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
Trang 1Surface nucleolin participates in both the binding and endocytosis
of lactoferrin in target cells
Dominique Legrand1, Keveen Vigie´1, Elias A Said2, Elisabeth Elass1, Maryse Masson1,
Marie-Christine Slomianny1, Mathieu Carpentier1, Jean-Paul Briand3, Joe¨l Mazurier1
and Ara G Hovanessian2
1 Unite´ de Glycobiologie Structurale et Fonctionnelle et Unite´ Mixte de Recherche n8576 du CNRS, Institut Fe´de´ratif de Recherche n118, Universite´ desScienceset Technologiesde Lille, Villeneuve d’Ascq, France; 2
Unite´ de Virologie et Immunologie Cellulaire, URA 1930 CNRS, Paris, France;3Institut 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 nM) 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: dominique.legrand@univ-lille1.fr
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)
Trang 2involvement 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% CO2at 37C 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 lg of the total RNA preparation from Jurkat cells was reverse transcribed into first-strand cDNA using oligo dTprimers (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/Pi) overnight at 20C with gentle 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] rhLfEGS,
Trang 3a 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/Pifor 1–6 h at 37C and according to the
manufac-turer’s instructions
Preparation and labeling of HB-19
The HB-19 pseudopeptide 5[Kw(CH2N)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(CH2N)PR, where
w(CH2N) represents a reduced peptide bond between lysine
and proline residues The synthesis of HB-19, and its
labeling with fluorescein 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· 106MDA-MB-231
cells were washed twice with NaCl/Piand lysed in NaCl/Pi,
1% Triton-X-100 (w/v) containing 1 mM of the protease
inhibitor Pefabloc [4-(2-aminoethyl)-benzenesulfonyl
fluor-ide] (Roche Diagnostics, Mannheim, Germany) for 1 h at
4C After centrifugation at 10 000 g for 30 min, the
supernatant was recovered, diluted 10-fold with NaCl/Pi
containing 1 mMPefabloc and incubated overnight at 4C
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/Pi The proteins
bound to the gel were sequentially eluted with two volumes
of 200 lL of 0.5M NaCl in 20 mM sodium phosphate
buffer, pH 7.4, two volumes of 200 lL of 1MNaCl in this
buffer, two volumes of 0.2Mglycine/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· 109 NaCl/Pi-washed Jurkat cells at 4C for 1 h in
25 mL of 20 mMTris/HCl, pH 7.6, 150 mM NaCl, 5 mM MgCl2, 5 mMb-mercaptoethanol, 0.5% (v/v) Triton X-100,
1 mM 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 4C using ice-cold buffers and columns
in the presence of 1 mMPefabloc and Complete protease inhibitor cocktail The cytoplasmic extract of Jurkat cells (25 mL) was diluted 10-fold with 20 mMsodium 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 mMsodium phosphate,
pH 7.0, elution of the adsorbed proteins was performed with 10 mL of the same buffer containing 1MNaCl The eluant was diluted 10-fold with 50 mM Tris/HCl, pH 7.9,
5 mM MgCl2, 0.1 mM EDTA, 1 mM 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 0.2M ammonium sulfate, and proteins were eluted in 50 lL fractions with 2 mL of buffer A containing 0.6M 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/Picontaining 1 mM Pefabloc at 4C for 2 h before 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 25C on
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 0.1M sodium acetate,
pH 5, at a flow rate of 10 lLÆmL)1, to a CM5 sensor chip that had been previously activated according to the manufacturer’s instructions Covalent binding resulted in a
Trang 4signal 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 nM, 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 lL of 10 mMHCl 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-TION3.1 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/Pi
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 15C for 45 min in 100 lL of cell culture
medium containing 1 lM HB-19-fluo and 0–8 lM hLf
After seven washes with NaCl/Pi, the cells were analyzed by
flow cytometry Binding specificity and reversibility controls
were performed with 0–50 lMunlabeled HB-19 For studies
on the nucleolin-binding site of Lf, CHO 618 cells were
incubated at 15C for 45 min in 100 lL of cell culture
media containing 1 lMHB-19-fluo and one of the
follow-ing: unlabeled HB-19 (50 lM), hTf, bLf, hLf or hLf and bLf
protein variants and peptides (8 lM) To assess potential
interactions between hLf and HB-19, hLf (1 lM) was
incubated with HB-19-biotin (1 lM) in 1 mL of NaCl/Pifor
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/Pi, 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 125I-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 4C with 250 lL of 0–3 lM125I-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 0.1M NaOH The cell lysates
were recovered for gamma counting For the binding
competition assays between hLf and HB-19, CHO cells were
incubated at 15C for 45 min with 1 lM125I-labeled hLf and 0–100 lM HB-19 Washes were performed five times with NaCl/Picontaining 1% BSA and twice with NaCl/Pi containing 0.3MNaCl, 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/Pi, the cells were resuspended in fresh Eagle’s medium containing 1% fetal bovine serum and incubated at 15C for 45 min with 1 lM HB-19-fluo, 1 lMhLf-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/Piand further incubated with streptavidin-FITC (1 : 2000) and FITC-labeled anti-rabbit IgG (1 : 4000), respectively After five washes with NaCl/Pi and two washes with NaCl/Pi containing 0.3M 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 37C for 1–14 h with 3 lMhLf-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
37C, the ligand-containing medium was replaced with fresh 37C-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/Piand twice with NaCl/Picontaining 0.3MNaCl, prior to fixation with 4% paraformaldehyde in NaCl/Pi(4C, 30 min) Cells were then washed with NaCl/Pi, permeabilized with 0.15% Triton-X-100 in NaCl/Pi (20C, 2 min), washed again, blocked by 1% ethanolamine in NaCl/Pi(4C, 20 min) and extensively washed with NaCl/Picontaining 1% BSA The treated cells were incubated (37C, 45 min) with FITC-conjugated streptavidin (1 : 800) and TRITC-FITC-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 (37C, 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
Trang 5invaginations 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/Pi 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
[3H]thymidine incorporation experiments, the 50%
inhibi-tion of cell proliferainhibi-tion was observed at 50 lgÆmL)1
(0.62 lM) 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 (IC50) value of 0.25 lM A complete
inhibi-tion of HIV infecinhibi-tion was observed at 2 lMLf (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 AZThad 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 0.5M 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
LELQGPR561
GYAFIEFASFEDAK537
TLVLSNLSYSATEETLQEVFEK508
Trang 6major 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 (kon) and dissociation rate
con-stant (koff) were 6.89 ± 0.46· 105M )1Æs)1 and 0.164 ±
0.001 s)1, respectively, using Langmuir’s one-site model,
which gave the best fit at all concentrations used (v2< 2)
The equilibrium dissociation constant (Kd), calculated from
the ratio of the kinetic rate constants (koff/kon), was
238 ± 15 nM, a value very similar to that calculated from
the extent of binding observed near equilibrium using a
Scatchard plot (249 ± 45 nM) (Fig 2B) Rmaxestimated 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 Kdof 0.43 ± 0.01· 10)6M, comparable with the one measured between hLf and purified nucleolin Hence, it can be assumed that the lower affinity sites on CHO K1 (Kd¼ 2.6 ± 0.1 · 10)6M and n¼ 555 000 ±
20 000) and CHO 677 (Kd¼ 2.1 ± 0.3 · 10)6M and
n¼ 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.
Trang 7Evidence 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 (0.3M
NaCl-containing NaCl/Pi 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) and CHO
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 M125I-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) or CHO 618 (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) or CHO 618 (r) cells without hLf.
Trang 8[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 20C before
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)4Nand rhLfEGSinhibited 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 (Kd¼ 45–123 nM) that represented 10% of the total binding (2.6–3.2· 105 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 hLf at 20 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].
Trang 9experimental conditions that avoid binding to
proteogly-cans; i.e washing with NaCl/Pithat contains 0.3MNaCl 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)3Nand hLf)4N, recombinant hLf mutated at residues 28–34 (rhLfEGS), 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.
Trang 10Previous 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 37C with
both biotinylated hLf and anti-nucleolin polyclonal Ig
Figure 8B,C shows that cells incubated with hLf-biotin at
37C 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 15C with hLf, at 37 C in the presence of 1 mM
sodium azide, or growth-arrested by overnight incubation
in medium containing 1% fetal bovine serum prior to incubation at 37C, 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 37C, but not
at 20C, indicating that it uses an active internalization process, consistent with other nucleolin-binding ligands [19,23] Endocytosis of hLf at 37C was also time 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 37C into CHO 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 lM 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
the presence of 50 l 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.