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Ganglioside Inhibition of Neurite Outgrowth Requires Nogo Receptor Function

2008; Elsevier BV; Volume: 283; Issue: 24 Linguagem: Inglês

10.1074/jbc.m802067200

1083-351X

Gareth Williams, Andrew Wood, Emma‐Jane Williams, Ying Gao, Mary Lynn T. Mercado, Alan H. Katz, Diane Joseph‐McCarthy, Brian Bates, Huai‐Ping Ling, Ann Aulabaugh, Joe Zaccardi, Yuhong Xie, Menelas N. Pangalos, Frank S. Walsh, Patrick Doherty,

Neuroblastoma Research and Treatments

Gangliosides are key players in neuronal inhibition, with antibody-mediated clustering of gangliosides blocking neurite outgrowth in cultures and axonal regeneration post injury. In this study we show that the ganglioside GT1b can form a complex with the Nogo-66 receptor NgR1. The interaction is shown by analytical ultracentrifugation sedimentation and is mediated by the sialic acid moiety on GT1b, with mutations in FRG motifs on NgR1 attenuating the interaction. One FRG motif was developed into a cyclic peptide (N-AcCLQKFRGSSC-NH2) antagonist of GT1b, reversing the GT1b antibody inhibition of cerebellar granule cell neurite outgrowth. Interestingly, the peptide also antagonizes neurite outgrowth inhibition mediated by soluble forms of the myelin-associated glycoprotein (MAG). Structure function analysis of the peptide point to the conserved FRG triplet being the minimal functional motif, and mutations within this motif inhibit NgR1 binding to both GT1b and MAG. Finally, using gene ablation, we show that the cerebellar neuron response to GT1b antibodies and soluble MAG is indeed dependent on NgR1 function. The results suggest that gangliosides inhibit neurite outgrowth by interacting with FRG motifs in the NgR1 and that this interaction can also facilitate the binding of MAG to the NgR1. Furthermore, the results point to a rational strategy for developing novel ganglioside antagonists. Gangliosides are key players in neuronal inhibition, with antibody-mediated clustering of gangliosides blocking neurite outgrowth in cultures and axonal regeneration post injury. In this study we show that the ganglioside GT1b can form a complex with the Nogo-66 receptor NgR1. The interaction is shown by analytical ultracentrifugation sedimentation and is mediated by the sialic acid moiety on GT1b, with mutations in FRG motifs on NgR1 attenuating the interaction. One FRG motif was developed into a cyclic peptide (N-AcCLQKFRGSSC-NH2) antagonist of GT1b, reversing the GT1b antibody inhibition of cerebellar granule cell neurite outgrowth. Interestingly, the peptide also antagonizes neurite outgrowth inhibition mediated by soluble forms of the myelin-associated glycoprotein (MAG). Structure function analysis of the peptide point to the conserved FRG triplet being the minimal functional motif, and mutations within this motif inhibit NgR1 binding to both GT1b and MAG. Finally, using gene ablation, we show that the cerebellar neuron response to GT1b antibodies and soluble MAG is indeed dependent on NgR1 function. The results suggest that gangliosides inhibit neurite outgrowth by interacting with FRG motifs in the NgR1 and that this interaction can also facilitate the binding of MAG to the NgR1. Furthermore, the results point to a rational strategy for developing novel ganglioside antagonists. Antibodies that bind to the ganglioside GT1b 2The abbreviations used are: GT1b, trisialoganglioside1b; NgR, nogo receptor; MAG, myelin-associated glycoprotein; GM1, monosialoganglioside 1; CHO, Chinese hamster ovary; AP, alkaline phosphatase. inhibit neurite outgrowth (1Vinson M. Strijbos P.J. Rowles A. Facci L. Moore S.E. Simmons D.L. Walsh F.S. J. Biol. Chem. 2001; 276: 20280-20285Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 2Williams G. Williams E.J. Maison P. Pangalos M.N. Walsh F.S. Doherty P. J. Biol. Chem. 2005; 280: 5862-5869Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 3Fujitani M. Kawai H. Proia R.L. Kashiwagi A. Yasuda H. Yamashita T. J. Neurochem. 2005; 94: 15-21Crossref PubMed Scopus (41) Google Scholar). Furthermore, passive immunization with anti-ganglioside antibodies directly inhibits axonal regeneration after injury in mice (4Lehmann H.C. Lopez P.H. Zhang G. Ngyuen T. Zhang J. Kieseier B.C. Mori S. Sheikh K.A. J. Neurosci. 2007; 27: 27-34Crossref PubMed Scopus (79) Google Scholar). A considerable body of evidence also suggests that autoimmune anti-ganglioside antibodies might contribute to the poor prognosis of some patients with Guillain-Barre syndrome (5Walsh F.S. Cronin M. Koblar S. Doherty P. Winer J. Leon A. Hughes R.A. J. Neuroimmunol. 1991; 34: 43-51Abstract Full Text PDF PubMed Scopus (147) Google Scholar) and other peripheral neuropathies (6Willison H.J. Yuki N. Brain. 2002; 125: 2591-2625Crossref PubMed Scopus (613) Google Scholar). A better understanding of the mechanisms whereby ganglioside antibodies inhibits neurite outgrowth and the development of agents to circumvent this might lead to novel therapeutic opportunities for some peripheral neuropathies. The myelin-associated glycoprotein (MAG) can inhibit neurite outgrowth (7McKerracher L. David S. Jackson D.L. Kottis V. Dunn R.J. Braun P.E. Neuron. 1994; 13: 805-811Abstract Full Text PDF PubMed Scopus (1020) Google Scholar, 8Mukhopadhyay G. Doherty P. Walsh F.S. Crocker P.R. Filbin M.T. Neuron. 1994; 13: 757-767Abstract Full Text PDF PubMed Scopus (944) Google Scholar). The response to soluble MAG and GT1b antibodies are not obviously different when compared side by side (1Vinson M. Strijbos P.J. Rowles A. Facci L. Moore S.E. Simmons D.L. Walsh F.S. J. Biol. Chem. 2001; 276: 20280-20285Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). MAG can bind directly to gangliosides including GT1b (9Collins B.E. Yang L.J. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), and the presence of complex gangliosides in neurons is required for MAG function (10Mehta N.R. Lopez P.H. Vyas A.A. Schnaar R.L. J. Biol. Chem. 2007; 282: 27875-27886Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This suggests that in some circumstances MAG inhibits neurite outgrowth by binding to and activating a GT1b receptor complex in an antibody-like manner. In support, the inhibitory response to soluble MAG and ganglioside antibodies requires activation of RhoA (Ras homolog gene family A) (1Vinson M. Strijbos P.J. Rowles A. Facci L. Moore S.E. Simmons D.L. Walsh F.S. J. Biol. Chem. 2001; 276: 20280-20285Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 3Fujitani M. Kawai H. Proia R.L. Kashiwagi A. Yasuda H. Yamashita T. J. Neurochem. 2005; 94: 15-21Crossref PubMed Scopus (41) Google Scholar). It follows that insights into how anti-ganglioside antibodies inhibit neurite outgrowth might be gleaned from understanding how MAG inhibits neurite outgrowth and vice versa. MAG can bind to a receptor complex in neurons that contain the Nogo-66 receptor NgR1 (11Fournier A.E. GrandPre T. Strittmatter S.M. Nature. 2001; 409: 341-346Crossref PubMed Scopus (993) Google Scholar, 12Domeniconi M. Cao Z. Spencer T. Sivasankaran R. Wang K. Nikulina E. Kimura N. Cai H. Deng K. Gao Y. He Z. Filbin M. Neuron. 2002; 35: 283-290Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, 13Liu B.P. Fournier A. GrandPre T. Strittmatter S.M. Science. 2002; 297: 1190-1193Crossref PubMed Scopus (528) Google Scholar, 14Wang K.C. Koprivica V. Kim J.A. Sivasankaran R. Guo Y. Neve R.L. He Z. Nature. 2002; 417: 941-944Crossref PubMed Scopus (834) Google Scholar), the p75 neurotrophin receptor (p75NTR) (15Wang K.C. Kim J.A. Sivasankaran R. Segal R. He Z. Nature. 2002; 420: 74-78Crossref PubMed Scopus (725) Google Scholar, 16Wong S.T. Henley J.R. Kanning K.C. Huang K.H. Bothwell M. Poo M.M. Nat. Neurosci. 2002; 5: 1302-1308Crossref PubMed Scopus (401) Google Scholar), and Lingo-1 (17Mi S. Lee X. Shao Z. Thill G. Ji B. Relton J. Levesque M. Allaire N. Perrin S. Sands B. Crowell T. Cate R.L. McCoy J.M. Pepinsky R.B. Nat. Neurosci. 2004; 7: 221-228Crossref PubMed Scopus (717) Google Scholar). The p75NTR receptor is the key signaling component of the complex, and GT1b also appears to be associated with the complex as antibodies to GT1b can immunoprecipitate p75NTR from neurons (3Fujitani M. Kawai H. Proia R.L. Kashiwagi A. Yasuda H. Yamashita T. J. Neurochem. 2005; 94: 15-21Crossref PubMed Scopus (41) Google Scholar, 18Yamashita T. Higuchi H. Tohyama M. J. Cell Biol. 2002; 157: 565-570Crossref PubMed Scopus (354) Google Scholar). These data suggest that GT1b could facilitate the interaction between MAG and the NgR1, and indeed enzymes that remove sialic acid from complex gangliosides inhibit soluble MAG binding to NgR1 and NgR2 in cells (19Venkatesh K. Chivatakarn O. Lee H. Joshi P.S. Kantor D.B. Newman B.A. Mage R. Rader C. Giger R.J. J. Neurosci. 2005; 25: 808-822Crossref PubMed Scopus (194) Google Scholar) and inhibit MAG function (20DeBellard M.E. Tang S. Mukhopadhyay G. Shen Y.J. Filbin M.T. Mol. Cell. Neurosci. 1996; 7: 89-101Crossref PubMed Scopus (166) Google Scholar). Recent studies using independently generated lines of mice that lack the NgR1 have clearly shown that the ability of soluble MAG to induce growth cone collapse from dorsal root ganglion neurons is dependent on this receptor (21Kim J.E. Liu B.P. Park J.H. Strittmatter S.M. Neuron. 2004; 44: 439-451Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 22Chivatakarn O. Kaneko S. He Z. Tessier-Lavigne M. Giger R.J. J. Neurosci. 2007; 27: 7117-7124Crossref PubMed Scopus (94) Google Scholar). However, the later study also provided conclusive evidence that the NgR1 is not required for the function of substrate bound MAG. In the present study we have used analytical ultracentrifugation sedimentation to demonstrate that GT1b can form higher order complexes with the NgR1. The binding required the presence of sialic acid on the ganglioside and was inhibited when any one of three independent FRG motifs in the NgR1 was mutated. One NgR1 sequence that contains an FRG motif (LQKFRGSS) lends itself well to the design of a cyclic peptide mimetic (N-Ac-CLQKFRGSSC-NH2). This mimetic peptide prevented GT1b antibodies from inhibiting neurite outgrowth. These data suggest that the inhibitory activity of anti-GT1b antibodies is dependent on NgR1 function. In support we show that GT1b antibodies do not inhibit neurite outgrowth from neurons isolated from mice that have the NgR1 gene genetically ablated from the germline. The same NgR1 peptide that inhibited the GT1b antibody response also antagonized the response stimulated by soluble MAG, with alanine scanning identifying the FRG sequence as the functional motif within the peptide. These data suggest that in some circumstances soluble MAG can inhibit neurite outgrowth through the ganglioside/NgR1 pathway. In support, mutations of the FRG motif that inhibit GT1b binding to the NgR1 also inhibit MAG binding to the receptor, and the inhibitory activity of soluble MAG was significant attenuated in neurons that do not express the NgR1. However, it is also clear that substrate-bound MAG can inhibit neurite outgrowth in the absence of NgR1 function (22Chivatakarn O. Kaneko S. He Z. Tessier-Lavigne M. Giger R.J. J. Neurosci. 2007; 27: 7117-7124Crossref PubMed Scopus (94) Google Scholar) and, in accord the NgR1 derived inhibitory peptides identified in this study, are not expected to, and indeed do not, inhibit the function of substrate-bound MAG. Neurite Outgrowth Assays—Cerebellar granule neurons isolated from post-natal day 2–3 rats or from day 3–5 mice were cultured over monolayers of 3T3 cells essentially as previously described (23Williams E. Williams G. Gour B.J. Blaschuk O.W. Doherty P. J. Biol. Chem. 2000; 275: 4007-4012Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Monolayers were established by seeding ∼80,000 3T3 cells into individual chambers of an 8-chamber tissue culture slide coated with poly-l-lysine and fibronectin. In general, co-cultures were established by removing the media from the monolayers and seeding ∼6000 dissociated cerebellar neurons into each well in SATO medium (Dulbecco's modified Eagle's medium supplemented with 0.062 mg/liter progester-one, 16.1 mg/liter putrescine, 0.4 mg/liter thyroxine, 0.039 mg/liter selenium, 0.337 mg/liter triiodothyronine, 10 mg/liter insulin (bovine pancreas), 100 mg/liter transferrin (human)) supplemented with 2% fetal calf serum. However, in some experiments the co-cultures were maintained in neurobasal medium + B27 + 1% l-glutamate + 1% penicillin/streptomycin + 25 mm KCl. Monolayers were established for 24 h before the addition of the neurons, and the cultures were maintained for ∼23–27 h before fixation with 4% paraformaldehyde. In general, the neurons were stained with a GAP-43 antibody, and the mean length of the longest neurite per cell was measured for ∼120–150 neurons, again as previously described (23Williams E. Williams G. Gour B.J. Blaschuk O.W. Doherty P. J. Biol. Chem. 2000; 275: 4007-4012Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, in some experiments neurons were labeled using TUJ1 (anti-βIII tubulin) followed by anti-mouse IgG-Alexa488, and nuclei were labeled with Hoechst. Mean total neurite length was calculated using the Neuronal Profiling bioapplication on a Cellomics ArrayScan. Similar results were obtained using both methods. For neurite outgrowth on substrate-bound MAG, 96-well plates were coated with a thin layer of nitrocellulose before incubating with 1 μg/ml MAG(d1–5) at 4 °C overnight. Wells were subsequently coated with 17 μg/ml of poly-d-lysine (Sigma) followed by incubation in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Rat cerebellar granule neurons were dissociated and seeded at a density of 1 × 104 cells per well. Cells were cultured for 18–20 h before being fixed with 4% paraformaldehyde and stained with a neuronal specific anti-βIII-tubulin antibody. The average of total neurite lengths from each neuron was measured automatically by the Meta-Xpress Neurite Outgrowth module (Molecular Devices) from at least 200 neurons per well, in triplicate wells per experiment. Results were repeated independently more than three times. Immunoprecipitations and Western Blots—Chinese hamster ovary (CHO) K1 cells (100 mm dishes) were transfected with p75NTR and various mutants of the NgR1. The cells were harvested after 24 h and lysed in 1 ml of radioimmune precipitation assay buffer (Sigma) supplemented with Complete protease inhibitor mixture (Roche Applied Science). After centrifugation at 14,000 × g for 15 min, the supernatants were collected, and a protein assay (Bio-Rad) was performed. Protein lysates (0.5 mg) were preincubated with protein G-Sepharose beads (GE Healthcare) at 4 °C for 1 h, then incubated with 2 μg of goat anti human NgR antibody (R&D Systems) plus protein G-Sepharose at 4 °C overnight. The beads were washed three times with radioimmune precipitation assay buffer and boiled in Laemmli sample buffer (Bio-Rad). Supernatants were subjected to 4–12% NuPAGE (Invitrogen), transferred onto nitrocellulose membrane (Bio-Rad), and probed with antibodies to the NgR and p75NTR (Promega). Western blot images were analyzed on a Storm gel imaging system using ImageQuant software (GE Healthcare). Whole brains from adult 129/lex, NgR1 knock-out, and NgR2 knock-out mice were homogenized and sonicated in radioimmune precipitation assay buffer (Sigma). Supernatants were collected after centrifugation, and a protein assay (Bio-Rad) was preformed. Protein lysates were subjected to 4–12% NuPAGE (Invitrogen), transferred onto nitrocellulose membrane (Bio-Rad), and probed with antibodies to NgR1 or NgR2 (R&D Systems). Western blot images (see supplemental Fig. 1b) were scanned and analyzed by Odyssey infrared imaging system (Li-Cor). Construction of NgR1 Mutants—Human NgR1 point mutants were constructed using the QuikChange XL site-directed mutagenesis kit (Stratagene) following the manufacturer's recommended protocol. The wild type human NgR1 cDNA (IMAGE:2121045 3) in a mammalian expression vector was used as a template to construct all the described mutants. Receptor Binding Assay—COS-7 cells were co-transfected with either wild type or mutant NgR1 constructs along with a cytomegalovirus-β-gangliosidase plasmid (pCMVb, BD Biosciences) as a transfection control. Transfection was performed in six-well plates using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. The next day cells were trypsinized and seeded at 30,000 cells per well in poly-lysine-coated 96-well plates (BD Biosciences). At that time, two sister plates were established, one of which was used in the binding assay, and the other was used to correct for transfection efficiency by measuring β-galactosidase activity (see below). Remaining cells were separately plated and assayed for surface expression of the NgR1 proteins by immunocytochemistry and for total NgR1 protein levels by Western blotting. All mutant proteins were expressed on the cell surface and produced in comparable amounts to the wild type protein (data not shown). The next day wells were rinsed once with Hanks' balanced salt solution containing 0.5 mg/ml bovine serum albumin, 0.1% NaN3, 20 mm HEPES, pH 7.0, at room temperature followed by incubation with 100 μl of fusion protein MAG-alkaline phosphatase (AP) diluted to a final concentration of 10 μg/ml in 20 mm HEPES, pH 7.0, for 90 min. After this, wells were washed 6 times with gentle shaking in 20 mm Hepes, pH 7.0, at room temperature, 5 min each wash. Cells were then fixed with acetone-formaldehyde (60-3%, in 20 mm HEPES, pH 7.0) for 15 s at room temperature then washed 3 times for 5 min each with Hanks' balanced salt solution. Binding of AP-tagged ligands was then measured using the Great EscAPe SEAP kit (BD Biosciences) following the manufacturer's recommended protocol. Briefly, after aspirating Hanks' balanced salt solution, a 60-μl dilution buffer was added to each well, and the plates were sealed and then incubated at 65 °C for 90 min. Plates were cooled on ice, and then 60 μl of assay buffer was added per well and incubated at room temperature for 5 min. Sixty microliters of diluted chemiluminescent alkaline phosphatase substrate was then added per well, incubated 10 min at room temperature, and then read on a LMAXII luminometer (Molecular Devices). Absolute binding numbers were corrected by subtracting average binding values obtained from mock-transfected controls. Binding was further corrected for sample-to-sample variations in transfection efficiency by normalizing to β-galactosidase activity. β-Galactosidase activity was measured using the luminescent β-galactosidase detection kit II (BD Biosciences) following the manufacturer's recommended protocol. Three independent binding experiments were conducted with at least six replicates per experiment. Background was subtracted, and β-galactosidase-corrected binding values were expressed relative to the wild type receptor. Preparation of AP-tagged Fusion Proteins—A fusion protein (Nogo66-AP) containing an N-terminal human placental AP and a C-terminal Nogo66 domain was constructed by ligating nucleotides encoding amino acids 1055–1120 of human NogoA (reticulon-4, NP_065393) to sequences encoding amino acids 23–511 of AP (NM_001632). This fusion was further modified by changing amino acid 47 of the Nogo66 sequence from cysteine to valine and introducing six consecutive histidine residues at the C terminus. The coding sequence was inserted into a mammalian expression vector and transiently transfected into HEK293GT cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). The next day serum-free medium (Free Style 293, Invitrogen) was added, and cells were incubated a further 48 h before collection of crude conditioned medium. Nogo66-AP concentration was determined by measuring alkaline phosphatase activity and by Western blotting for alkaline phosphatase. A stable CHO cell line expressing a fusion protein containing an N-terminal human myelin-associated glycoprotein (human MAG; NM_002361; amino acids 1–516) and a C-terminal AP domain (amino acids 23–511) bearing six C-terminal histidine residues was created (referred to as MAG-AP). Cells were incubated in serum-free medium for 48 h; the conditioned medium was collected, and the fusion protein was purified using TALON cobalt affinity chromatography (Clontech) following the manufacturer's protocol. MAG-AP concentration was determined by measuring alkaline phosphatase activity and by Western blotting for alkaline phosphatase and MAG. Analytical Ultracentrifugation—Sedimentation velocity experiments were performed on a Beckman XLI/XLA analytical ultracentrifuge. NgR1(310)-fc (0.21 or 0.38 μm final) was added to ganglioside at increasing ganglioside concentrations from 0 to 48 μm. Mutant protein used in the sedimentation velocity experiments corresponded to the column fraction of greatest purity based on SDS gel analysis. Wild type or mutant NgR(310)-Fc was added to Tris-buffered saline (TBS) buffer or TBS buffer containing GT1b to a final concentration of 16–30 μg/ml protein and 0 or 22 μm GT1b in a microcentrifuge tube. The solution, 400 μl, was loaded into 2-channel (1.2-cm path length) carbon-Epon centerpieces in an An-50-Ti rotor. Scans were recorded at 20 °C with a rotor speed of 35,000 rpm, and the signal was detected at 230 nm with a spacing of 0.006 in the continuous mode. Sedimentation profiles were analyzed by the program Sedfit (24Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3152) Google Scholar) to obtain the sedimentation coefficient distributions. The solvent density (1.006) and partial specific volume (0.72) were calculated using the program Sednterp (25Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar). Neuraminidase Treatment—CHO parental cells and NgR1 stable cells were seeded at 30,000 cells per well in 96-well plates the night before the assay. Various concentrations of Vibrio cholerae neuraminidase (Roche Applied Science) in growth medium (Dulbecco's modified eagle medium containing 10% fetal bovine serum) were incubated with cells for 1 h at 37 °C. Medium was replaced with affinity-purified MAG-AP or Nogo66-AP in Hanks' balanced salt solution supplemented with 1% fetal bovine serum and 20 nm HEPES and incubated at room temperature for 90 min. Cells were then washed four times with supplemented Hanks' balanced salt solution. Alto-Phos (0.6 mg/ml) (Promega) was added to indicate of bound ligands. After a 30-min incubation at room temperature, the plates were read at emission/excitation wavelength of 400 nm/505 nm by FlexStationII384 (Molecular Devices). Reagents—Synthetic peptides were all obtained from a commercial supplier (Multiple Peptide Systems). All peptides were purified to the highest grade by reverse-phase high performance liquid chromatography and obtained at the highest level of purity (>97%). With all peptides there was no indication of higher molecular weight species. Where peptide sequences are underlined, this denotes a peptide that has been cyclized via a disulfide bond between the given cysteine residues. All peptides were acetylated and amide-blocked. Recombinant MAG-Fc chimera was obtained from R&D Systems and used at a final concentration of 5–25 μg/ml. The monoclonal antibody to GT1b (clone GMR5) was obtained from Seikagaku America and was used at a final concentration of 20 μg/ml. All reagents were diluted into the co-culture media and in general added to the cultures just before plating of the neurons. GT1b and GM1 were a kind gift of Dr. Gino Toffano. Asialo-GM1 was obtained from Sigma. The recombinant NgR1(310)-Fc chimera and the extracellular portion of MAG(d1–5) were expressed and purified in-house. Pharmacological reagents were obtained from Calbiochem and/or Sigma. Generation and Characterization of NgR1 and NgR2 Knockout Mice—Targeting vectors (see supplemental Fig. 1a) were introduced into mouse embryonic stem cells of the 129SvBrd background via electroporation. Homologously targeted integrants were selected in G418 (0.4 g/liter) and identified by Southern blotting using probes external to the targeting vector on both the 5′ and 3′ sides. Retention of the 5′-most loxP site in the targeted cells was determined by PCR of genomic DNA using the following primers: for NgR1 allele, 5′-GGTCTAGGGATGCATCTCAG and 5′-ACATCTGAAGGCCTTCTGG; for the NgR2 allele, 5′-GTTGGTGGGGTTCTTGTCTCAGG and 5′-GGCCTGGCCCCCTCCCTTCAC. Embryonic stem cells were used to establish mouse lines and animals bearing the NgR1f and NgR2f alleles were crossed to Prm-cre transgenic mice (26O'Gorman S. Dagenais N.A. Qian M. Marchuk Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14602-14607Crossref PubMed Scopus (388) Google Scholar). The Prm-cre transgene directs expression of cre recombinase in the male germline. Therefore, males harboring the transgene along with the NgR1f or NgR2f allele transmit the non-functional NgR1d or NgR2d alleles, respectively, to their offspring. Cre-mediated recombination of the floxed (f) alleles to the deleted (d) alleles was detected by PCR of genomic DNA using the following primers: for NgR1, 5′-GGTCTAGGGATGCATCTCAG and 5′-GTGGTCTGTGTGGCTCCTGC; for NgR2, 5′-GTTGGTGGGGTTCTTGTCTCAGG and 5′-CCCCCCTGCCCCAGCTACGC. All alleles were maintained on the 129SvBrd background. Binding Motifs on the NgR1—There are two published crystal structures of the NgR1, Protein Data Bank accessions 1OZN (27He X.L. Bazan J.F. McDermott G. Park J.B. Wang K. Tessier-Lavigne M. He Z. Garcia K.C. Neuron. 2003; 38: 177-185Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and 1P8T (28Barton W.A. Liu B.P. Tzvetkova D. Jeffrey P.D. Fournier A.E. Sah D. Cate R. Strittmatter S.M. Nikolov D.B. EMBO J. 2003; 22: 3291-3302Crossref PubMed Scopus (196) Google Scholar), but currently no ligand-receptor complex structure has been solved. Detailed mutagenesis studies have recently mapped the residues critical for the binding of MAG to the receptor (29Lauren J. Hu F. Chin J. Liao J. Airaksinen M.S. Strittmatter S.M. J. Biol. Chem. 2007; 282: 5715-5725Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and these are illustrated in Fig. 1a. Small ligand binding sites show up as cavities and can be revealed by the clustering of a small probe under the influence of a van der Waals potential. In Fig. 1b we show the two lowest energy clusters for a probe with van der Waals radius of 3.5 Å. The potential binding pockets lie on the convex side of the protein, and interestingly, both neighbor FRG triplet motifs that can be found in the other NgRs (discussed in detail below). Sialic acid residues on gangliosides and possibly other glycoconjugates bind directly to an FRG motif in MAG itself (30Tang S. Woodhall R.W. Shen Y.J. deBellard M.E. Saffell J.L. Doherty P. Walsh F.S. Filbin M.T. Mol. Cell. Neurosci. 1997; 9: 333-346Crossref PubMed Scopus (103) Google Scholar). These observations have led us to develop the hypothesis that gangliosides can interact with FRG motifs in the NgR and that this interaction might facilitate MAG binding to the receptor. Binding of MAG, but Not Nogo66, to NgR1 Is Partially Sensitive to Neuraminidase—In neurons soluble MAG binds to the NgR1 and NgR2 in a sialic acid-dependent manner (19Venkatesh K. Chivatakarn O. Lee H. Joshi P.S. Kantor D.B. Newman B.A. Mage R. Rader C. Giger R.J. J. Neurosci. 2005; 25: 808-822Crossref PubMed Scopus (194) Google Scholar). In the present study we confirmed the neuraminidase sensitivity of MAG binding to the NgR1 expressed in CHO cells. The data show that over a wide range of concentrations (2.5–20 μg/ml) the specific binding of the MAG-AP fusion protein to NgR1-expressing cells is partially inhibited (55%) by treating the CHO cells with neuraminidase. The effect was dependent upon the concentration of neuraminidase, and even at the highest concentration Nogo66-AP binding remained completely unaffected (Fig. 2). These data suggest that MAG binding to the NgR1 is dependent at least in part on sialic acid binding to the receptor. Effects of Loop 2 and Additional FRG Mutations on GT1b Binding to the NgR1—GT1b is a sialic acid-containing ganglioside that has previously been reported to be a key component of the MAG receptor complex (18Yamashita T. Higuchi H. Tohyama M. J. Cell Biol. 2002; 157: 565-570Crossref PubMed Scopus (354) Google Scholar, 31Vyas A.A. Patel H.V. Fromholt S.E. Heffer-Lauc M. Vyas K.A. Dang J. Schachner M. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8412-8417Crossref PubMed Scopus (247) Google Scholar). We have tested whether GT1b can bind directly to the ectodomain of the NgR1 using analytical ultracentrifugation. In the absence of GT1b, the dimeric NgR1(310)-Fc migrates with a sedimentation coefficient of ∼6.5 S (Fig. 3). In the presence of low μm concentrations of GT1b, the 6.5 S species decreases, and additional peaks with higher sedimentation coefficients appear in a dose-dependent manner (Fig. 3a). In this assay, GM1 can also interact with NgR1 (Fig. 3b), which suggests that the interaction might be dependent on sialic acid. In support, no change in sedimentation coefficient of the NgR1(310)-Fc is observed in the presence of asialo-GM1. No effect was observed upon the addition of 22 μm GT1b to anti-hNgR AF 1208 antibody from R&D, which provides additional evidence that the interaction of GT1b with NgR1 is specific (not shown). We next determined if the binding of GT1b to the NgR1 was sensitive to mutation of the FRG motifs. Importantly, based on the relative ratios of the ∼6.7- and ∼11-S peaks, it can be estimated that mutation of the arginine 279 to an aspartic acid reduced binding to ∼56% that of wild type NgR, suggesting this site plays a role in mediating the interaction (Fig. 3d). Mutation of arginine 151 (Fig. 3e) or arginine 199 (Fig. 3f) also reduced GT1b binding to 49 and 33% that of wild type, respectively. These data su