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: 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) 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 [...]... together with the respective phase contrast ionic interactions of hLf with DNA and glycosaminoglycans [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. .. 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. .. 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... 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. .. FEBS 2003 Nucleolin is a cell surface lactoferrin -binding site (Eur J Biochem 271) 313 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... 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 Ó FEBS 2003 Nucleolin is a cell surface lactoferrin -binding site (Eur J Biochem 271) 315 Fig 10 Prediction of nucleolin- binding sites in. .. 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 antiproliferative effect of Lf on cancerous mammary gland cells [7,16] Cell growth arrest was connected to both inhibition of Cdk2 and Cdk4 activities and increase of Cdk inhibitor... the 30 kDa N- and 50 kDa C-terminal tryptic fragments of human lactotransferrin Biochem J 236, 839–844 31 Legrand, D., Mazurier, J., Colavizza, D., Montreuil, J & Spik, G (1990) Properties of the iron -binding site of the N-terminal lobe of human and bovine lactotransferrins Importance of the glycan moiety and of the non-covalent interactions between the N- and C-terminal lobes in the stability of the. .. proteoglycans The observation that endocytosis of hLf into different types of cells occurs by an active process is consistent with the requirement of both heparansulfate 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. .. 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 . Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells Dominique Legrand 1 , Keveen Vigie ´ 1 ,. 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