Cell surface nucleolin on developing muscle is a potential ligand for the axonal receptor protein tyrosine phosphatase-r Daniel E Alete1, Mark E Weeks2, Ara G Hovanession3, Muhamed Hawadle1 and Andrew W Stoker1 Neural Development Unit, Institute of Child Health, University College London, UK Molecular Oncology, CRUK, Barts and The London School of Medicine and Dentistry, John Vane Centre, UK UPR 2228 CNRS, UFR Biomedicale-Universite Rene Descartes, Paris, France Keywords affinity chromatography; axon targeting; nucleolin; RAP assay; receptor protein tyrosine phosphatases Correspondence A W Stoker, Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK Fax: +44 207 78314366 Tel: +44 207 9052244 E-mail: a.stoker@ich.ucl.ac.uk (Received 21 April 2006, revised August 2006, accepted 15 August 2006) doi:10.1111/j.1742-4658.2006.05471.x Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases and receptor tyrosine phosphatases, plays an essential part in cell signaling during axonal development Receptor protein tyrosine phosphatase-r has been implicated in the growth, guidance and repair of retinal axons This phosphatase has also been implicated in motor axon growth and innervation Insect orthologs of receptor protein tyrosine phosphatase-r are also implicated in the recognition of muscle target cells A potential extracellular ligand for vertebrate receptor protein tyrosine phosphatase-r has been previously localized in developing skeletal muscle The identity of this muscle ligand is currently unknown, but it appears to be unrelated to the heparan sulfate ligands of receptor protein tyrosine phosphatase-r In this study, we have used affinity chromatography and tandem MS to identify nucleolin as a binding partner for receptor protein tyrosine phosphatase-r in skeletal muscle tissue Nucleolin, both from tissue lysates and in purified form, binds to receptor protein tyrosine phosphatase-r ectodomains Its expression pattern also overlaps with that of the receptor protein tyrosine phosphatase-r-binding partner previously localized in muscle, and nucleolin can also be found in retinal basement membranes We demonstrate that a significant amount of muscle-associated nucleolin is present on the cell surface of developing myotubes, and that two nucleolin-binding components, lactoferrin and the HB-19 peptide, can block the interaction of receptor protein tyrosine phosphatase-r ectodomains with muscle and retinal basement membranes in tissue sections These data suggest that muscle cell surface-associated nucleolin represents at least part of the muscle binding site for axonal receptor protein tyrosine phosphatase-r and that nucleolin may also be a necessary component of basement membrane binding sites of receptor protein tyrosine phosphatase-r Vertebrate nervous system development relies on a multitude of guidance cues to stimulate axonal extension and stable synaptic contacts with targets such as muscles Interpretation of these environmental signals by growth cones involves multiple receptor classes such as cell adhesion molecules (CAMs) [1], DCC and neu- Abbreviations AP, alkaline phosphatase; CAM, cell adhesion molecule; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; HB-19, 5[Kw(CH2N)PR]-TASP; HSPG, heparin sulfate proteoglycan; PLAP, placental alkaline phosphatase; PTP, protein tyrosine phosphatase; RAP, receptor affinity probe; RPTP, receptor protein tyrosine phosphatase; RTK, receptor protein tyrosine kinase 4668 FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS D E Alete et al ropilins [2,3], and enzymes involved in phosphotyrosine signaling, such as the receptor protein tyrosine kinases (RTKs) [4] and receptor protein tyrosine phosphatases (RPTPs) [5–7] Evidence for the role of phosphotyrosine signaling during axon growth and guidance has come from studies showing that signaling by fibroblast growth factor (FGF) receptor, an RTK acting alongside neural CAMs, promotes neurite growth [8,9] Also, members of the Eph family of RTKs regulate retinal axon guidance in direct response to graded patterns of their ligands, the ephrins [10–12] It is therefore unsurprising that the counterbalancing enzymes, the RPTPs, are also implicated in many of these processes There are 21 recognized human RPTPs [13], with homologs and orthologs throughout the vertebrates and invertebrates RPTPs show strong developmental expression in the central and peripheral nervous systems, coinciding with significant events such as axonogenesis, target contact, synaptogenesis and plasticity [14,15] The type IIa subfamily exemplifies these neural RPTPs These enzymes have two cytoplasmic phosphatase domains and an extracellular region consisting of immunoglobulin-like domains and fibronectin type III repeats, similar to the NCAM family of cell adhesion molecules [15] Members of the type IIa RPTPs include vertebrate LAR, PTPd and PTPr, leech hmLAR and Drosophila DLAR and DPTP69D Studies have implicated PTPd and PTPr in retinal axon development in both chick and Xenopus [16–18] In Drosophila, DLAR and DPTP69D have roles in photoreceptor and commissural axon guidance [19–21] Mouse LAR deficiency leads to reduced size of basal forebrain cholinergic neurons and diminished hippocampal innervation [22], whereas PTPd deficiency causes impaired learning and enhanced hippocampal long-term potentiation [23] PTPr deficiency leads to the most extreme defects, with hypomyelination of peripheral nerves, abnormalities in development of the hypothalamus and pituitary, and ataxias [24,25] Type IIa RPTPs have also been implicated in the development of the neuromuscular system In Drosophila, DLAR and DPTP69D are required for guidance of motor axons [26,27], and DLAR has also been implicated in neuromuscular synaptic plasticity [28– 31] LAR-related RPTPs also influence synaptogenesis in muscles in other species [32,33] A recent study of mice with a double gene deficiency in PTPr and PTPd demonstrated that these two RPTPs are critical for the generation of a branched innervation pattern in the diaphragm, and subsequent motor neuron survival [34] In the chick embryo, there is strong expression of PTPr in vertebrate spinal and cranial motor neurons Nucleolin is a potential ligand for PTPr [35,36], and there is evidence from affinity probe assays of a potential PTPr ligand in developing muscle [37] Recent studies using gene knockdown in the chick spinal cord have also shown that type IIa RPTPs play a role in motor axon growth [38] These collective data show that vertebrate type IIa RPTPs, like DLAR in the fly, are involved in neuromuscular development Relatively little is known about the signaling mechanisms of these type IIa RPTPs and how such signaling is regulated One way to understand these events might be to identify the extracellular ligands of the RPTPs in the neuromuscular system For example, it is known that PTPd and an isoform of LAR can interact homophilically [39] and that LAR can also bind heterophilically to the laminin–nidogen complex [40] Heparin sulfate proteoglycans (HSPGs) have also been identified as potential ligands for PTPr [41], and recently syndecan and dallylike, both HSPGs, have been reported as functional ligands for DLAR in Drosophila motor axons and neuromuscular synaptogenesis [28,30,31] Nevertheless, the potential ligand of PTPr within developing muscle of the chick embryo appears not be HSPG-related, and only interacts with the short protein isoform of PTPr expressed in motor neurons [37] Given the interest in PTPr in neuromuscular development, we have undertaken the identification of this potential muscle ligand using an affinity chromatography approach We report that chick nucleolin, expressed on the cell surface of developing muscle cells, is a PTPr-binding protein Nucleolin expression correlates with the location of PTPr-binding sites on developing muscle, and nucleolin-binding proteins and peptides can perturb this PTPr binding These data demonstrate that cell surface nucleolin is likely to be part of the PTPr-binding site in developing skeletal muscles We also show that nucleolin can be found in retinal basement membranes and that this nucleolin may also be necessary, alongside HSPGs, for PTPr interactions Results Nucleolin is a PTPr-binding protein We have shown previously that PTPr binds to an unidentified ligand, or ligand complex, in developing muscle of the chick [37] To identify PTPr-binding proteins and potential ligands, an immobilized fusion protein consisting of the first six subdomains of the PTPr ectodomain fused to alkaline phosphatase (AP) (termed FN3d–AP; Fig 1A,B) was used to perform affinity chromatography on solubilized muscle tissue FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4669 Nucleolin is a potential ligand for PTPr A D E Alete et al B D C E Fig Affinity chromatography isolation of protein tyrosine phosphatase-r (PTPr)-binding and lactoferrin-binding proteins (A) Schematic diagram of PTPr-derived proteins Shown are the two main isoforms of PTPr, PTPr1 and PTPr2 The first six subdomains of the PTPr ectodomain were fused to placental alkaline phosphatase (AP) to generate the fusion construct FN3d–AP [41] Circles, immunoglobulin-like domains; squares, fibronectin type III domains; PTP, phosphatase catalytic domains; black dots, protease cleavage sites (B) SDS ⁄ PAGE separation of FN3d–AP purified from conditioned media using anti-placental alkaline phosphatase (PLAP) agarose (C) SDS ⁄ PAGE and silver stain of proteins isolated from AP sepharose (lane 1) and FN3d–AP sepharose (lane 2) A protein band of approximately 95 kDa is present exclusively in the FN3d–AP eluate (arrowhead) (D) Immunoblot of proteins isolated from AP sepharose (lane 1) and FN3d–AP sepharose (lane 2) using polyclonal rabbit anti-nucleolin serum (E) Immunoblot of column eluates from blank sepharose (lane 1) and lactoferrin sepharose (lane 2) using polyclonal rabbit anti-nucleolin serum A 100 kDa nucleolin band is present (arrow) Several lower molecular weight bands corresponding to proteolytic fragments of nucleolin are also present from 10-day-old (E10) chick embryos To identify specifically retained proteins that interact with PTPr, we performed (as a negative control) chromatography on AP-conjugated sepharose Detergent extracts of chick muscle tissue were loaded onto these two columns, and proteins were eluted using a high-salt buffer Eluted proteins were compared after SDS gel electrophoresis, and this revealed a complex pattern of protein bands The only reproducible difference observed was a 95 kDa band identified as being present in the eluate from the PTPr column, but absent in the control eluate (Fig 1C) For protein identification, multiple affinity runs were performed, and eluates were concentrated, separated by SDS gel electrophoresis and stained with Coomassie The band of interest was excised from the gel, digested with trypsin, and analyzed by tandem MS As shown in Fig 2A, 21 peptides were sequenced and found to correspond to chicken nucleolin (SwissProt accession number P15771) All 21 peptides could be identified within the C-terminal region of the nucleolin sequence (Fig 2B), 4670 with no peptide sequence tags being obtained from the N-terminal part of nucleolin This is probably due to the clustering of glutamic acid residues within the N-terminal region, preventing the formation of reasonably sized peptides for MS ⁄ MS analysis The calculated mass of nucleolin, based on its sequence, is 76 kDa; however, it migrates at approximately 100 kDa in SDS gel electrophoresis, due to post-translational modifications and a high content of negatively charged amino acids [42] The identity of the 95 kDa protein band as nucleolin was confirmed by immunoblotting eluates using antibody to nucleolin (Fig 1D) This revealed a band at approximately 95 kDa present in the PTPr eluate only These data confirm that nucleolin is a binding partner for PTPr under these conditions PTPr can bind directly to nucleolin To investigate whether nucleolin can bind directly to PTPr, we carried out solid-phase binding assays using FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS D E Alete et al Nucleolin is a potential ligand for PTPr A B Fig Tandem MS analysis of in-gel tryptic digest of 95 kDa protein band isolated using FN3d–AP sepharose (A) Peptides determined by sequencing of relevant mass peaks are shown These were found to correspond with a high degree of certainty to chicken nucleolin (SwissProt accession number P15771) Thirty-one per cent of the chicken nucleolin sequence is covered by MS ⁄ MS analysis (B) Location of sequenced peptides within the nucleolin sequence The primary structure of chicken nucleolin is indicated by the single-letter amino acid code sequence) Tryptic peptides are printed in bold and underscored recombinant, myc-tagged chick nucleolin purified from transfected 293T cells Purification of nucleolin is challenging, because it is accompanied by a high degree of protein degradation (D Alete & A Hovanession, unpublished results) Although our purified chick nucleolin was similarly only around 20% intact (data not shown), it was considered to be of sufficient quality for initial solid-phase overlay assays The purified nucleolin was immobilized on charged microtiter plates and incubated with varying concentrations of conditioned media containing FN3d–AP The data revealed significant binding of PTPr to nucleolin above that of the BSA control (Fig 3), demonstrating that PTPr can bind to nucleolin directly Nevertheless, this assay was near the limit of detection for this interaction, as successive dilution of the probe soon led to loss of signal The low yields of the AP probe and of nucleolin, together with the inevitable partial degradation of nucleolin, mean that calculations of binding affinity are unrealistic at this stage Nucleolin localization in muscle is analogous to PTPr ligand localization To determine whether the nucleolin identified by affinity chromatography is potentially a muscle ligand for PTPr, we compared the expression pattern of nucleolin with the distribution of the muscle binding sites for PTPr receptor affinity probes (RAPs) [43,44] (Fig 4) E10 cranial tissue sections were cut and stained using FN3d–AP fusion protein (Fig 4A,C) or antibody to nucleolin (Figs 4B.D) The strongest binding of the FN3d–AP fusion protein was localized to myotubes of developing muscle (Fig 4A and arrow in Fig 4C), FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4671 Nucleolin is a potential ligand for PTPr D E Alete et al produced a closely overlapping staining pattern to that seen in the RAP assay, with most of the staining localized to developing muscle (Fig 4B) Increased magnification showed that the most intense staining was, as with the RAP stain, within the myotubes and on myotube surfaces (arrow in Fig 4D) Technical limitations meant that we could not directly demonstrate how much of the RAP stain and nucleolin fluorescence directly overlapped Nevertheless, these data demonstrate that the developmental expression of nucleolin within developing muscle is largely coincident with the location of the muscle binding sites for PTPr Fig Solid-phase binding assay of protein tyrosine phosphatase-r (PTPr) to purified nucleolin Fifteen micrograms of BSA or affinityisolated nucleolin were bound to charged plates and incubated with various concentrations of FN3d–AP supernatant Binding of FN3d– AP was determined by alkaline phosphatase (AP) activity measured at 405 nm The reaction rate (change in absorbance per min) over h was used to quantify FN3d–AP concentrations Filled circles represent nucleolin; open circles represent BSA Error bars show standard error of the mean (n ¼ 3) *P £ 0.03, **P £ 0.005 with binding also being observed in motor nerves and other scattered cells and matrix No AP signal at all is generated when only soluble AP probes are used, or when nonbinding forms of PTPr–AP are used [37,41] Immunofluorescence staining using nucleolin antibody A C 4672 HB-19 pseudopeptide and lactoferrin bind nucleolin and perturb PTPr binding to developing muscle To ascertain whether FN3d–AP binds to nucleolin in chick muscle tissue, we tested whether the pentameric pseudopeptide 5(Kw(CH2N)PR)-TASP (referred to as HB-19 [45]) could perturb the binding of FN3d–AP HB-19 is a potent inhibitor of HIV entry into the cell, acting by specifically binding to, and forming complexes with, cell surface nucleolin [46–48] It has been shown to exert this effect independently of HSPGs, by binding the C-terminal tail of nucleolin containing the RGG domain, consisting of residues 656–707 [49] HB19 (10 lm) or BSA (0.5 mgỈmL)1) were prebound to chick sections and washed, and RAP analysis was then performed as described earlier (Fig 5) Prebinding of B D Fig Immunohistochemical and receptor affinity probe (RAP) analysis of chick E10 sections (A,C) RAP staining using the FN3d–AP fusion construct, demonstrating protein tyrosine phosphatase-r (PTPr) affinity for its ligand in the developing muscle (B, D) Immunofluorescence detection of nucleolin expression using polyclonal rabbit anti-nucleolin serum Analogous muscle staining patterns are observed in nucleolin and RAP-stained serial sections Both the FN3d–AP and nucleolin antibody stain the periphery of myotubes [arrows in (C) and (D)] DO, dorsal oblique eye muscle Scale bar ¼ 0.1 mm (A,B), and 25 lm (C,D) FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS D E Alete et al Nucleolin is a potential ligand for PTPr A B C D E F G Fig HB-19 and lactoferrin perturb protein tyrosine phosphatase-r (PTPr) binding to its ligand in developing muscle E10 cranial tissue sections were preincubated with 0.5 mgỈmL)1 BSA (A,C,E), 10 lM HB-19 peptide (B) or 0.5 mgỈmL)1 lactoferrin (D,F) before RAP assays using FN3d–AP (G) Fluorescent staining of retinal basement membrane using biotinylated HB-19 peptide Preincubation with lactoferrin and HB-19 abolishes FN3d–AP binding to muscle tissue [asterisks in (A) and (B), and (C) and (D), respectively] Lactoferrin and HB-19 prebinding also perturbs retinal basement membrane staining [(E) and (F), and data not shown] pe, Pigmented epithelium; nr, neural retina; vc, vitreous chamber; is, interorbital septum; oc, optic chiasma Scale bar ¼ 0.3 mm (A, B,C,D), and 0.15 mm (E,F,G) HB-19 prevented subsequent FN3d–AP binding to both muscle and basement membranes (Fig 5A,B and data not shown) We were surprised that HB-19 blocked the basement membrane sites, as these were thought to be only HSPG-dependent Nevertheless, direct detection of nucleolin with a biotinylated form of HB-19 revealed binding to basement membranes in the retina (Fig 5G) This may explain why HB-19 could block FN3d–AP binding to its HSPG ligands No nonspecific disruptive effects of HB-19 were observed on the binding of antibodies to the antigens laminin and myosin (data not shown) Further perturbation experiments were carried out using the protein lactoferrin Lactoferrin, an ironbinding protein of the transferrin family, is present in external secretions and the secondary granules of FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4673 Nucleolin is a potential ligand for PTPr D E Alete et al polymorphonuclear leukocytes [50] Lactoferrin is a highly basic protein [51] that binds to, and is internalized by, cell surface nucleolin, with the binding site located within the C-terminal RGG domain of nucleolin [52] We preincubated sections of E10 chick embryos with 0.5 mgỈmL)1 lactoferrin This preincubation with lactoferrin effectively blocked FN3d–AP binding to muscle tissue (Fig 5C,D) Like HB-19, lactoferrin also perturbed PTPr binding to the basement membrane of the retina (Fig 5E,F) Lactoferrin is also an HSPG-binding protein [50,53], and may therefore be directly interfering competitively with PTPr binding to basement membrane-associated HSPGs It is unlikely, however, that lactoferrin blocks PTPr binding in muscles through an effect on HSPGs, as our previous work has shown that the muscle binding site is not HSPG-related [41] To confirm that lactoferrin can bind to nucleolin from developing muscle, we carried out affinity chromatography of muscle lysates using immobilized lactoferrin Nucleolin was specifically retained on the lactoferrin column (Fig 1E) Immunoblots using antibodies against actin and myosin, as a control for nonspecific binding, were negative (data not shown) Prebinding of lactoferrin to muscle sections also showed no nonspecific, disruptive effect on the binding of antibodies to antigens such as laminin and myosin (data not shown) Both HB-19 and lactoferrin bind to the C-terminal RGG domain of nucleolin Therefore, if PTPr also binds to this domain, we would predict that an antibody raised to a sequence outside this domain might have little effect on the RAP assay signal This was tested with an antibody to nucleolin raised against amino acids 271–520 No effect on PTPr binding to muscle was observed (data not shown) There is currently no RGG-specific antibody to test directly whether it can block PTPr binding These data demonstrate first that two known nucleolin-binding components (HB-19 and lactoferrin) can specifically inhibit PTPr binding to its muscle ligand Second, binding of PTPr to nucleolin is likely to be mediated through the RGG domain of nucleolin Nucleolin is present on the surface of developing muscle tissue In order to function as a potential ligand for PTPr, nucleolin must be present on the surface of the target cells Originally, nucleolin was reported to be exclusively nuclear [54]; however, more recent studies have shown that nucleolin is present on the surface of a variety of cell lines [42,48,52,55,56] and on the surface of endothelial cells [57,58] To address whether nucleo4674 lin is also present on the surface of developing muscle cells, we carried out immunofluorescence analysis of primary chick muscle cells isolated from E10 embryos (Fig 6) These were grown in 6% serum, as this has been reported to promote the cell surface localization of nucleolin in MDAMB-435 carcinoma cells [57] Live, nonpermeabilized cells and fixed, semipermeabilized cells were costained with antibodies to nucleolin and myosin Anti-nucleolin staining of the live nonpermeabilized cells showed punctate patches on the outside of the cells (Fig 6B) No myosin staining was observed in nonpermeabilized cells, which confirms the integrity of the membrane By contrast, semipermeabilized cells showed high levels of nucleolin staining within the cytoplasm, with myosin staining also being observed (Fig 6A) As paraformaldehyde was used for only partial permeabilization, we did not expect to see nucleolar localization of nucleolin in these experiments To determine more precisely if the staining observed on the nonpermeabilized cells was present on myotubes, the live cells were first incubated with anti-nucleolin sera, and then fixed, permeabilized and treated with myosin antibody (Fig 6C,D) We observed punctate staining of nucleolin on the surface of myotubes (arrow in Fig 6D), as well as on the surface of nonmyosinexpressing cells Some of the punctate localization also occurred at the cell–cell interface between these cells and myotubes (arrowheads in Fig 6D) It is of interest that a punctate pattern of nucleolin is also seen on the surface of Hela cells after treatment of live cells with a nucleolin ligand, midkine [59] It is possible that the punctate pattern in muscle cells is in part caused by clustering of nucleolin by the antibodies in the live cells To investigate further which cells express surface nucleolin in muscle, we performed confocal microscopy on chick muscle sections costained with nucleolin and myosin antibodies (Fig 6E,F) In a flattened confocal stack (Fig 6F), intracellular overlap was observed as yellow staining, but in addition we observed punctate regions of nucleolin on myotube surfaces (arrows in Fig 6E) Furthermore, three-dimensional rendering of the myotubes using the green channel (myosin) to differentiate between intracellular nucleolin signal (yellow, arrowhead in Fig 6F) and surface nucleolin (red) showed nucleolin patches on the surface of developing myotubes (arrows in Fig 6F) This form of nucleolin should therefore be accessible to cell surface PTPr Discussion Studies carried out previously have identified two distinct tissue binding sites for PTPr ectodomains, within FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS D E Alete et al Nucleolin is a potential ligand for PTPr A Fig Nucleolin is on the cell surface of developing myotubes Immunofluorescence staining of permeabilized (A) and live unpermeabilized (B) chick primary muscle cultures (nonconfocal images) Cells were stained using anti-nucleolin (red), and anti-myosin (green), and counterstained with DAPI (blue) Cell surface staining is indicated in the unpermeabilized cells [arrows in (B)] (C,D) Live unpermeabilized cells were incubated with anti-nucleolin serum, permeabilized, and then incubated with anti-myosin serum Cell surface nucleolin is shown on a myosin-expressing cell [arrow in (C)], and at the interface of a cell juxtaposed to a myotube [arrowheads in (D)] (E,F) Confocal images of a tissue section through developing chick skeletal muscle, stained for myosin (green) and nucleolin (red) Regions of colocalization are shown in yellow (E) Flattened image stack through myotubes Nucleolin (red) present on the surface of myotubes is indicated (arrows) (F) Three-dimensional reconstruction of confocal images using VOLOCITY software Cell surface nucleolin (red patches) is indicated (arrows) Scale bars ¼ 20 lm B C D E F basement membranes [60] and in developing skeletal muscle [37] The basement membrane-associated ligands have been identified as HSPGs Here, using affinity chromatography, tandem MS and RAP affinity assays, we identified the multifunctional protein nucleolin as a potential new ligand present in developing muscle It was confirmed that nucleolin and the PTPr ectodomain could directly interact Furthermore, we demonstrated for the first time that nucleolin is expressed on the surface of developing myotubes, and that its localization in muscle overlaps that of the previously characterized PTPr interactor Nucleolin was first described as a major nuclear protein consisting of a negatively charged N-terminal domain, an RNA-binding domain and a C-terminal domain rich in RGG motifs [54] Nucleolin has been reported to be involved in a diverse array of cellular processes, including cell proliferation and growth, cytokinesis, replication, embryogenesis and nucleogene- sis [61] More recently, numerous studies have reported nucleolin as being present on the cell surface [57,58,62,63] and to function as a ligand ⁄ receptor for a number of different proteins, including lactoferrin [52], pleiotrophin [48], achran sulfate [55], HIV [47], l-selectin [42] and midkine [64] Nucleolin does not have a classic secretion signal and it is therefore not known how it reaches the cell surface The clustering of nucleolin on cell surfaces is nonetheless dependent on an intact actin cytoskeleton, to which it must attach through an unidentified, integral membrane protein [56] Nucleolin can also reach cell surfaces without endogenous HSPG production [59], and nucleolin has even been reported to function as a shuttle between the cell surface and the nucleus [65] Our study has now shown that nucleolin is found on the surface of developing myotubes Indeed, the overall expression of nucleolin is specifically elevated in embryonic muscle, although its non-nucleolar FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4675 Nucleolin is a potential ligand for PTPr D E Alete et al localization was also observed in several other tissue sites The patch-like or punctate pattern of nucleolin on myotubes in culture and also in muscle sections may also indicate that nucleolin has a role at localized areas of the developing muscle membrane It is noteworthy that several binding partners of nucleolin are also found in a punctate pattern on cell surfaces, and in one case pleiotrophin can copatch nucleolin [48] Our previous data suggest that the PTPr ligand in muscle does not localize selectively with developing neuromuscular junctions [37] Direct examination of the localization of nucleolin and acetylcholine receptor also indicates that nucleolin is not notably enriched in neuromuscular junctions (D Alete & A Stoker, unpublished data) It is possible that nucleolin is interacting with motor axon-associated PTPr prior to neuromusclular junction formation, and it also cannot be ruled out that nucleolin associates with PTPr on sensory axons [66], in particular the mechanosensory afferents In the blocking experiments with lactoferrin and HB19, our data also showed a blockade of PTPr interactions with other known ligand sites, in particular those of HSPGs in retinal basement membranes It is not clear what this means at present, as the basement membrane interactions of PTPr are absolutely dependent on HSPGs If they are also dependent on nucleolin, then this might invoke a receptor complex containing both HSPGs and nucleolin, both of which might be necessary for a functional interaction with PTPr in the retinal inner basement membrane It is interesting to note that of the molecules known to bind to surface nucleolin, pleiotrophin, midkine, lactoferrin and PTPr also bind to HSPGs [48,53] For example, both midkine and PTPr have been reported to bind to the HSPG agrin [41,67] Although this may be coincidental, it suggests that these molecules share some binding properties and may therefore interact with nucleolin, or a complex of nucleolin and HSPGs, in a similar fashion Having said this, the situation in muscle is still distinct, since PTPr binding occurs independently of heparan sulfate [37] Furthermore, nucleolin does not require HSPGs to reach the cell surface, at least in CHO cells [59] The mechanism of molecular interaction between PTPr and the muscle-associated nucleolin remains to be determined However, in light of the facts that HB19 and lactoferrin bind to the RGG domain at the C-terminal tail of nucleolin, and that both these components perturb the interaction between PTPr and its muscle ligand, it is plausible to suggest that PTPr interacts with the RGG domain of surface nucleolin 4676 The biological significance of the interaction between PTPr and nucleolin in muscle has also yet to be elucidated Although both PTPr and PTPd influence motor axon growth and branching within the target field [34,38], nucleolin itself has not so far been implicated in muscle or neuromuscular development Furthermore, although nucleolin may act as a coreceptor for HIV, for example, the normal molecular function of cell surface nucleolin in any type of cell is still relatively unclear From the present study, we could hypothesize that nucleolin might serve as part of a receptor complex on the surface of developing muscle, recognizing adhesive molecules such as PTPr present on incoming growth cones of motor neurons or mechanosensory afferent axons Recent studies have indeed shown that cell surface nucleolin can function as a cell adhesion molecule [68] To address further the function of nucleolin in muscles, function-blocking C-terminal antibodies would be advantageous, and methods need to be developed for isolating larger amounts of nondegraded, cell surface nucleolin If nucleolin is involved in an RPTP recognition complex, it will then be possible to test more directly what the cellular and biochemical consequences are of PTPr–nucleolin interactions Materials and methods Fusion protein constructs and fusion protein production The FN3d–AP protein represents a truncated ectodomain region of cPTPr1 (amino acids 1–597), fused at its C-terminus to the placental AP gene in vector pBG as described previously [41] The FN3d–AP expression vector was transfected into 293T cells (grown in, DMEM, 10% fetal bovine serum, 1% penicillin ⁄ streptomycin mixture; Sigma Aldrich, Gillingham, UK) using calcium phosphate Conditioned medium containing the secreted FN3d–AP fusion protein was collected after 6–7 days, sterile filtered, buffered to pH 7.4 with 20 mm Hepes, and stored at °C RAP assays were carried out on unfixed tissue cryosections as described previously [44] Purification of fusion protein One milliliter of anti-placental alkaline phosphatase-agarose (anti-PLAP; Sigma) was packed into an FPLC column (Amersham Biosciences, Chalfont St Giles, UK) Purification of FN3d–AP was carried out using an AKTA FPLC system (Amersham Biosciences) The column was equilibrated using five column volumes of 0.05 m Tris and 0.5 m NaCl (pH 8.0) at a flow rate of 0.5 mLỈmin)1, and FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS D E Alete et al flow-through absorbance was measured at 280 nm Conditioned media was centrifuged at 400 g for on a Sorvall Legend RT with a 7500 6445 rotor, and the supernatant was recovered and loaded onto the column The column was then washed with five column volumes of the equilibration buffer to remove unbound components Bound components were eluted using a mixture containing 0.05 m glycine and 0.5 m NaCl (pH 2.8) Fractions (500 lL) were collected directly into tubes containing 50 lL of a 1.0 m Tris ⁄ HCl (pH 9.0) solution Purified fusion constructs (determined by the absorbance at 280 nm) were pooled, and desalted using a PD10 desalting column (Amersham Biosciences), and their purity was determined by gel electrophoresis Affinity chromatography Limb and chest muscle tissue (1 g) was dissected from E10 chick embryos and homogenized with a glass homogenizer in a mixture containing 10 mL of 4% Chaps, 100 mm KH2PO4 (pH 7.5), 5% glycerol, and protease inhibitor cocktail (Roche, Lewes, UK) The lysate was vortexed for h at °C and centrifuged at 2000 g on a Sorvall Legend RT with a 7500 6445 rotor, and the supernatant was recovered and diluted : in NaCl ⁄ Pi and incubated with affinity matrix (1 mL of CnBr sepharose covalently coupled to mg of FN3d–AP, AP or lactoferrin) overnight at °C Chromatography was carried out on an AKTA FPLC system (Amersham Biosciences) under the following conditions The column was washed with five column volumes of NaCl ⁄ Pi containing 0.1% Tween and 30 mm EDTA Bound components were eluted using NaCl ⁄ Pi containing 0.5 m NaCl (2 mL) into 200 lL fractions, separated by PAGE (6% gel) under reducing conditions, and visualized by silver staining [69] Protein identification by tandem MS (LC-MS ⁄ MS) Bands from a Coomassie-stained gel were cut out and subjected to digestion with trypsin as follows Gel pieces were washed three times in 30 lL of 50% CH3CN with agitation The gel pieces were dried in a vacuum centrifuge for 10 min, and reduced with a mixture containing 10 mm dithiothreitol and 10 mm NH4HCO3 (pH 8.0) (15 lL) for 45 at 50 °C; this was followed by alkylation with 50 mm idoacetamide and 10 mm NH4HCO3 for h at room temperature in the dark Gel pieces were washed three times in 30 lL of 50% CH3CN and vacuum-dried before being reswollen with 50 ng of modified trypsin (Promega, Southampton, UK) in lL of 10 mm NH4HCO3 The pieces were then overlaid with 10 mm NH4HCO3 (10 lL) and incubated for 16 h at 37 °C The samples were centrifuged at 16 000 g on a Heraeus Biofuge and the supernatant was recovered Peptides were further extracted twice with 10 lL of 5% trifluoroacetic acid in Nucleolin is a potential ligand for PTPr 50% CH3CN and the supernatants were pooled Peptide extracts were vacuum-dried and resuspended in lL of double-distilled H2O containing 20 mm NH4HPO4 Digested peptide mixtures were separated by nanoHPLC (Ultimate; LC Packings, Amsterdam, Holland) equipped with a PepMap column (75 lm · 15 cm; LC Packings) at a flow rate of 300 nLỈmin)1 Eluting peptides were analysed by ESI-MS ⁄ MS in a quadrupole ⁄ orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer (Micromass, Wythenshaw, Manchester, UK), using a nanoelectrospray ion source and ESI emitters with a 15 lm tapered end (New Objective, Woburn, MA, USA) Proteins were identified using the SwissProt database with the MASCOT search engine (http://www.matrix-science.com) A parent ion tolerance of ± m ⁄ z, a peptide ion tolerance of ± m ⁄ z, one missed cleavage, fixed carbamidomethylation of cysteines and variable oxidation of methionines were specified Purification of nucleolin A myc-tagged chick nucleolin expression vector [gift from E A Nigg, Swiss institute for experimental cancer research (ISREC), Epalinges, Switzerland] was transfected into 293T cells Cells were cultured for days, washed in cold NaCl ⁄ Pi and solubilized in a mixture containing 4% Chaps, 100 mm KH2PO4 (pH 7.5), 5% glycerol, and protease inhibitor cocktail (Roche) The lysate was centrifuged at 1500 g on a Sorvall Legand RT with a 7500 6445 rotor, and the supernatant was recovered Myc-tagged nucleolin was purified using anti-myc agarose (Sigma), as described for fusion protein purification Solid-phase binding assay Fifteen micrograms of nucleolin and BSA were immobilized on a 96-well microtiter plate for h at room temperature The remaining binding sites were saturated by overnight incubation in NaCl ⁄ Pi containing 2% goat serum (Dako, Glostrup, Denmark) Wells were incubated for h at room temperature with conditioned media containing AP fusion proteins (FN3d) After four washes in NaCl ⁄ Pi and one in SEAP buffer (0.5 mm MgCl2, m diethanolamine, pH 9.8), the bound AP activity was determined by adding 200 lL of SEAP buffer containing 10 mm p-nitrophenyl phosphate Progress curves were recorded for h at room temperature, at 405 nm, using a Dynex MRX microplate reader (Dynex, Worthing, UK) Immunoblot analysis Affinity-purified samples were separated on a 6% Tris ⁄ glycine gel by SDS ⁄ PAGE and transferred onto PVDF membrane for 40 at 120 V Membranes were blocked with FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4677 Nucleolin is a potential ligand for PTPr D E Alete et al NaCl ⁄ Pi containing 5% milk powder for h and incubated with rabbit polyclonal anti-nucleolin serum (1 : 5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in NaCl ⁄ Pi, 5% milk and 0.05% Tween-20 overnight at °C After extensive washing, the membranes were incubated with peroxidase-coupled goat anti-rabbit serum (1 : 10 000; Dako), and bound antibody was detected using ECL (Amersham Biosciences) Immunohistochemistry and immunofluorescence analysis E10 chick embryo heads were frozen in Tissue-Tek OCT (optimal cutting temperature) compound (Sakura Finetek, Torrance, CA, USA), cryosectioned (10–12 lm) and mounted on superfrost plus slides (VWR, Lutterworth, UK) For immunohistochemistry, sections were fixed for with )20 °C methanol and then blocked with 1% goat serum and 0.05% Tween in NaCl ⁄ Pi for 30 at room temperature Primary antibodies used were rabbit anti-nucleolin (Santa Cruz) at : 100 dilution, and mouse anti-myosin (F59; Developmental Studies Hybridoma Bank) used at : 100 Antibodies were diluted in NaCl ⁄ Pi containing 1% goat serum and 0.05% Tween-20, and incubated on sections for h at room temperature Sections were then washed three times in NaCl ⁄ Pi and 0.05% Tween-20, and secondary antibodies [goat anti-rabbit biotin conjugated, : 100 (Dako); goat anti-mouse fluorescein isothiocyanate (FITC) conjugated : 100 (Dako)] were incubated for h at room temperature After three final washes, FITC-labeled sections were mounted with Vectashield HardsetTM mounting medium with 4’-6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA, USA) For biotinlabeled sections, slides were incubated for a further 30 in NaCl ⁄ Pi and 0.05% Tween, containing streptavidin-conjugated Cy3 (1 : 400; Amersham Biosciences), and washed and mounted as described For HB-19 peptide staining, biotinylated HB-19 peptide [49] was diluted in NaCl ⁄ Pi containing 1% goat serum and 0.05% Tween-20 to a final concentration of 10 lm and incubated for h at room temperature Sections were washed and incubated with streptavidin-conjugated Cy3 as described above Sections were analyzed using an Axiophot fluorescence microscope (Zeiss, Welwyn Garden City, UK) and photographed with a Leica DC500 digital camera (Leica, Milton Keynes, UK) Myotube cultures Embryonic myotube cultures were established from E10 chick muscle tissue E10 trunk tissue was dissociated enzymatically and plated at 106 cells per 60 mm plate on fibronectin-coated glass coverslips in a mixture containing DMEM, 2% chick serum, 4% fetal bovine serum and 1% penicillin ⁄ streptomycin (Sigma) for 72 h For cell surface 4678 staining, primary antibodies [rabbit anti-nucleolin, : 100; mouse anti-myosin (F59), : 100] were diluted in growth medium and incubated with the cells for 30 at °C Cells were then washed three times with cold NaCl ⁄ Pi and fixed with 4% paraformaldehyde for 15 at °C Secondary antibodies (anti-rabbit biotin conjugated, anti-mouse FITC conjugated) were diluted : 100 in a mixture containing NaCl ⁄ Pi, 1% goat serum and 0.05% Tween-20, and incubated with the cells for h at room temperature Cells were washed, mounted and analyzed as described above To semipermeabilize cells for intracellular staining, cells were incubated in 4% paraformaldehyde for 30 at °C in the first instance and then stained as described above Confocal microscopy and imaging E10 chick head sections were stained as described earlier with antibodies against myosin and nucleolin with FITC and Cy3 secondary antibodies, respectively The sections were examined using a Leica TCS 4D laser scanning confocal microscope The 488 nm line of the laser was used to visualize the FITC–myosin and the 568 nm line was used for the Cy3–nucleolin With use of these wavelengths, separation of the fluorescent signals from the two fluorophores was almost complete A series of optical sections lm apart were taken through a depth of 20 lm All images were stored digitally, and three-dimensional reconstruction and visualization were carried out using the volocity software (Improvision Ltd, Coventry, UK) The parameters were set as follows: green channel 100% density, red channel 1% density, medium noise filter Acknowledgements We thank Clare Faux and Juan Pedro Martinez-Barbera for critical reading of the manuscript The research was funded by the Wellcome Trust (071418) References Van Vactor D (1998) Adhesion and signaling in 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secreted recombinant monoclonal antibody productivities Proteomics 5, 4689–4704 FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4681 ... anti-placental alkaline phosphatase (PLAP) agarose (C) SDS ⁄ PAGE and silver stain of proteins isolated from AP sepharose (lane 1) and FN3d–AP sepharose (lane 2) A protein band of approximately 95 kDa is. .. nucleolin, mean that calculations of binding affinity are unrealistic at this stage Nucleolin localization in muscle is analogous to PTPr ligand localization To determine whether the nucleolin identified... This revealed a band at approximately 95 kDa present in the PTPr eluate only These data confirm that nucleolin is a binding partner for PTPr under these conditions PTPr can bind directly to nucleolin