Myelin-associated Glycoprotein Interacts with Ganglioside GT1b
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m100345200
ISSN1083-351X
AutoresMary Vinson, Paul J. L. M. Strijbos, Alison Rowles, Laura Facci, Stephen E. Moore, David L. Simmons, Frank S. Walsh,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoMyelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG. Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG. myelin-associated glycoprotein sialic acid-binding Ig-related lectin α-methyl sialic acid Neu5Acα2–3Galβ1–4Glc tetramethylrhodamine isothiocyanate enzyme-linked immunosorbent assay fetal calf serum phosphate-buffered saline monoclonal antibody; ganglioside nomenclature is that of Suennerholm (69Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1313) Google Scholar). Expression of myelin-associated glycoprotein (MAG; siglec 4a),1 is restricted to myelinating glial cells on myelin membrane adjacent to the axon and is required for maintenance of myelin integrity (1Li C.M. Tropak M.B. Gerlai R. Clapoff S. Abramownewerly W. Trapp B. Peterson A. Roder J. Nature. 1994; 369: 747-750Crossref PubMed Scopus (325) Google Scholar, 2Montag D. Giese K.P. Bartsch U. Martini R. Lang Y. Bluthmann H. Karthigasan J. Kirschner D.A. 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Cell. Neurosci. 1997; 9: 333-346Crossref PubMed Scopus (103) Google Scholar), involving activation of Rho GTPase, a key signaling step for the inhibitory effect of myelin on regeneration of neurons in vivo (9Lehmann M. Fournier A. Selles-Navarro I. Dergham P. Sebok A. Leclerc N. Tigyi G. McKerracher L. J. Neurosci. 1999; 19: 7537-7547Crossref PubMed Google Scholar). MAG is therefore thought to contribute to the inhibitory properties of myelin, which is in part responsible for the lack of regenerative capacity of the central nervous system after injury or disease (10Johnson A.R. Bioessays. 1993; 15: 807-813Crossref PubMed Scopus (48) Google Scholar, 11Huang D.W. McKerracher L. Braun P.E. David S. Neuron. 1999; 24: 639-647Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Like other siglecs, MAG binds to sialic acid residues at the termini of glycans on opposing cells through a sialic acid binding site located in the N-terminal V-set Ig domain (12Kelm S. Pelz A. Schauer R. Filbin M.T. Tang S. Debellard M.E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 13Freeman S.D. Kelm S. Barber E.K. Crocker P.R. Blood. 1995; 85: 2005-2012Crossref PubMed Google Scholar, 14Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar, 15Patel N. Brinkman-Van der Linden E.C.M. Altmann S.W. Gish K. Balasubramanian S. Timans J.C. Peterson D. Bell M.P. Bazan J.F. Varki A. Kastelein R.A. J. Biol. Chem. 1999; 274: 22729-22738Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 16Nicoll G. Ni J. Liu D. Klenerman P. Munday J. Dubock S. Mattei M.G. Crocker P.R. J. Biol. Chem. 1999; 274: 34089-34095Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 17Angata T. Varki A. Glycobiology. 2000; 10: 431-438Crossref PubMed Scopus (100) Google Scholar, 18Floyd H. Ni J. Cornish A.L. Zeng Z.Z. Liu D. Carter K.C. Steel J. Crocker P.R. J. Biol. Chem. 2000; 275: 861-866Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 19Vinson M. Vandermerwe P.A. Kelm S. May A. Jones E.Y. Crocker P.R. J. Biol. Chem. 1996; 271: 9267-9272Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 20Vandermerwe P.A. Crocker P.R. Vinson M. Barclay A.N. Schauer R. Kelm S. J. Biol. Chem. 1996; 271: 9273-9280Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 21May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 22Crocker P.R. Vinson M. Kelm S. Drickamer K. Biochem. J. 1999; 341: 355-361Crossref PubMed Scopus (54) Google Scholar). MAG binds specifically to terminal sialic acid residues in α2–3 linkage to galactose, which occurs in glycans linked β1–3 to GalNAc or GlcNAc or β1–4 to GlcNAc (12Kelm S. Pelz A. Schauer R. Filbin M.T. Tang S. Debellard M.E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 23Yang L.J.S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (270) Google Scholar, 24Kelm S. Brossmer R. Isecke R. Gross H.J. Strenge K. Schauer R. Eur. J. Biochem. 1998; 255: 663-672Crossref PubMed Scopus (144) Google Scholar, 25Brinkman-Van der Linden E.C.M. Varki A. J. Biol. Chem. 2000; 275: 8625-8632Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Use of sialic acid analogues has identified specific groups on sialic acid essential for interaction with MAG (26Strenge K. Schauer R. Bovin N. Hasegawa A. Ishida H. Kiso M. Kelm S. Eur. J. Biochem. 1998; 258: 677-685Crossref PubMed Scopus (57) Google Scholar), consistent with interactions seen between sialic acid and conserved amino acids in the siglec 1 crystal structure (21May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). It is also thought that the core glycan structure on which the terminal sialic acid is presented plays a role in recognition by MAG (23Yang L.J.S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (270) Google Scholar, 26Strenge K. Schauer R. Bovin N. Hasegawa A. Ishida H. Kiso M. Kelm S. Eur. J. Biochem. 1998; 258: 677-685Crossref PubMed Scopus (57) Google Scholar, 27Collins B.E. Ito H. Sawada N. Ishida H. Kiso M. Schnaar R.L. J. Biol. Chem. 1999; 274: 37637-37643Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The ability of MAG to bind specific gangliosides bearing terminal α2–3-linked sialic acid has been well documented. Gangliosides bind to MAG with the relative potencies GQ1bα > GT1aα, GD1α > GD1a, GT1b ≫ GM3, GM4, whereas GM1, GD1b, GD3, and GQ1b do not support adhesion (23Yang L.J.S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (270) Google Scholar, 27Collins B.E. Ito H. Sawada N. Ishida H. Kiso M. Schnaar R.L. J. Biol. Chem. 1999; 274: 37637-37643Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 28Collins B.E. Yang L.J.S. 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 (156) Google Scholar). Although the binding of MAG to sialylated glycans and gangliosides is well characterized, the functional importance of these interactions to the inhibition of neurite outgrowth by MAG is unclear. MAG binding to neurons is dependent on the presence of cell surface sialic acid (7Debellard M.E. Tang S. Mukhopadhyay G. Shen Y.J. Filbin M.T. Mol. Cell. Neurosci. 1996; 7: 89-101Crossref PubMed Scopus (166) Google Scholar). Furthermore, addition of exogenous sugars or neuraminidase treatment of neurons reduces the effect of MAG on neurite outgrowth (7Debellard M.E. Tang S. Mukhopadhyay G. Shen Y.J. Filbin M.T. Mol. Cell. Neurosci. 1996; 7: 89-101Crossref PubMed Scopus (166) Google Scholar), suggesting that the interaction between MAG and sialylated cell surface receptors results in inhibition of neurite outgrowth. However, mutation of arginine 118, an amino acid which is predicted to form hydrogen bonds with the carboxylate group of sialic acid (21May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), failed to inactivate the protein (29Tang S. Shen Y.J. Debellard M.E. Mukhopadhyay G. Salzer J.L. Crocker P.R. Filbin M.T. J. Cell Biol. 1997; 138: 1355-1366Crossref PubMed Scopus (130) Google Scholar). This led to the suggestion that a second, sialic acid-independent, site on MAG interacts with an unknown counter-receptor on neurons, triggering the intracellular signaling cascade leading to inhibition of neurite outgrowth (29Tang S. Shen Y.J. Debellard M.E. Mukhopadhyay G. Salzer J.L. Crocker P.R. Filbin M.T. J. Cell Biol. 1997; 138: 1355-1366Crossref PubMed Scopus (130) Google Scholar). In this report, we investigate the roles of sialic acid and ganglioside recognition by MAG in the inhibition of neurite outgrowth. We show that, in the absence of arginine 118, the potency of MAG is significantly reduced, but the residual inhibitory activity also involves carbohydrate recognition. We then show that interaction of MAG with ganglioside GT1b is a potential mechanism for the inhibitory effect of MAG on neurite outgrowth. These results strongly suggest that GT1b represents a potential receptor for MAG, mediating inhibition of neurite outgrowth. Unless otherwise specified, all reagents were purchased from Sigma Chemical Co. (UK). Tissue culture media and B27 supplement were from Life Technologies (Paisley, UK). BCA protein quantification kit was purchased from Pierce (Chester, UK). Anti-MAG antibody (MAB1567) and isotype control antibody were purchased from Chemicon (Harrow, UK). SDS-polyacrylamide gel electrophoresis gels were purchased from Bio-Rad. ECL reagents were from Amersham Pharmacia Biotech (UK). Neu5Ac, 3′-sialyllactose, and purified gangliosides were purchased from Sigma. Anti-GT1b (clone GMR5, an IgM) and biotinylated anti-GT1b and anti-GD1a (clone GMR17, an IgM) were purchased from Seikagaku America (Falmouth, MA). Anti-GM1 (an IgG) was from Cambio (Cambridge, UK) and A2B5 (an IgM that recognizes uncharacterized ganglioside(s) (30Eisenbarth G.S. Walsh F.S. Nirenberg M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4913-4917Crossref PubMed Scopus (819) Google Scholar, 31Fredman P. Magnani J.L. Nirenberg M. Ginsburg V. Arch. Biochem. Biophys. 1984; 233: 661-666Crossref PubMed Scopus (104) Google Scholar)) was from Roche Molecular Biochemicals(Mannheim, Germany). TRITC-conjugated anti-mouse immunoglobulin was from Dako (Cambridge, UK) and streptavidin-Texas red was from Amersham Pharmacia Biotech. Y27632 was from Tocris (Bristol, UK). The constructs MAG-Fc/pIg and MAGR118A-Fc/pIg have been described elsewhere (29Tang S. Shen Y.J. Debellard M.E. Mukhopadhyay G. Salzer J.L. Crocker P.R. Filbin M.T. J. Cell Biol. 1997; 138: 1355-1366Crossref PubMed Scopus (130) Google Scholar) and were kindly provided by Prof. M. T. Filbin. The rat SIRP-Fc/pIg construct consisted of the three extracellular N-terminal Ig-like domains fused to human IgG1 and was provided by Dr. L. Vernon-Wilson. Recombinant protein was produced by transient transfection of COS-7 cells as described previously (12Kelm S. Pelz A. Schauer R. Filbin M.T. Tang S. Debellard M.E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). ELISA was carried out using standard methods (19Vinson M. Vandermerwe P.A. Kelm S. May A. Jones E.Y. Crocker P.R. J. Biol. Chem. 1996; 271: 9267-9272Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar,29Tang S. Shen Y.J. Debellard M.E. Mukhopadhyay G. Salzer J.L. Crocker P.R. Filbin M.T. J. Cell Biol. 1997; 138: 1355-1366Crossref PubMed Scopus (130) Google Scholar). Briefly, a 96-well plate was coated with 10 μg/ml goat anti-human IgG overnight at 4 °C. After washing, Fc proteins were applied at varying concentrations and incubated at 37 °C for 2 h. Wells were washed and incubated with anti-MAG antibody at 10 μg/ml for 1 h. This was followed by visualization using anti-mouse horseradish peroxidase and O-phenylenediamine substrate. The hippocampi of gestational day 18 rat embryos were dissected out, incubated in trypsin (0.08%, 30 min at 37 °C), and dissociated mechanically (32Skaper S.D. Facci L. Milani D. Leon A. Toffano G. Conn P.M. Methods in Neuroscience. 2. Academic Press, San Diego1990: 17-33Google Scholar). Hippocampal cells were resuspended in neurobasal medium supplemented with B27, anti-oxidants, 1 mm glutamine, 25 μmglutamate, and 1 mm pyruvate, and plated at a density of 3000 cells/well into 96-well dishes that had previously been coated with poly-d-lysine followed by 10% FCS. Cerebellar granule neurons were prepared from postnatal day 8 Harlan Sprague-Dawley rat pups. Cerebella were enzyme-digested, triturated, and plated into poly-l-lysine-coated 96-well plates at a density of 20,000 cells/well in Eagle's basal medium supplemented to contain 25 mm KCl, 10% FCS, and 50 μg/ml gentamicin (32Skaper S.D. Facci L. Milani D. Leon A. Toffano G. Conn P.M. Methods in Neuroscience. 2. Academic Press, San Diego1990: 17-33Google Scholar). One hour after plating primary neuronal cells, Fc proteins or anti-ganglioside antibodies at various concentrations were added in equal volumes of PBS to triplicate wells. All antibodies used in these assays were previously dialyzed against cell culture medium. For preincubation experiments, 10 μg/ml anti-MAG or control antibody or various concentrations of sugars were incubated with MAGR118A-Fc for 1 h at room temperature prior to addition to cells. For ganglioside preincubation experiments, gangliosides were reconstituted at 25 mg/ml in chloroform:methanol (1:1) and stored at −20 °C. Fresh stock solutions of 250 μg/ml ganglioside in 1% fatty acid-free bovine serum albumin in PBS in glass tubes were prepared by vigorous vortexing immediately before each experiment. Gangliosides were diluted to 2 μg/ml into Fc protein solution in sterile glass tubes and incubated for 1 h at room temperature before being added to cultures as above. After 24 h (cerebellar granule neurons) or 48 h (hippocampal cells), cells were fixed with 4% paraformaldehyde for 1 h on ice, washed with PBS, and stained using Coomassie Blue (11Huang D.W. McKerracher L. Braun P.E. David S. Neuron. 1999; 24: 639-647Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Briefly, 50 μl of 0.1% Coomassie Blue R-250 (in 40% methanol, 10% acetic acid) were added per well and incubated for 30 s. Stain was tipped off and the wells were washed three times with PBS. Assays were quantified using a KS300 image analysis system (Imaging Associates, UK). For each cell measured, the length from the edge of the cell to the end of the longest neurite was measured for 100 cells/well for each treatment in triplicate. Results are expressed as a percentage of the length of neurites of cells treated with control-treated with PBS alone. The length of neurites from control-treated neurons varied between experiments (between 35 and 50 μm for hippocampal neurons after 48 h of culture and 20–40 μm for cerebellar neurons cultured for 24 h). Therefore control-treated cells were included on every 96-well plate and results for each treatment in an experiment were expressed as a percentage of the length of control-treated cells. This allowed data from three independent experiments to be pooled. Data points therefore represent mean and S.E. of data pooled from three independent experiments. Primary neuronal cells were plated onto 8-well chamber slides coated as described above. After 24 h (cerebellar granule neurons) or 48 h (hippocampal neurons), cells were fixed as above and stained by standard immunocytochemistry techniques. 10 μg/ml primary antibody (or isotype control) or biotinylated cholera toxin in PBS/10% FCS was incubated overnight at 4 °C. Secondary reagents were then incubated at room temperature for 1 h as follows: for biotinylated anti-GT1b and cholera toxin, 1:200 dilution of streptavidin-Texas Red; for anti-GD1a and A2B5, 1:30 dilution of anti-mouse-TRITC. To assess their structural integrity, MAG-Fc and MAGR118A-Fc recombinant proteins were tested in an ELISA assay using the anti-MAG monoclonal antibody, which recognizes a conformation-dependent epitope. MAG-Fc and MAGR118A-Fc reacted identically in a dose-dependent manner (Fig.1 a) indicating that both proteins were correctly folded. To investigate the role of arginine 118 in inhibition of neurite growth by MAG, neurite outgrowth experiments were carried out in primary cultured hippocampal neurons. Consistent with previous reports, MAG-Fc potently inhibited neuronal outgrowth (Fig. 1 b). SIRP-Fc (consisting of the three extracellular N-terminal Ig-like domains of rat SIRP (33Fujikowa Y. Matozaki T. Noguchi T. Iwamatsu A. Yamao T. Takahashi N. Tsuda M. Takada T. Kasguga M. Mol. Cell. Biol. 1996; 16: 6887-6899Crossref PubMed Scopus (392) Google Scholar) fused to the Fc region of human IgG1) had no effect on neurite outgrowth from hippocampal neurons (Fig. 1 b), consistent with previous observations using cerebellar granule neurons (34Prinjha R. Moore S.E. Vinson M. Blake S. Morrow R. Christie G. Michalovich D. Simmons D.L. Walsh F.S. Nature. 2000; 403: 383-384Crossref PubMed Scopus (550) Google Scholar), therefore showing that inhibition of outgrowth by MAG-Fc was specific for MAG and not a result of the presence of the Fc region. MAGR118A-Fc also inhibited neurite outgrowth but with lower potency compared with the unmutated form (Fig. 1 b). This inhibition was specific when compared with negative control Fc protein SIRP-Fc, and was reversed with the anti-MAG antibody mAb 513 but not mouse IgG1 isotype control. A similar effect was seen for neurite outgrowth from cerebellar granule neurons (data not shown). To investigate the basis for the residual activity of MAGR118A-Fc, we carried out neurite outgrowth assays using MAGR118A-Fc in the presence of mono- and trisaccharides. The sugars tested did not affect neurite length when added to concentrations up to 5 mm in the absence of MAGR118A-Fc (data not shown). Inhibition of neurite outgrowth by MAGR118A-Fc was blocked by preincubation with α-methyl sialic acid (Neu5Ac) and the sialylated trisaccharide 3′-sialyllactose (Neu5Acα2–3Galβ1–4Glc) (Fig.2); however, incubation with 0.1 mm 6′-sialyllactose did not significantly reverse inhibition (data not shown). This suggests that MAGR118A-Fc inhibits neurite outgrowth via recognition of sialylated glycans on the surface of neurons. Specific gangliosides are known to bind MAG via their carbohydrate epitopes with relative potencies GQ1bα > GT1aα, GD1α > GT1b, GD1a ≫ GM1. The recent availability of anti-ganglioside antibodies (35Kotani M. Ozawa H. Kawashima I. Ando S. Tai T. Biochim. Biophys. Acta. 1992; 1117: 97-103Crossref PubMed Scopus (145) Google Scholar, 36Ozawa H. Kotani M. Kawashima I. Tai T. Biochim. Biophys. Acta. 1992; 1123: 184-190Crossref PubMed Scopus (78) Google Scholar) has provided the opportunity to investigate the functional significance of these interactions. Although MAG binds to the α-series gangliosides with higher affinity, their expression in brain is very low (0.5, 0.9, and 0.3 mg/kg for GQ1bα, GT1aα, and GD1α, respectively) compared with that of GD1a and GT1b (abundance of GD1a is 1200 mg/kg), which are among two of the four major brain gangliosides (27Collins B.E. Ito H. Sawada N. Ishida H. Kiso M. Schnaar R.L. J. Biol. Chem. 1999; 274: 37637-37643Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). If gangliosides are involved in neurite outgrowth inhibition in response to MAG, GD1a and GT1b would be predicted to be involved, because MAG affects all neuronal types tested. To examine whether GD1a and GT1b could compete with endogenous neuronal MAG receptors for MAG binding, we preincubated Fc protein with gangliosides prior to addition into neurite outgrowth assays (37Probstmeier R. Pesheva P. Glycobiology. 1999; 9: 101-114Crossref PubMed Scopus (53) Google Scholar). Preincubation of MAG-Fc with a mixture of brain gangliosides, purified GT1b or GD1a but not asialo GM1 or GM1, blocked the inhibitory action of MAG (Fig. 3 a). The inhibitory activity of MAGR118A-Fc was also partially reversed by GT1b or GD1a preincubation (Fig. 3 b). Immunocytochemistry using anti-ganglioside antibodies confirmed the expression of GT1b and GD1a on the surface of surface of primary cerebellar and hippocampal neurons (Fig.4). Both these antibodies showed most intense staining associated with cell bodies. Weaker staining was observed on some neurites. Ganglioside GM1 and the A2B5 antigen (an uncharacterized epitope carried by several gangliosides) were also found to be expressed on the surface of these cells (Fig. 4). A lack of fluorescence for cells stained with secondary antibody alone confirmed specificity (data not shown). The effect of anti-ganglioside antibodies on neurite outgrowth was then assessed. If MAG inhibits neurite outgrowth by binding to either GD1a or GT1b on the surface of neurons, antibodies recognizing these gangliosides may be expected to mimic the effect of MAG on neurite outgrowth. Anti-GT1b antibody, but not isotype control or antibodies recognizing GD1a, GM1, or the A2B5 antigen inhibited neurite outgrowth in a dose-dependent manner (Fig.5, a and b). The specificity of this effect was demonstrated by preincubation of anti-GT1b antibody with purified gangliosides. Preincubation with GT1b but not with other gangliosides blocked the inhibition of neurite outgrowth by the anti-GT1b antibody (Fig. 5 c). Rho GTPase has been shown to be a key signaling step for the inhibitory effect of myelin on regeneration of neurons in vivo (9Lehmann M. Fournier A. Selles-Navarro I. Dergham P. Sebok A. Leclerc N. Tigyi G. McKerracher L. J. Neurosci. 1999; 19: 7537-7547Crossref PubMed Google Scholar). Furthermore, inhibition by MAG has been shown to be blocked by C3 exoenzyme, a specific inhibitor of Rho (9Lehmann M. Fournier A. Selles-Navarro I. Dergham P. Sebok A. Leclerc N. Tigyi G. McKerracher L. J. Neurosci. 1999; 19: 7537-7547Crossref PubMed Google Scholar). We investigated the effect of Y27632, a specific inhibitor of Rho kinase, a downstream effector of Rho, on inhibition of neurite outgrowth by MAG and anti-GT1b antibody. Y27632 blocked inhibition of neurite outgrowth by both MAG and anti-GT1b antibody from cerebellar and hippocampal neurons (Fig.6). The blockade of the inhibitory activity of MAGR118A-Fc with sialic acid, 3′-sialyllactose, and specific gangliosides shown in this study suggests that this mutated form of MAG retains the ability to bind carbohydrate ligands and that these sugars and gangliosides, when added exogenously, can compete with endogenous neuronal receptors. Although these findings do not rule out interactions between MAG and other neuronal cell surface molecules, they strongly suggest that MAG inhibits neuronal outgrowth by recognition of sialylated glycans on the neuronal cell surface. From the crystal structure of siglec 1 (sialoadhesin) complexed with 3′-sialyllactose, it is evident that amino acids other than arginine 118 are involved in binding sialic acid (21May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). These are conserved in MAG and include tryptophan 2, a key amino acid for sialic acid recognition (21May A.P. Robinson R.C. Vinson M. Crocker P.R. Jones E.Y. Mol. Cell. 1998; 1: 719-728Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Furthermore, components of the core glycan on which sialic acid is presented are able to increase the binding affinity of MAG for sialic acid either through direct contacts or more favorable presentations of sialic acid, increasing the affinity of MAG for sialic acid itself (23Yang L.J.S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (270) Google Scholar, 26Strenge K. Schauer R. Bovin N. Hasegawa A. Ishida H. Kiso M. Kelm S. Eur. J. Biochem. 1998; 258: 677-685Crossref PubMed Scopus (57) Google Scholar). The reduced potency of inhibition of MAGR118A-Fc compared with the unmutated protein is therefore consistent with a reduced affinity of binding due to the loss of arginine 118. Inhibitors of neurite outgrowth in myelin may prevent aberrant sprouting under steady-state conditions (38Buffo A. Zagrebelsky M. Huber A.B. Skerra A. Schwab M.E. Strata P. Rossi F. J. Neurosci. 2000; 20: 2275-2286Crossref PubMed Google Scholar). The activity of these molecules may require regulation to prevent unwanted neuronal retraction. Linking carbohydrate recognition by MAG to biological function raises the possibility that the effect of MAG on neurons during steady-state or disease conditions could be regulated by changes in lectin activity. Two mechanisms of regulation of lectin activity for other siglecs have been identified. First, "cis " interactions between siglecs (including MAG) and sialylated glycans on the same cell surface (and perhaps on siglecs themselves) have been shown to block binding to opposing cells (13Freeman S.D. Kelm S. Barber E.K. Crocker P.R. Blood. 1995; 85: 2005-2012Crossref PubMed Google Scholar, 14Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar, 29Tang S. Shen Y.J. Debellard M.E. Mukhopadhyay G. Salzer J.L. Crocker P.R. Filbin M.T. J. Cell Biol. 1997; 138: 1355-1366Crossref PubMed Scopus (130) Google Scholar, 39Razi N. Varki A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7469-7474Crossref PubMed Scopus (219) Google Scholar, 40Barnes Y.C. Skelton T.P. Stamenkovic I. Sgroi D.C. Blood. 1999; 93: 1245-1252Crossref PubMed Google Scholar). In the case of siglec 2 (CD22), blocking of lectin activity by cisinteractions on resting B-lymphocytes can be "unmasked" on pharmacological or physiological activation, demonstrating the potential for this type of regulation in physiological processes (39Razi N. Varki A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7469-7474Crossref PubMed Scopus (219) Google Scholar). Second, the ability of siglecs to bind sialic acid may be regulated intracellularly. In the case of siglecs containing immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic domains (siglecs 2, 3, 5, 6, and 7), inside-out regulation of the ability to bind sialylated glycans occurs through the recruitment of SHP-1 and SHP-2 (41Taylor V.C. Buckley C.D. Douglas M. Cody A.J. Simmons D.L. Freeman S.D. J. Biol. Chem. 1999; 274: 11505-11512Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Regulation of sialic acid binding therefore appears to be an important aspect of siglec biology and may rep
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