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• W2070711271 abstract "HB-GAM (heparin-binding growth-associated molecule, also designated as pleiotrophin) and midkine form a two-member family of extracellular matrix proteins that bind tightly to sulfated carbohydrate structures such as heparan sulfate. These proteins are used by developing neurons as extracellular cues in axonal growth and guidance. HB-GAM was recently reported to enhance differentiation of neural stem cells. Based on the solution structure of HB-GAM, we have recently shown that HB-GAM consists of two β-sheet domains flanked by flexible lysine-rich N- and C-terminal tails with no apparent structure. These domains are homologous to thrombospondin type I repeats present in numerous extracellular proteins that interact with the cell surface. Our findings showed that the two β-sheet domains fold independently. We showed that the domains (but not the lysine-rich tails) in HB-GAM are required and sufficient for interaction with hippocampal neurons. The individual domains bind heparan sulfate weakly and fail to produce significant biological effects in neurite outgrowth and long term potentiation assays. The amino acids in the linker region joining the two domains may be replaced with glycines with no effect on protein function. These results suggest a co-operative action of the two β-sheet domains in the biologically relevant interaction with neuron surface heparan sulfate. HB-GAM (heparin-binding growth-associated molecule, also designated as pleiotrophin) and midkine form a two-member family of extracellular matrix proteins that bind tightly to sulfated carbohydrate structures such as heparan sulfate. These proteins are used by developing neurons as extracellular cues in axonal growth and guidance. HB-GAM was recently reported to enhance differentiation of neural stem cells. Based on the solution structure of HB-GAM, we have recently shown that HB-GAM consists of two β-sheet domains flanked by flexible lysine-rich N- and C-terminal tails with no apparent structure. These domains are homologous to thrombospondin type I repeats present in numerous extracellular proteins that interact with the cell surface. Our findings showed that the two β-sheet domains fold independently. We showed that the domains (but not the lysine-rich tails) in HB-GAM are required and sufficient for interaction with hippocampal neurons. The individual domains bind heparan sulfate weakly and fail to produce significant biological effects in neurite outgrowth and long term potentiation assays. The amino acids in the linker region joining the two domains may be replaced with glycines with no effect on protein function. These results suggest a co-operative action of the two β-sheet domains in the biologically relevant interaction with neuron surface heparan sulfate. The formation of neuronal connections in the developing brain is regulated by interactions of the cell surface with extracellular matrix molecules, soluble growth factors, and cell surface adhesion molecules. Similar mechanisms are implicated in the control of plastic changes in the adult nervous system. Heparan sulfate (HS)-mediated interactions of growing neuronal processes with proteins of the extracellular matrix are involved in axonal path finding during development and plasticity. Understanding the molecular mechanisms of heparin/HS binding by these proteins is therefore of key importance in molecular neurobiology.HB-GAM 3The abbreviations used are: HB-GAMheparin-binding growth-associated moleculeTSRthrombospondin type 1 repeatPBSphosphate-buffered salinefEPSPfield excitatory postsynaptic potentialLTPlong term potentiationBSAbovine serum albuminHSQCheteronuclear single quantum coherenceHSheparan sulfate.3The abbreviations used are: HB-GAMheparin-binding growth-associated moleculeTSRthrombospondin type 1 repeatPBSphosphate-buffered salinefEPSPfield excitatory postsynaptic potentialLTPlong term potentiationBSAbovine serum albuminHSQCheteronuclear single quantum coherenceHSheparan sulfate. has been described as a heparin-binding protein expressed at the extracellular matrix of axonal tracts in the developing brain (1Rauvala H. Huttunen H.J. Fages C. Kaksonen M. Kinnunen T. Imai S. Raulo E. Kilpeläinen I. Matrix Biol. 2000; 19: 377-387Crossref PubMed Scopus (129) Google Scholar, 2Rauvala H. Vanhala A. Castren E. Nolo R. Raulo E. Merenmies J. Panula P. Brain Res. Dev. Brain Res. 1994; 79: 157-176Crossref PubMed Scopus (94) Google Scholar, 3Kinnunen A. Kinnunen T. Kaksonen M. Nolo R. Panula P. Rauvala H. Eur. J. Neurosci. 1998; 10: 635-648Crossref PubMed Scopus (67) Google Scholar, 26Rauvala H. EMBO J. 1989; 8: 2933-2941Crossref PubMed Scopus (352) Google Scholar). In addition, the protein has been implicated in hippocampal synaptic plasticity (4Lauri S.E. Rauvala H. Kaila K. Taira T. Eur. J. Neurosci. 1998; 10: 188-194Crossref PubMed Scopus (42) Google Scholar, 5Lauri S.E. Taira T. Kaila K. Rauvala H. Neuroreport. 1996; 7: 1670-1674Crossref PubMed Scopus (29) Google Scholar) and bone development (6Imai S. Kaksonen M. Raulo E. Kinnunen T. Fages C. Meng X. Lakso M. Rauvala H. J. Cell Biol. 1998; 143: 1113-1128Crossref PubMed Scopus (101) Google Scholar). Neurite outgrowth and osteoblast migration induced by HB-GAM depend on the neuronal cell surface heparan sulfate proteoglycan N-syndecan, also designated as syndecan-3 (6Imai S. Kaksonen M. Raulo E. Kinnunen T. Fages C. Meng X. Lakso M. Rauvala H. J. Cell Biol. 1998; 143: 1113-1128Crossref PubMed Scopus (101) Google Scholar, 7Raulo E. Chernousov M.A. Carey D.J. Nolo R. Rauvala H. J. Biol. Chem. 1994; 269: 12999-13004Abstract Full Text PDF PubMed Google Scholar, 8Kinnunen T. Raulo E. Nolo R. Maccarana M. Lindahl U. Rauvala H. J. Biol. Chem. 1996; 271: 2243-2248Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). HB-GAM is also described as a developmentally regulated cytokine pleiotrophin (9Li Y.S. Milner P.G. Chauhan A.K. Watson M.A. Hoffman R.M. Kodner C.M. Milbrandt J. Deuel T.F. Science. 1990; 250: 1690-1694Crossref PubMed Scopus (452) Google Scholar).We have suggested in our previous work (10Kilpeläinen I. Kaksonen M. Avikainen H. Fath M. Linhardt R.J. Raulo E. Rauvala H. J. Biol. Chem. 2000; 275: 13564-13570Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) that HB-GAM and mid-gestation kidney protein MK are structurally similar heparin-binding proteins homologous to the thrombospondin type 1 repeat (TSR) (Fig. 1). The TSR repeat is found in a larger superfamily of extracellular matrix-associated and cell surface proteins, such as thrombospondins 1 and 2, F-spondin, mindin, semaphorins F and G, and TRAP (thrombospondin-related anonymous protein from malaria parasite Plasmodium falciparum) (for review, see Ref. 11Adams J.C. Tucker R.P. Dev. Dyn. 2000; 218: 280-299Crossref PubMed Scopus (263) Google Scholar). A common feature for proteins in this superfamily is the function in cell surface and matrix binding that is dependent on heparin-type polysaccharides. Indeed, HB-GAM has been shown to bind the cell surface receptor N-syndecan via the heparan sulfate side chains of N-syndecan (7Raulo E. Chernousov M.A. Carey D.J. Nolo R. Rauvala H. J. Biol. Chem. 1994; 269: 12999-13004Abstract Full Text PDF PubMed Google Scholar, 8Kinnunen T. Raulo E. Nolo R. Maccarana M. Lindahl U. Rauvala H. J. Biol. Chem. 1996; 271: 2243-2248Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This interaction leads to signaling by N-syndecan to cortactin/Src kinase pathway and reorganization of the cytoskeletal network resulting in the neuronal growth cone and osteoblast migration (6Imai S. Kaksonen M. Raulo E. Kinnunen T. Fages C. Meng X. Lakso M. Rauvala H. J. Cell Biol. 1998; 143: 1113-1128Crossref PubMed Scopus (101) Google Scholar, 12Kinnunen T. Kaksonen M. Saarinen J. Kalkkinen N. Peng H.B. Rauvala H. J. Biol. Chem. 1998; 273: 10702-10708Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Similarly, mid-gestation kidney protein MK has been shown to interact with syndecans 1 and 4 earlier in development (13Kojima T. Katsumi A. Yamazaki T. Muramatsu T. Nagasaka T. Ohsumi K. Saito H. J. Biol. Chem. 1996; 271: 5914-5920Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In addition to N-syndecan, HB-GAM interacts with the receptor-type protein tyrosine phosphatase RPTPβ/ζ at the cell surface (14Maeda N. Nishiwaki T. Shintani T. Hamanaka H. Noda M. J. Biol. Chem. 1996; 271: 21446-21452Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 15Milev P. Chiba A. Haring M. Rauvala H. Schachner M. Ranscht B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 6998-7005Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 16Milev P. Monnerie H. Popp S. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 21439-21442Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). We have recently shown that HB-GAM inhibits proliferation and induces differentiation of neural stem/progenitor cells. These activities of HB-GAM are proposed to occur through competition with fibroblast growth factor-2 for binding to cell surface heparan sulfate (17Hienola A. Pekkanen M. Raulo E. Vanttola P. Rauvala H. Mol. Cell. Neurosci. 2004; 26: 75-88Crossref PubMed Scopus (61) Google Scholar).The present paper shows that the TSR domains in HB-GAM fold independently, and they do not interact with each other in solution conditions. Individual TSR domains derived from HB-GAM bind weakly to heparin/HS and fail to influence neurite outgrowth and plasticity. The domain structure of HB-GAM, in which both TSR domains are present, is required for heparin/HS binding and interaction with hippocampal neurons.EXPERIMENTAL PROCEDURESPCR Mutagenesis of TSR Domain Proteins—The TSR domains of HB-GAM as individual recombinant proteins and as a di-domain polypeptide (TABLE ONE) were produced in Escherichia coli using the glutathione S-transferase fusion vector pGEX-2T (Amersham Biosciences) as described previously (10Kilpeläinen I. Kaksonen M. Avikainen H. Fath M. Linhardt R.J. Raulo E. Rauvala H. J. Biol. Chem. 2000; 275: 13564-13570Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The domain boundaries were predicted from previous NMR structural data (10Kilpeläinen I. Kaksonen M. Avikainen H. Fath M. Linhardt R.J. Raulo E. Rauvala H. J. Biol. Chem. 2000; 275: 13564-13570Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18Iwasaki W. Nagata K. Hatanaka H. Inui T. Kimura T. Muramatsu T. Yoshida K. Tasumi M. Inagaki F. EMBO J. 1997; 16: 6936-6946Crossref PubMed Scopus (142) Google Scholar). The lysine-rich tails were excluded from the domain constructs. The single domain spans were as follows: N-terminal domain, amino acids Ser13-Asn58; C-terminal domain, amino acids Ala65-Gly110. The di-domain construct spanned amino acids Ser13-Gly110. Mismatch primers were used to generate differentially truncated forms of rat HB-GAM cDNA coding region as presented in TABLE ONE. To study the contribution of the linker region between the two TSR domains in heparin binding and biological activity constructs, G-linked di-TSR, TSR-N+Linker, and TSR-C+Linker were generated. All DNA constructs were sequenced to exclude PCR-born mutations.TABLE ONEMutants produced by PCR mutagenesis Open table in a new tab Heparin Affinity Chromatography of HB-GAM Domains—The bacterial lysates were subjected to heparin affinity chromatography. A linear NaCl gradient from 0 to 2 m in 20 mm sodium phosphate buffer, pH 7.5, was used to compare the elution profiles of separate domains with the intact HB-GAM produced similarly in E. coli. All recombinant products described above were purified by this method and verified for >90% homogeneity on SDS-PAGE. The proteins were subjected to matrix-assisted laser desorption ionization mass spectroscopy to verify molecular weight before they were used in the subsequent studies. The 15N- and 15N/13C-labeled protein samples for NMR were produced by growing the bacteria in a minimal media as described previously (10Kilpeläinen I. Kaksonen M. Avikainen H. Fath M. Linhardt R.J. Raulo E. Rauvala H. J. Biol. Chem. 2000; 275: 13564-13570Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), followed by normal workup.NMR Measurements of HB-GAM-derived TSR Domains—All the spectra were acquired at 30 °C on Varian Inova 600- and 800-MHz spectrometers using protein concentrations of 0.2-1 mm. The spectra were processed with Vnmr (Varian Inc., Palo Alto, CA) and analyzed manually with Felix 97 software (Biosym/Molecular Simulations, Inc., San Diego, CA). The backbone assignments for the individual domains were carried out by recording a typical set of triple resonance spectra (CBCA(CO)NH, HNCACB, HNCA and HN(CO)CA, (19Cavanagh J. Fairbrother W.J. Palmer A.J. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice. Academic Press Limited, London1968: 478-518Google Scholar)) followed by normal spectral interpretation/analysis. The possible interaction of the individual domains was followed by titrating a 15N-labeled N-terminal domain sample (1 mm, residues 13-58) to a 5-fold excess of unlabeled C-domain (residues 65-110), and vice versa. The heparin interaction experiments were carried out by titrating the protein samples with a purified 14-meric heparan sulfate preparate (20Fath M. VanderNoot V. Kilpelainen I. Kinnunen T. Rauvala H. Linhardt R.J. FEBS Lett. 1999; 454: 105-108Crossref PubMed Scopus (24) Google Scholar).Ligand-binding Assays—HB-GAM domain binding to heparin was analyzed by surface plasmon resonance using the IAsys instrument (Thermo Electron Corporation). Heparin-derivatized surfaces were prepared by immobilizing heparin-BSA (Sigma) to one cell of the double-well planar aminosilane cuvette according to the manufacturer's instructions. Briefly, 0.2 mg/ml heparin-BSA in 15 mm Na2HPO4, pH 7.4, was coupled via amino groups to the aminosilane surface activated with polymerized glutaraldehyde, resulting in an immobilization level of ∼0.9 ng of heparin-BSA/mm2 planar surface. The second cell was coated with BSA alone to generate a control surface. Unbound molecules were removed by washing the cuvettes with the immobilizing buffer and 2 m NaCl in phosphate-buffered saline (PBS). Remaining activated sites were blocked with β-casein. The binding data for each ligand were obtained by successive additions of increasing ligand concentrations in PBS and measuring the equilibrium response after each addition. Between the measurements for different ligands, the cuvette was regenerated by washing with 2 m NaCl in PBS. All ligands displayed low or negligible binding to the control BSA surface, as was monitored simultaneously in the control cell. The response in the control cell was subtracted from the value measured in the heparinized cell to obtain the specific response R, which was used to calculate the dissociation constant Kd by fitting in the equation r = Rmax[L]/(Kd + [L]), where [L] is the ligand concentration and Rmax corresponds to the R value at saturation.Binding to Heparan Sulfate—Recombinant rat N-syndecan ectodomain was expressed as an Fc-fusion protein in 293T cells using the pIG expression vector (21Simmons D.C. Cellular Interactions in Development: a Practical Approach. 1993; (Hartley, D. A., ed) pp., Oxford University Press, Oxford: 93-127Google Scholar). The construct was transfected to semiconfluent 293T cells using FuGENE® transfection reagent (Roche Diagnostics, Gmbh, Mannheim, Germany). Three days post-transfection the medium was harvested. The medium was centrifuged to remove debris and used as a reagent in binding assays. As a control, a mock medium transfected with FuGENE® alone was prepared. Wells of Nunc maxisorb plates were coated with 1 μg/ml protein A (Amersham Biosciences) and blocked with 1% BSA in PBS, 0.05% Tween 20. Conditioned medium from transfected 293T cells was added to the wells. After washing three times with 0.1% BSA, 0.05% Tween 20 in PBS, biotinylated di-TSR was added with or without competing unlabeled ligands. The level of biotin bound to the wells was monitored with horseradish peroxidase-conjugated streptavidin using the ortho-phenylenediamine chromogenic substrate according to the manufacturer's instructions (Sigma). To control the binding through heparan sulfate glycosaminoglycans, heparinase II (Sigma) was used. 0-2 IU/ml heparinase II was added in the assay medium to cleave heparan sulfate glycosaminoglycan side chains of the recombinant N-syndecan-Fc-fusion protein.Heparin-binding Assay—96-well Nunc maxisorb plates were coated with 100 μl of 1 μg/ml HB-GAM for an hour. The wells were blocked with 1% BSA in PBS, 0.05% Tween 20 for 1 h. After washing the wells three times with the blocking buffer, 0.1 μg/ml biotin-BSA-heparin (Sigma) with 20 μg/ml competing unlabeled ligands in 0.1% BSA in PBS, 0.05% Tween 20 was incubated in the wells for 1 h at +4 °C. The level of biotin bound to the wells after three washing steps was monitored with horseradish peroxidase-conjugated streptavidin using the ortho-phenylenediamine chromogenic substrate according to the manufacturer's instructions (Sigma).Cells and Neurite Outgrowth Assays—Hippocampal primary cultures were prepared essentially as described previously (22Brewer G.J. Cotman C.W. Brain Res. 1989; 494: 65-74Crossref PubMed Scopus (324) Google Scholar). For neurite outgrowth assays, neurons were plated at 12,500/well density in microwells coated with recombinant HB-GAM and different TSR domains. For the counting of neurite outgrowth, images were taken from living cells using randomly selected microscopic fields, and the extensions exceeding 10 μm in length were considered as neurites. The induction of neurite outgrowth was calculated as percentage of cells growing neurites in the 48-h assay.The ability of different TSR domains to inhibit neurite outgrowth on HB-GAM-coated wells when presented as soluble factors was monitored as described previously (23Raulo E. Julkunen I. Merenmies J. Pihlaskari R. Rauvala H. J. Biol. Chem. 1992; 267: 11408-11416Abstract Full Text PDF PubMed Google Scholar). Briefly, neurons were plated at 12,500/well density in microwells coated with HB-GAM (1 μg/ml). The cells were grown for 48 h in assay medium containing different concentrations of soluble domains and analyzed for neurite outgrowth.Transfilter Migration Assays—Migration assays were performed with NMRI mice strain embryonic day 14-15 forebrain neurons prepared as described previously (24Rauvala H. Merenmies J. Pihlaskari R. Korkolainen M. Huhtala M.L. Panula P. J. Cell Biol. 1988; 107: 2293-2305Crossref PubMed Scopus (76) Google Scholar). The cells were plated at the density of 150,000 cells/well on 12-mm COSTAR transfilter plates with a 12-μm pore size. The outer surfaces of the filters were precoated with 1 mm HB-GAM. Because the di-TSR polypeptide was apparently unable to coat the wells, we performed the assay in the presence of 1 mm HB-GAM or di-TSR as soluble competitors in the assay medium (BSA at 10 mg/ml, Dulbecco's modified Eagle's medium). The inhibitory effect on migration after 16 h was followed.In Vitro Electrophysiology—Transverse hippocampal slices (400 μm thick) from adult Wistar rats (1.5-2 months old) were cut using Vibratome 3000 Plus for in vitro electrophysiological experiments. Slices were allowed to recover at room temperature for at least 60 min before the experiments. Recordings were made in the interface-type chamber (volume 1 ml; perfusion rate 1 ml/min) at +32 °C. Artificial cerebrospinal fluid containing (in mm) NaCl (124), KCl (3Kinnunen A. Kinnunen T. Kaksonen M. Nolo R. Panula P. Rauvala H. Eur. J. Neurosci. 1998; 10: 635-648Crossref PubMed Scopus (67) Google Scholar), CaCl2 (2Rauvala H. Vanhala A. Castren E. Nolo R. Raulo E. Merenmies J. Panula P. Brain Res. Dev. Brain Res. 1994; 79: 157-176Crossref PubMed Scopus (94) Google Scholar), NaHCO3 (25Anderson W.W. Collingridge G.L. J. Neurosci. Methods. 2001; 108: 71-83Crossref PubMed Scopus (345) Google Scholar), NaH2PO4 (1.1), Mg SO4 (1.3), glucose (10Kilpeläinen I. Kaksonen M. Avikainen H. Fath M. Linhardt R.J. Raulo E. Rauvala H. J. Biol. Chem. 2000; 275: 13564-13570Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), was equilibrated with the mixture of 5% CO2 and 95% O2 to yield a pH of 7.4.Extracellular recordings from stratum radiatum of the CA1 area of the hippocampi were obtained using glass capillary microelectrodes filled with 150 mm NaCl. Field excitatory postsynaptic potentials (fEPSPs) were elicited by stimulating Schaffer collaterals with a bipolar stimulation electrode. The stimulus intensity was adjusted to gain a half-maximal fEPSP amplitude. Base-line synaptic transmission was monitored at 0.05 Hz, pulse length 0.1 ms. The slope of fEPSP was used as an indicator of synaptic efficacy and was calculated between 20 and 80% of the maximal amplitude. Long term potentiation (LTP) was induced by high frequency stimulation (100 Hz/1 s), during which the pulse length was doubled. TSR domains were dissolved in PBS (0.1 mm NaHPO4, 150 mm NaCl, pH 7.4) at a concentration of 200 μg/ml. Because of the limited amounts of TSR domains, they were pressure-injected (∼0.2 μl) into the dendritic area of the hippocampal CA1 region close to the recording site 10 min before LTP induction (as described in Ref. 4Lauri S.E. Rauvala H. Kaila K. Taira T. Eur. J. Neurosci. 1998; 10: 188-194Crossref PubMed Scopus (42) Google Scholar). Control experiments with PBS injections of the same volume were carried out to ensure that the procedure did not interfere with the base-line synaptic response or induction of LTP.LTP program, version 230d (25Anderson W.W. Collingridge G.L. J. Neurosci. Methods. 2001; 108: 71-83Crossref PubMed Scopus (345) Google Scholar), was used for data acquisition and analysis. Student's t test was used for statistical analysis of the data. Changes were considered to be significant at p values <0.05.RESULTS AND DISCUSSIONTSR Domains of HB-GAM Fold Independently—The 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the isolated domains coincide with the corresponding spectrum of full-length HB-GAM (Fig. 2). Only very minor changes are observed in the chemical shifts, indicating that the structures of the isolated domains are similar to those in the native protein. Also the di-TSR 1H-15N HSQC spectrum coincides fully with the spectrum of the whole protein (data not shown), indicating a proper fold.FIGURE 21H-15N HSQC spectrum (750 MHz) of HB-GAM (purple) and the N-terminal (green) and C-terminal (blue) TSR domain overlaid. The spectra of the individual TSR domains essentially align with the residues in HB-GAM spectra. This indicates that the domains fold independently and do not interact with each other in the intact protein. ppm, parts/million.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Domain-domain titration experiments were carried out to gain information on possible domain-domain interactions. In these titrations, a sample of the 15N-labeled domain was titrated with an unlabeled second domain. The titrations were carried out in both ways, i.e. the N-terminal domain was titrated with the C-domain, and vice versa. However, no changes in chemical shifts were observed in either case. Therefore, it is evident that the two domains fold independently and they do not have domain-domain interactions in solution state.Binding of Heparin to HB-GAM as Followed by NMR Spectroscopy—We have previously shown (10Kilpeläinen I. Kaksonen M. Avikainen H. Fath M. Linhardt R.J. Raulo E. Rauvala H. J. Biol. Chem. 2000; 275: 13564-13570Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) that during the titration of full-length HB-GAM with heparin, the 1H-15N HSQC resonances of the structural areas of the protein (i.e. N- and C-terminal domain) gradually disappear from the spectrum, but the resonances of the non-structural, lysine-rich tails (residues 1-12 and 111-136) remain in the spectrum with high intensity and show no significant changes in their chemical shifts. In this respect, the di-TSR (Gly13-Ser110) protein behaves in an exactly similar way as the full-length protein. During the titration with heparin, the 1H-15N resonances of the protein gradually broaden under the detection limit (data not shown).The individual N- and C-terminal domains interact with heparin in fast exchange in the NMR timescale, as shown in Fig. 3A for the N-terminal domain (spectral data for C-terminal not shown). The titration data, as a function of amino acid sequence for the isolated domains, are presented in Fig. 3, B and C. The binding data show that individual domains bind the 14-meric heparin fragment with a stoichiometry of 1:1. The Kd values for the separate N- and C-terminal domains were 53 and 20 μm, respectively, as calculated from the chemical shift changes during the titration. In the heparin titration experiments, residues 14-18, 39-43, and 52-54 in the N-terminal domain and residues 66-69 and 89-93 in the C-terminal domain show significant changes in their chemical shifts, indicating that these residues are playing a role in the interaction with heparin. However, these findings should be confirmed with mutation experiments.FIGURE 3A, an expansion of the 1H-15N HSQC spectra of the N-terminal domain of HB-GAM during heparin titration (600 MHz). TSR-N without heparin (black), TSR-N:heparin (1:0.2, red), TSR-N:heparin (1:0.5, orange), TSR-N:heparin (1:1, blue), TSR-N:heparin (1:2, blue), and TSR-N:heparin (1:5, green) are shown. F1, indirectly detected dimension (15N chemical shifts); F2, detected dimension (1H chemical shifts). B and C, the titration data as a function of amino acid sequence for the isolated domains to a ratio of 1:5. Residues 14-18, 39-43, and 52-54 in the N-terminal domain and residues 66-69 and 89-93 in the C-terminal domain show significant changes in their chemical shifts upon binding to heparin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A titration of a sample containing the N- and C-terminal domains in a 1:1 ratio with heparin shows a similar effect (data not shown), as seen with the whole protein and the di-TSR sample. However, the interaction of heparin with the two domains appears in intermediate exchange in the NMR timescale (not slow, as in the case of the intact protein). The titration was carried out by first titrating a sample of the 15N-enriched N-terminal domain with 15N-enriched C-terminal domain to a ratio of 1:1, followed by titration with heparin 14-mer and observing changes in the 1H-15N HSQC spectrum. During the course of titration with heparin, the original signals from the domains in solution gradually disappear from the spectrum. With an excess of heparin, a part of the signals becomes visible, but a large number of signals remains below detection limit, even in the presence of a large excess of heparin. Further, a good number of signals are just weakly visible at the end of the titration. Thus, it was not possible to obtain reliable assignments for the (TSR-N·TSR-C·heparin) complex. Similar results were obtained with commercial low molecular weight heparin. The disappearance of the signals of the structural domains of the protein when both domains are present in the solution cannot solely be explained by the molecular weight of the complex. It seems that HB-GAM undergoes a conformational change in the complex form, which causes the broadening of the signals. This is in agreement with the circular dichroism spectroscopy data described previously (10Kilpeläinen I. Kaksonen M. Avikainen H. Fath M. Linhardt R.J. Raulo E. Rauvala H. J. Biol. Chem. 2000; 275: 13564-13570Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar).The NMR titration data shows that the N- and C-terminal domains interact with heparin in a co-operative manner. The domains do not interact with each other in the absence of heparin, but the timescale of the heparin interaction changes when both domains are present, even when as separate domains.Both TSR Domains Are Required for High Affinity Interaction with Heparin/HS—The affinity of different HB-GAM domains for heparin was analyzed by surface plasmon resonance using a heparin-BSA-coated cuvette. The dissociation constants were determined by equilibrium titration (TABLE TWO). Intact HB-GAM displayed high affinity for heparin (Kd of 176 ± 37 nm). Removal of the lysine-rich tails had no effect on heparin binding of HB-GAM, as the Kd for the di-TSR remained nearly identical (153 ± 40 nm). However, a dramatic change was observed for the individual TSR domains. The C-terminal domain of HB-GAM displayed affinity for heparin (Kd of 13.0 ± 2.5 μm) that was two orders of magnitude lower than that of the intact protein. The N-terminal domain of HB-GAM bound heparin even more weakly. At the concentrations used in our assay, the saturation could not be reached, indicating a Kd value >70 μm. This would indicate that, although the individual domains of HB-GAM can interact with heparin, cooperation between the N-terminal and C-terminal domain results in the optimal heparin binding. The lysine-rich tails do not appear to be directly involved in the HB-GAM interaction with heparin.TABLE TWODissociation constants for ligand binding to immobilized heparin-BSA determined by equilibrium titrationIntact HB-GAMDi-TSRTSR-NTSR-Cnmnmμmμm176 ± 37153 ± 40>7013.0 ± 2.5" @default.
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• W2070711271 title "The Two Thrombospondin Type I Repeat Domains of the Heparin-binding Growth-associated Molecule Bind to Heparin/Heparan Sulfate and Regulate Neurite Extension and Plasticity in Hippocampal Neurons" @default.
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