reson-
ance plots of the raw data, from a representative experiment
out of three, are shown in Fig. 2A. As shown, both
association and dissociation phases were fairly rapid. The
association rate constant (k
on
) and dissociation rate con-
stant (k
off
)were6.89±0.46· 10
5
M
)1
Æs
)1
and 0.164 ±
0.001 s
)1

, respectively, using Langmuir’s one-site model,
which gave the best fit at all concentrations used (v
2
<2).
The equilibrium dissociation constant (K
d
), calculated from
the ratio of the kinetic rate constants (k
off
/k
on
), was
238 ± 15 n
M
, a value very similar to that calculated from
the extent of binding observed near equilibrium using a
Scatchard plot (249 ± 45 n
M
) (Fig. 2B). R
max
estimated at
2005 ± 50 RU, correlates well with the maximal bind-
ing expected for hLf to sensorchip-immobilized nucleolin
(3200 RU). These results demonstrated that hLf binds with
fast kinetics and medium affinity to nucleolin. It should be
noted that under similar conditions, hTf did not bind
nucleolin (data not shown).
Evidence for hLf binding to proteoglycan-independent
sites on cells
CHO mutant cells [27], wild-type CHO K1 cells, and

mutant cell lines defective in the expression of heparan-
sulfate (CHO 677 cells) or both heparan- and chondroitin-
sulfate proteoglycans (CHO 618 cells), are convenient cell
lines for using to determine the role of proteoglycans in the
mechanism of interaction of a given ligand with its cell
surface receptor(s). As the expression of surface nucleolin is
not modified in these cell lines, they have been recently used
to demonstrate that the anti-HIV pseudopeptide, HB-19,
binds surface nucleolin [24,26]. As shown in Fig. 3A, the
highest hLf binding was observed on CHO K1 cells, which
express both heparan- and chondroitin-sulfate proteogly-
cans (660 000 ± 60 000 sites), while  50% binding was
noted on CHO 677 cells (294 000 ± 42 000 sites) and only
about 15% on proteoglycan-free CHO 618 cells (105 000 ±
8000 sites). Interestingly, Scatchard plots of the binding
curves showed the presence of at least two classes of binding
sites on CHO K1 and 677 cells, the highest affinity class
being the only one still remaining on CHO 618 cells
(Fig. 3B). These proteoglycan-independent sites on CHO
618 cells have a K
d
of 0.43 ± 0.01 · 10
)6
M
, comparable
with the one measured between hLf and purified nucleolin.
Hence, it can be assumed that the lower affinity sites on
CHO K1 (K
d
¼ 2.6 ± 0.1 · 10

)6
M
and n ¼ 555 000 ±
20 000) and CHO 677 (K
d
¼ 2.1 ± 0.3 · 10
)6
M
and
n ¼ 190 000 ± 10 000) cells are relevant to the presence
of proteoglycans.
Fig. 2. Surface plasmon resonance sensorgram of the binding of human
lactoferrin (hLf) to human extranuclear nucleolin. The raw data shown
are representative of a set of three experiments. Human nucleolin,
purified from nucleus-free extracts of Jurkat cells, was immobilized
onto a CM5 sensorchip. Human Lf, at different concentrations
(40–2560 n
M
), was incubated with immobilized nucleolin and analyzed
on a BIAcore 3000 apparatus. (A) Surface plasmon resonance sen-
sorgrams. (B) Binding curve and the Scatchard plot derived from these
data at equilibrium (insert). RU, response unit.
308 D. Legrand et al. (Eur. J. Biochem. 271) Ó FEBS 2003
Evidence that nucleolin is the major proteoglycan-
independent hLf-binding site on cells
The presence of proteoglycan-independent hLf-binding sites
on CHO cells led us to investigate the possible involvement
of surface nucleolin. Hence, competition experiments for
the binding to cell surface nucleolin were performed
between hLf and the nucleolin-specific pseudopeptide,

HB-19 [23–26], under experimental conditions that prevent
nonspecific HB-19 binding to proteoglycans (0.3
M
NaCl-
containing NaCl/P
i
washes) [26]. Those conditions were
found not to influence hLf binding to the high-affinity sites
on CHO K1, 677 and 618 cells (not shown). It should also
be noted that we found no specific or nonspecific interaction
between Lf and HB-19 (data not shown).
As demonstrated in Fig. 4A, up to 75% inhibition of
hLf binding to the three CHO lines was obtained with
100-molar excesses of HB-19. Interestingly, the inhibitory
effect was also observed, although to a slightly lower extent
(70%), on proteoglycan-free CHO 618 cells. This provides
direct evidence that competition between the two molecules
occurs for binding to surface nucleolin. Competition
between hLf and HB-19 for nucleolin binding was further
confirmed by experiments using HB-19-fluo (Fig. 4B). In
these experiments, HB-19 binding to cells was efficiently
inhibited by hLf. Indeed, inhibition rates increased to 62, 90
and 75% for CHO K1, 677 and 618 cells, respectively, with
maximal inhibitions at much lower concentrations (2–4
molar excesses) of the hLf competitor. Such efficient
inhibition could be attributed, in part, to a larger steric
hindrance effect exerted by hLf ( 80 kDa) as compared to
HB-19 ( 0.3 kDa). Lastly, it can be noted that HB-19 was
more significantly displaced by hLf at lower concentrations
on CHO 618 than on CHO K1 and 677 cells (Fig. 4B). This

is probably a result of the fact that the cell-surface binding
of HB-19 is mostly due to nucleolin [24,26], whereas the
binding of Lf to the cell surface implicates several molecules,
including mainly proteoglycans and nucleolin. Taken
together, our results suggest that nucleolin is the major
proteoglycan-independent Lf-binding site on CHO cells. As
the C-terminal tail of nucleolin is the site for HB-19 binding
Fig. 3. The presence, on cells, of human lactoferrin (hLf)-binding site(s)
different from proteoglycans. Binding experiments were performed by
incubating wild-type Chinese hamster ovary (CHO) K1 and the
mutant cell lines CHO 677 (heparan sulfate-deficient proteoglycans)
and CHO 618 (heparan and chondroitin sulfate-deficient proteogly-
cans), with
125
I-labeled hLf at concentrations ranging from 0 to 3 l
M
.
(A) Specific binding of hLf to CHO K1 (d), CHO 677 (j)andCHO
618 (r) cells. (B) Scatchard analysis of the data showing two classes of
hLf-binding sites on CHO K1 and CHO 677 cells in contrast to a single
class on CHO 618 cells. Data shown represent mean values ± SEM of
three experiments conducted in duplicate.
Fig. 4. Competition between human lactoferrin (hLf) and HB-19 for
binding to Chinese hamster ovary (CHO) wild-type and mutant cell lines.
(A) Inhibition, by HB-19, of hLf binding to CHO cells. CHO cells were
incubated (45 min, 15 °C) with 1 l
M
125
I-labeled hLf and 0–100 molar
excesses of HB-19. Data are expressed as percentages ± SEM from

radioactivity bound to CHO K1 (d), CHO 677 (j)orCHO618(r)
cells without HB-19. (B) Inhibition of HB-19 binding to the CHO
mutant cells by hLf. CHO cells were incubated (45 min, 15 °C) with
1 l
M
HB-19-fluo and 0–8 l
M
hLf. The intensity of green fluorescence
associated with the cells was measured by flow cytometry. Data are
expressed as mean percentages ± SEM for three separate experiments,
performed in duplicate, from total HB-19 bound to CHO K1 (d),
CHO 677 (j)orCHO618(r) cells without hLf.
Ó FEBS 2003 Nucleolin is a cell surface lactoferrin-binding site (Eur. J. Biochem. 271) 309
[26], our results, showing the efficient competition between
Lf and HB-19 for binding to nucleolin, suggest that the
C-terminal tail of nucleolin should be implicated in the
mechanism of binding of Lf to nucleolin.
In general, the crosslinking of a ligand leads to the clus-
tering or capping of its surface receptor. Accordingly, we
investigated the distribution of surface nucleolin following
the crosslinking of bound hLf using rabbit polyclonal
antibodies (Fig. 5). For these studies we used a human T
lymphocyte cell line, MT-4, which is suitable for studies
investigating the capping of surface antigens [19,26]. The
binding of hLf to MT-4 cells was carried out at 20 °Cbefore
washing of the cells and further incubation with anti-Lf Ig to
induce the lateral aggregation of surface-bound hLf.
Partially fixed cells were then incubated with the mAb, D3
(specific for human nucleolin), to reveal the steady state
distribution of nucleolin at the plasma membrane. Under

these experimental conditions, the nucleolin signal was
patched at one pole of the cell, which coincided with the hLf
signal (Fig. 5A). On the other hand, in control cells treated
similarly, but in the absence of hLf, the nucleolin signal was
evenly distributed in the plasma membrane in a diffused
state (Fig. 5B). Such a ligand-dependent capping of surface
nucleolin is a specific event because the distribution of
another surface protein, CD45, was not affected (data not
shown; see Fig. 1 in ref. [26]).
The two lobes of Lf, but not its basic N-terminal
region, bind to surface nucleolin
The basic sequences 2RRRR5 and 28RKVR31, located at
the N terminus of hLf, have been reported to contribute to
most of the ionic hLf interactions, particularly with
proteoglycans and nucleic acids [6,37,38]. The sequence
28RKVR31 was also proposed as a candidate for the
binding of hLf to its hypothetical receptor expressed on
lymphocytes [6]. In order to investigate the domain in Lf
implicated in its interaction with nucleolin, we investigated
the capacity of various Lf constructs and derivatives to
inhibit the binding of HB-19-fluo to CHO 618 cells
(Fig. 6A,B). Consistent with the proteoglycan-independent
binding of HB-19 to the cell-surface expressed nucleolin
[24,26], hLf
)2N
,hLf
)3N
,hLf
)4N
and rhLf

EGS
inhibited the
binding of HB-19-fluo to CHO 618 cells to an extent similar
to that of the native hLf. The noninvolvement of sequence
28RKVR31 of hLf was further confirmed by the lack of
inhibition of HB-19 binding to cells by synthetic peptide hLf
20–37 and its bovine counterpart, bLfc. Interestingly, a
strong inhibition of HB-19 binding was observed with hLf
N-t, hLf C-t or hLf N2 fragments, thus suggesting the
presence of nucleolin-binding patterns in both lobes of hLf
and, more particularly, in the N2 domain. Furthermore,
possible nonspecific ionic interactions between Lf and
nucleolin were ruled out by the observation that the basic
hen egg lysozyme (pI 10.5–11) had no effect on the binding
of HB-19. Similarly, the iron-binding hTf had no effect. On
the other hand, bLf inhibited the binding of HB-19 to cells.
In accordance with the involvement of both lobes of hLf in
the mechanism of Lf binding to nucleolin, the bLf N-t and,
at a somewhat lower extent, the bLf C-t, were also inhibitory
(Fig. 6B). These observations further confirm the inter-
action of Lf with surface nucleolin and illustrate that the
nucleolin-binding sequences in Lf are not species specific.
Lf binds to nucleolin at the surface of human cancerous
mammary gland cells
A previous study on MDA-MB-231 cells demonstrated the
presence of low affinity hLf-binding sites corresponding to
heparan-sulfate proteoglycans and higher affinity sites
(K
d
¼ 45–123 n

M
)thatrepresented 10% of the total
binding (2.6–3.2 · 10
5
sites per cell) [7]. We investigated
whether surface nucleolin is expressed on MDA-MB-231
cells and if it accounts for the proteoglycan-unrelated
binding of hLf to cells. To achieve this, flow cytometry cell-
binding experiments were performed using HB-19-fluo in
Fig. 5. Capping of surface nucleolin as a result of surface-bound human lactoferrin (hLf). (A) MT-4 cells were incubated in the presence (+ Lf) of
1 l
M
hLfat20 °C for 30 min before further incubation (20 °C for 60 min) in the presence of rabbit immune serum (1 : 50) raised against hLf. After
partial fixation in 0.25% paraformaldehyde, the co-aggregation of hLf with nucleolin was investigated using the murine mAb D3 against human
nucleolin [23]. (B) The same experiment as described in (A) but without hLf as a control. The rabbit antibodies were revealed by Texas Red dye (TR)
conjugated donkey anti-rabbit Ig, whereas the murine antibody was revealed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse
IgG. A cross-section for each staining is shown with the merge of the two colors and the respective phase contrast. Experimental conditions have
been described previously [19,26].
310 D. Legrand et al. (Eur. J. Biochem. 271) Ó FEBS 2003
experimental conditions that avoid binding to proteogly-
cans; i.e. washing with NaCl/P
i
that contains 0.3
M
NaCl. In
addition, the expression of surface nucleolin and its fate
following binding with Lf were investigated by confocal
microscopy using the biotin-labeled hLf (hLf-biotin)
and polyclonal antibodies against the C-terminal part of
nucleolin (residues 345–706).

The cell surface expression of nucleolin in MDA-MB-
231 cells was illustrated by flow cytometry experiments
using anti-nucleolin Ig (Fig. 7A) and HB-19-fluo (Fig. 7B).
A marked binding competition occurred between either
HB-19-fluo and 8-molar excesses of hLf (84% inhibition of
HB-19-fluo binding) (Fig. 7B) or hLf-biotin and 50-molar
excesses of HB-19 (87% inhibition) (Fig. 7C). Therefore,
nucleolin is present on MDA-MB-231 cells and acts as a
functional hLf-binding site. It is interesting to note that
only a slight effect on hLf binding was observed in the
presence of the polyclonal antibodies against nucleolin
(Fig. 7C), suggesting that these antibodies do not react
with the extreme conserved C-terminal tail of nucleolin
that constitutes the binding site for ligands of nucleolin,
such as HB-19 [26], midkine [19] and Lf (the results
herein). In view of their efficient binding to surface nucleo-
lin and to their weak competition with hLf, these antibodies
were further used in our microscopy co-localization
experiments.
Lf complexed to nucleolin is internalized into cancerous
human mammary gland cells
The binding of both hLf and anti-nucleolin polyclonal Ig
to MDA-MB-231 cells was studied by fluorescence micro-
scopy (Fig. 8A). Interestingly, fluorescence staining revealed
that both hLf and anti-nucleolin Ig were located at common
and distinct clusters on cells (Fig. 8A). When hLf-biotin
was incubated in competition with a 20–50 molar excess of
unlabeled HB-19, no distinct hLf clusters were detected on
the surface of cells (not shown).
Fig. 6. Location of nucleolin-binding sites in both lobes, but not in the basic N-terminus of lactoferrin. (A) Schematic linear representation of the

human lactoferrin (hLf) derivatives used in binding competition studies with HB-19 on Chinese hamster ovary (CHO) 618 cells. Numbers at the
ends of the strips correspond to the first and last amino acid residues of polypeptides. The dotted lines show the approximate locations of the four
structural domains: N1 and N2 domains (N-t lobe) and C1 and C2 domains (C-t lobe) [52]. The black boxes in the strips show the location of basic
sequences 1-GRRRR-5 and 28-RKVRGPP-34. (B) CHO 618 cells were incubated (45 min, 15 °C) with 1 l
M
HB-19-fluo and 8 l
M
hLf derivatives:
intact hLf, the N-terminally deleted proteins hLf
)2N
,hLf
)3N
and hLf
)4N
, recombinant hLf mutated at residues 28–34 (rhLf
EGS
), the 30 kDa (hLf
N-t), 50 kDa (hLf C-t) and 18 kDa (hLf N2) hLf tryptic fragments, and a synthetic octadecapeptide corresponding to residues 20–37 of hLf (hLf
20–27); bLf polypeptides: intact bLf, the 30 kDa (bLf N-t) and 50 kDa (bLf C-t) tryptic fragments of bLf and bovine lactoferricin (bLfc); control
molecules: HB-19 (50 l
M
), hTf and chicken egg white lysozyme (8 l
M
). The intensity of green fluorescence associated with the cells was measured
by flow cytometry. Data are expressed as mean percentages ± SEM for three separate experiments, performed in duplicate, from the total HB-19
bound to CHO 618 cells without hLf.
Ó FEBS 2003 Nucleolin is a cell surface lactoferrin-binding site (Eur. J. Biochem. 271) 311
Previous studies showed a growth arrest effect on
several cancerous mammary gland cell lines incubated for
12–24 h with hLf [16,39], but its possible endocytosis was

not investigated. Double immunofluorescence microscopy
was therefore performed on cells incubated at 37 °Cwith
both biotinylated hLf and anti-nucleolin polyclonal Ig.
Figure 8B,C shows that cells incubated with hLf-biotin at
37 °C exhibited intense intracellular fluorescent punctu-
ated green patterns that were found randomly throughout
the cell. Furthermore, fluorescent clusters were also
observed in the nucleus of some cells (shown with arrows
in Fig. 8B), suggesting the translocation of Lf into the
nucleus. Such nuclear signals became prominent in the
nucleus of most of cells upon longer incubation periods
(12 h) with hLf-biotin (data not shown). Cells incubated
at 15 °CwithhLf,at37°C in the presence of 1 m
M
sodium azide, or growth-arrested by overnight incubation
in medium containing 1% fetal bovine serum prior to
incubation at 37 °C, exhibited no or very little endocytosis
of hLf (not shown). Interestingly, the results indicate that
anti-nucleolin Ig co-localize with hLf in most of the
endocytic vesicles. To investigate the nature of the
endosome compartment containing nucleolin-bound hLf,
co-localization experiments were performed with markers
specific for clathrin-dependent and -independent endocy-
tosis pathways. The results presented in Fig. 8C demon-
strate that most of the hLf-containing vesicles co-localized
with EEA1, a marker specifically associated with clathrin
in early endosomes. Consistent with this, the hLf-
containing vesicles co-localized with the transferrin recep-
tor, CD71 (data not shown). On the other hand, hLf did
not co-localize with caveolin-1 (data not shown), a major

protein constituent of caveolae implicated in endocytosis
via a clathrin-independent pathway [40]. Our results
demonstrate that hLf complexed with surface nucleolin
undergo active endocytosis into MDA-MB-231 cells via
the clathrin-dependent pathway.
Endocytosis of hLf requires both surface-expressed
nucleolin and proteoglycans
In a series of experiments using confocal immunofluores-
cence laser microscopy, we demonstrated that endocytosis
of hLf occurs in different types of cells (HeLa, MDA-MB-
231 and MT-4). Such endocytosis occurs at 37 °C, but not
at 20 °C, indicating that it uses an active internalization
process, consistent with other nucleolin-binding ligands
[19,23]. Endocytosis of hLf at 37 °Cwasalsotime
dependent, reaching saturation at 60–90 min (data not
shown). The results presented in Figs 3 and 4 suggest that
both proteoglycans and nucleolin are implicated in the
overall amount of hLf in CHO cell lines that bind to the cell
surface. In view of this, we investigated endocytosis of hLf
in CHO cell lines, the wild-type K1 cells and the proteo-
glycan-deficient cell lines CHO 677 and 618. Consistently,
we found that hLf becomes internalized at 37 °CintoCHO
K1 cells but not into CHO 677 cells deficient in heparan-
sulfate expression (Fig. 9A) or into CHO 618 cells deficient
in both heparan- and chondroitin-sulfate expression (data
not shown). Under similar experimental conditions, hLf was
found at the plasma membrane in both CHO K1 and CHO
677 cells (Fig. 9B), as expected from the results shown in
Figs 3 and 4. Therefore, despite efficient binding to the cell
surface, heparan-sulfate proteoglycan expression is required

for hLf endocytosis (Fig. 9). In addition, expression of
heparan-sulfate proteoglycans in CHO K1 cells is not
sufficient for hLf endocytosis, as the nucleolin-binding HB-
19 pseudopeptide at a concentration of 1 l
M
completely
prevents endocytosis in these cells (data not shown, similar
to that presented for CHO 677 cells in Fig. 9A). In further
experiments, we demonstrated that endocytosis of hLf in
CHO K1 cells is significantly enhanced or reduced in serum-
activated or serum-starved cells, respectively. Interestingly,
the expression of surface nucleolin is also enhanced or
decreased with serum stimulation or starvation, respectively
[23]. Our results suggest that both heparan sulfates and
nucleolin are involved in the endocytosis of hLf into CHO
K1 cells.
Fig. 7. Binding of human lactoferrin (hLf) to nucleolin expressed on
MDA-MB-231 cells. The figure shows the green fluorescence intensity
bound to cells in a set of three representative flow cytometry experi-
ments. (A) Binding of rabbit polyclonal antibodies, directed against
residues 345–706 of nucleolin, to MDA-MB-231 cells. The figure dis-
plays a typical profile of nonstained cells (none) and of those incubated
with antibodies to nucleolin (1 : 200 dilution) (Anti-Nucl pAb). The
control is represented by cells incubated with nonimmune rabbit serum
(1 : 200 dilution) and immunostained as described for the other sam-
ples (Non-immune pAb control). Immunostaining was achieved with
fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG. (B) Binding
of HB-19 to MDA-MB-231 cells and its inhibition by hLf. The figure
displays a typical profile of cells incubated without HB-19-fluo (none),
with 1 l

M
HB-19-fluo alone (HB-19-fluo) or with 1 l
M
HB-19-fluo in
thepresenceof50l
M
HB-19 (HB-19-fluo + HB-19) or 8 l
M
hLf (HB-
19-fluo + hLf). (C) Binding of hLf to MDA-MB-231 cells and its
inhibition by HB-19. The figure displays a typical profile of cells
incubated with 1 l
M
hLf-biotin alone (hLf-biotin) or in the presence of
50 l
M
HB-19 (hLf-biotin + HB-19) or 8 l
M
hLf (hLf-biotin + hLf)
or anti-nucleolin polyclonal immunoglobulin (1 : 200 dilution) (hLf-
biotin + anti-Nucl pAb). Fluorescence staining was achieved with
streptavidin-FITC. Controls with cells incubated without proteins
(None) and with streptavidin-FITC only (Avidine-FITC control) are
shown.
312 D. Legrand et al. (Eur. J. Biochem. 271) Ó FEBS 2003
Discussion
Lf appears to participate in host defense mechanisms
against various infections and cancer. Although the pro-
posed mechanisms for the antiproliferation properties of Lf
in cancer are still controversial, its nuclear targeting has

been reported [14,15]. Consistent with this, a potential
nuclear import signal in hLf (residues 2RRRR5) has
recently been described [41]. This feature and its localization
in the nucleus of leukemia K562 cells, along with its capacity
to bind specific DNA sequences [14,15], suggest that Lf
might trigger gene expression through direct interaction
with target DNA sequences.
Here we report, for the first time, the binding of Lf to a
nuclear protein – nucleolin – that also exists as a cell surface
receptor. Indeed, our results from Lf-binding studies on
CHO wild-type and proteoglycan-deficient cell lines, and
competition with the nucleolin-binding HB-19 pseudopep-
tide, highlight nucleolin as an important Lf-binding site
alongside proteoglycans. In the CHO 618 cells that are
deficient in proteoglycan expression, Lf binds cells with a K
d
of  430 n
M
, which is similar to that estimated by biosensor
studies using purified nucleolin (K
d
¼ 240 n
M
). This affinity
is also similar to that reported for the binding of HB-19 to
the lymphoid cell line, CEM [26], and should account for the
marked competition between hLf and HB-19 for binding to
surface nucleolin. Previous studies have identified a poten-
tial hLf-binding protein of 105 kDa from T lymphocytes
and platelets [6,8,9]. Although the molecular weight of this

protein is similar to that of nucleolin, they should represent
two distinct proteins because hLf residues 28–34 were
reported to be essential for binding to the 105 kDa receptor
[6]. Further investigations are needed to confirm that
nucleolin and the 105 kDa receptor are distinct proteins
and, if need be, to assess the relative importance of both
proteins in cells.
Although hLf binding to nucleolin occurs with average
affinity and dissociates with elevated salt concentrations,
our results are not in favor of simple ionic interactions
between basic hLf and the N-terminal anionic domain of
nucleolin [36]. In accordance with this, we show that the hLf
sequences 2RRRR5 and 28RKVR31, responsible for most
Fig. 8. Colocalization of human lactoferrin (hLf) with nucleolin, both on the surface of MDA-MB-231 cells and in vesicles of the recycling/degradation
pathway. A series of 10 optical sections at 0.4 lm was performed through cells. The figure shows cross-sections towards the middle of representative
pairs of cells for streptavidin-fluorescein isothiocyanate (FITC) bound to hLf-biotin (green), tetrarhodamine isothiocyanate (TRITC)- or Alexa
Fluor 546-stained antibodies bound to nucleolin or vesicle markers (red) with the merge of the two colors (yellow). Staining of cells with either
streptavidin-FITC or the red-labeled secondary antibodies alone was not significantly different from background. Bars, 5 lm. (A) Binding of hLf-
biotin and rabbit polyclonal anti-nucleolin Ig to MDA-MB-231 cells. Cells were incubated for 1 h at 15 °Cwith3l
M
biotin-labeled hLf (green) and
rabbit anti-nucleolin Ig (1 : 100 dilution) (red). (B) Endocytosis of hLf-biotin and rabbit polyclonal anti-nucleolin immunoglobulin into MDA-MB-
231 cells. Cells were incubated for 2 h at 37 °Cwith3 l
M
hLf-biotin (green) and rabbit antiserum to nucleolin (1 : 100 dilution) (red). White arrows
show clusters that were fluorescent through one to three successive planes in the middle of cells and inside the nucleus. (C) Co-localization of hLf-
biotin with EEA1, the early endosome antigen 1. Cells were incubated for 2 h at 37 °Cwith3l
M
biotin-labeled hLf. Fixed cells were then incubated
with mouse mAbs to EEA1 for 45 min at 37 °C.

Ó FEBS 2003 Nucleolin is a cell surface lactoferrin-binding site (Eur. J. Biochem. 271) 313
ionic interactions of hLf with DNA and glycosamino-
glycans [6,37,38], are not necessary for the binding of hLf
to surface nucleolin. We have previously shown that the
specific binding of HB-19 to surface nucleolin occurs
through the arginine-rich basic C-terminal tail of nucleolin
[26]. The fact that hLf and HB-19 compete with each other
for binding to surface nucleolin suggests that they should
share a common binding site in nucleolin, i.e. the C-terminal
tail containing nine RGG repeats. The conservation of this
nucleolin domain among various species [22] probably
accounts for the binding of hLf and HB-19 to either rodent
(CHO) or human (MDA-MB-231, HeLa, MT-4) cells.
Previously, the RGG domain in nucleolin has been reported
to bind RNA [42], rDNA [43], the cytokine midkine [19,32]
and subset of ribosomal proteins [44]. Accordingly, studies
using a combination of CD and infrared spectroscopy have
provided evidence that repeated b-turns are a major
structural component of the RGG domain and might play
a role in the formation of protein–protein interactions [42].
It should also be noted that the RGG domain contains five
phenylalanine residues that potentially can establish
cation–p interactions [45] with arginine and lysine residues
that could be accessible in Lf. Whatever the case, our results
clearly illustrate that both the N- and C-terminal lobes of Lf
have the potential to interact with nucleolin.
The stable binding of Lf to cells could mostly be
coordinated on the one hand by nucleolin and on the other
hand by surface proteoglycans, which are able to interact
with the basic residues at the N-terminal end of Lf. Indeed,

hLf binding is about twofold higher in wild-type CHO K1
cells compared with CHO 677 cells deficient in the
expression of heparan-sulfate proteoglycans (Fig. 3),
although both of these cell types express similar levels of
surface nucleolin [26]. In contrast, the inhibition of hLf
binding to these CHO cell lines by low concentrations of the
nucleolin-specific HB-19 pseudopeptide is much more
efficient in CHO 677 cells, i.e. in those cells that do not
express heparan-sulfate proteoglycans (Fig. 4). A cooper-
ative mechanism between proteoglycans and nucleolin
could account for this latter effect. Similarly, a cooperative
mechanism appears also to be operational for the endo-
cytosis of hLf (Fig. 9). Indeed, endocytosis is prevented by
either the nucleolin-binding HB-19 pseudopeptide or the
absence of heparan-sulfate proteoglycans. It should be
noted that active internalization of the cytokine, midkine,
following binding to surface nucleolin does not require
proteoglycans, as midkine can be internalized at a similar
extent into CHO cell lines expressing or not expressing
heparan-sulfate or heparan/chondroitin-sulfate proteogly-
cans [19]. Therefore, the mechanism implicated in the
internalization of Lf should be different from that of
midkine, although both of these ligands require the presence
of accessible surface nucleolin. Interestingly, both Lf and
midkine are internalized at 37 °C, but not at 20 °C. As
fibroblast growth factor 2, which uses heparan-sulfate
proteoglycans as a low affinity receptor, is internalized,
even at 4 °C [19], endocytosis could occur either by an active
process through nucleolin or by a passive process through
heparan sulfate proteoglycans. The observation that endo-

cytosis of hLf into different types of cells occurs by an active
process is consistent with the requirement of both heparan-
sulfate proteoglycans and nucleolin for its endocytosis.
To our knowledge, and with the exception of bacterial
receptors [46], this is the first study reporting interaction
sites located in both lobes of Lf. In an attempt to delineate
the nucleolin-binding sites on Lf, we searched for solvent-
accessible sequences that were conserved in both lobes of
hLf, but absent from hTf. As significant binding inhibition
was achieved with the hLf N2 fragment, attention was paid
to sequences 91–255 and 433–603 of hLf and the corres-
ponding homologous regions of bLf and hTf. The only
sequence complying with the above-mentioned require-
ments was GENK, residues 178–181 and 514–517, which is
contained in a highly solvent-accessible area of the molecule,
stabilized by a set of two disulfide bridges (Fig. 10).
According to the information given in the PdbSum database
(Protein Data Bank accession numbers: 1LFG and 1BLF
for hLf and bLf, respectively), GENK forms b-turn loops in
both lobes of hLf that are also present in both lobes of bLf.
Fig. 9. The binding, but not endocytosis, of human lactoferrin (hLf)
occurs in heparan sulfate-deficient cells. Wild-type Chinese hamster
ovary (CHO) K1 cells, and the heparan sulfate-deficient CHO 677
cells, were incubated (for 90 min) at either 37 °C(A)or20°C(B)in
fresh culture medium containing 10% fetal bovine serum and hLf
(1 l
M
). Cells were then washed and fixed with 3.7% paraformaldehyde
and permeabilized with Triton X-100 (A) or fixed with 3.7% parafor-
maldehyde without permeabilization (B) [19]. The primary antibody

was rabbit immune serum (1 : 50 dilution) raised against hLf, whereas
the secondary antibody was Texas Red-labeled donkey anti-rabbit IgG.
A scan corresponding to a cross-section towards the middle of the cell
monolayer is shown, together with the respective phase contrast.
314 D. Legrand et al. (Eur. J. Biochem. 271) Ó FEBS 2003
However, while loop GENQ of the bLf N2 domain is very
close to its hLf counterpart, GLDK is not. This feature
could explain the least inhibitory effect of HB-19 binding to
CHO 618 cells by bLf C-t. Further experiments, using
mutated Lfs, are required to assess the involvement, if any,
of these loops in Lf–nucleolin interactions.
Evidence for binding of Lf to surface nucleolin
may enlighten on the multifunctional properties of Lf.
MDA-MB-231 cells were previously used for studying the
antiproliferativeeffectofLfoncancerousmammarygland
cells [7,16]. Cell growth arrest was connected to both
inhibition of Cdk2 and Cdk4 activities and increase of Cdk
inhibitor p21 expression [7]. Whether some of these
responses are induced following interaction of Lf with
nucleolin remains to be elucidated. Our results show that
hLf co-localizes with nucleolin at the surface of MDA-MB-
231 cells and that both molecules undergo endocytosis by an
active process into vesicles of the classical endocytosis
pathway. In light of our results, it is probable that nucleolin
is responsible, at least in part, for the endocytosis of Lf and
its nuclear targeting. Internalization of specific antibodies
bound to surface nucleolin [23,29], which then gain access to
the nucleus, has been reported previously [29]. It has also
been reported that the pleiotropic activities of a number of
growth factors are mediated not only through receptor-

induced signaling but also following internalization via an
endosomal-like pathway across the endoplasmic pathway
and nuclear retrotransport [47]. A growth factor, such as
prolactin, is then able to complex with cyclophilin B and to
act as a transcriptional inducer [48]. Another example has
been provided with midkine [49]. Comparable nuclear
retrotranslocation mechanisms could lead to the nuclear
localization of Lf in MDA-MB-231 cells, alone or com-
plexed to nucleolin, and its possible activity as a transcrip-
tion factor. Lastly, one can speculate on the presence of
accessory molecules accounting for specificity, as nucleolin
is a ubiquitous protein found in the nuclei of eukaryotic cells
and also expressed at the surface of many, if not all, dividing
cells. On the other hand, proteoglycans that have been
shown to be essential for the Lf antiproliferative effect on
MDA-MB-231 cells [7] can be considered as potential
candidates, as they differ from one cell type to the other.
Moreover, proteoglycans have been shown to modulate
receptor binding and cellular responses of growth factors
and chemokines [50].
HIV infects target cells by the ability of its envelope
glycoproteins, the gp120–gp41 complex, to attach cells and
induce the fusion of virus and cell membranes. The receptor
complex for HIV entry consists of the CD4 molecule and at
least one of the members of the chemokine receptor family,
mainly CXCR4 or CCR5 [51]. Although both CD4 and
CXCR4/CCR5 are essential for the HIV entry process, the
initial association of HIV particles to cells (referred to as
attachment) could occur in the absence or blockade of these
receptors. Several observations have pointed out that the

attachment of HIV particles to the cell surface occurs, on
the one hand, through the coordinated interactions with
heparan-sulfate proteoglycans and, on the other hand, with
the cell surface-expressed nucleolin [26]. Consequently,
targeting any one of these components could result in the
inhibition of HIV attachment. Indeed, HIV attachment
could be blocked either by the fibroblast growth factor 2
that binds heparan-sulfate proteoglycans, or by the anti-
HIV pseudopeptide HB-19 that binds nucleolin [24,26].
Therefore, the capacity to bind proteoglycans and surface
nucleolin makes Lf a potent inhibitor of HIV attachment
and thus infection. Previously, several laboratories had
reported the capacity of Lf to inhibit HIV infection with an
IC
50
value of 0.4–1.5 l
M
[3,4]. Here, we show that the
purified hLf inhibits HIV infection, in the experimental
model of HeLa P4 cells, with an estimated IC
50
value of
0.25 l
M
. Moreover, we show that the mechanism of this
inhibition is a result of the marked blockade of HIV
attachment to cells.
In conclusion, our results show that nucleolin, in addition
to proteoglycans, is a major Lf-binding site at the surface of
cells. As nucleolin is a ubiquitous multiligand protein

known for its shuttle function between the cell surface and
the nucleus, it may be expected that many of the biological
functions of Lf are relevant to this binding. Some of these
Fig. 10. Prediction of nucleolin-binding sites in human lactoferrin (hLf).
Sequences of the N2 and C2 domains of hLf, bovine lactoferrin (bLf)
and human transferrin (hTf) were aligned and compared for similar-
ities and divergences using the
CLUSTALW
and
EDTALN
programs from
the
FASTA
package ( Fur-
thermore, accessibility of residues to solvent was assessed using
SWISS
PDBVIEWER
3.7 software (Glaxo Wellcome Experimental Research).
(A) Sequences 154–190 and 490–526 of hLf are aligned with their
counterparts in bLf and hTf. Sequence GEN in bold characters (resi-
dues 178–180 and 514–516) was the only solvent-accessible consensus
sequence found in both domains of hLf and in the N2 domain of bLf,
but absent in hTf. Brackets indicate positions of the two disulfide
bridges. (B) a-Backbone ribbon folding representation of hLf (PDB Id.
1LFG) performed using the
VIEWERLITE
software (el-
rys.com). Positions of sequences 154–190, 178–180, 490–526, 514–516,
and basic sequences 1–5 and 28–34 of the N1 domain, are indicated.
Ó FEBS 2003 Nucleolin is a cell surface lactoferrin-binding site (Eur. J. Biochem. 271) 315

functions might rely on the capacity of Lf to enter cells, as
we have illustrated in cancerous mammary gland cells.
Other functions, such as the anti-HIV activity of Lf, might
be the consequence of competition for binding to surface
nucleolin and to proteoglycans. Further investigations are
necessary in order to elucidate the participation of Lf–
nucleolin and Lf–proteoglycan interactions in the overall
mechanism of action of Lf.
Acknowledgements
This work was supported, in part, by the Universite
´
des Sciences et
Technologies de Lille, the Centre National de la Recherche Scientifique
(UMR n°8576; Director: Dr J. -C. Michalski), and Agence Nationale
de la Recherche sur le SIDA (research grant to A. G. Hovanessian). We
thank S. Baveye for participating in preliminary studies. We thank
C. Slomianny and E. Perret for confocal microscopy and J P.
Decottignies, M. Benaı
¨
ssa and J. Svab for excellent technical assistance.
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