A radioimmunoassay for the myelin-associated glycoprotein

Abstract: The myelin-associated glycoprotein (MAG) was purified from rat brain and radioiodinated with Bolton-Hunter reagent for use in a double-antibody radioimmunoassay. The conditions of the assay were adjusted to measure between 2 and 30 ng of MAG. The antigenic sites of MAG in tissue samples were exposed by solubilization in 1% (wt/vol) sodium dodecyl sulfate (SDS), and the final assay was done in a mixture of 0.25% SDS and 0.25% Triton X-100. The presence of the Triton X-100 overcame the inhibitory effect of SDS alone on the immune reactions. Application of the assay to whole homogenates of developing rat brain revealed the expected increase of MAG with the progression of myelination. Adult brain homogenate and purified myelin contained 2.7 and 7.4 μg of MAG/mg protein, respectively. Sciatic nerve contained a lower level of MAG, and cross-reacting material was not detected in nonneural tissues. This assay makes possible for the first time the analysis of MAG in whole tissue without prior myelin isolation or glycoprotein separation.     

Synthesis of soluble myelin-associated glycoprotein in insect and mammalian cells

The myelin-associated glycoprotein (MAG) has an extracellular domain containing five sequences which are homologous to the immunoglobulin-fold motif. Adhesive interactions mediated by the MAG extracellular domain are involved in the development of the myelin sheath. The MAG cDNA has been modified to introduce a stop codon immediately before the transmembrane domain. Expression of the modified cDNA in insect cells and murine NIH-3T3 cells resulted in secretion of the soluble MAG extracellular domain. Treatment of soluble MAG with glycopeptidase F and endoglycosidase H showed significant differences in glycosylation for the insect and mammalian cell-expression systems. The soluble form of MAG has been purified from insect-cell supernatants by adsorption to a lentil-lectin support. The soluble MAG will provide a powerful new approach for studies of MAG-adhesive interactions during brain development.     

Developmental and pathophysiological aspects of the myelin-associated glycoprotein

1. A glycoslylated sulfate-containing protein known as myelin-associated glycoprotein (MAG) appears to be unique to the central and peripheral nervous systems. This component has been characterized and cDNA clones have been isolated. 2. MAG is a member of the immunoglobulin superfamily. The principal form of MAG synthesized in brain during active myelination has an apparent molecular weight of 100,000. Alternate exon splicing leads to an additional 5000-dalton smaller form with a different C terminus. 3. In patients with multiple sclerosis, MAG is rapidly lost in areas of active disease. It is immunologically reactive in patients with benign monoclonal gammopathy associated with peripheral neuropathy. 4. The role of MAG in the formation of the myelin sheath and its participation in autoimmune neurological disorders are outlined.     

The relationship between glycosylation and glycoprotein metabolism of mouse neuroblastoma N18 cells.

Two inhibitors of glycosylation, glucosamine and tunicamycin, were utilized to examine the effect of glycosylation inhibition in mouse neuroblastoma N18 cells on the degradation of membrane glycoproteins synthesized before addition of the inhibitor. Treatment with 10 mM-glucosamine resulted in inhibition of glycosylation after 2h, as measured by [3H]fucose incorporation into acid-insoluble macromolecules, and in a decreased rate of glycoprotein degradation. However, these results were difficult to interpret since glucosamine also significantly inhibited protein synthesis, which in itself could cause the alteration in glycoprotein degradation [Hudson & Johnson (1977) Biochim. Biophys. Acta 497, 567-577]. N18 cells treated with 5 microgram of tunicamycin/ml, a more specific inhibitor of glycosylation, showed a small decrease in protein synthesis relative to its effect on glycosylation, which was inhibited by 85%. Tunicamycin-treated cells also showed a marked decrease in glycoprotein degradation in experiments with intact cells. The inhibition of glycoprotein degradation by tunicamycin was shown to be independent of alterations in cyclic AMP concentration. Polyacrylamide-gel electrophoresis of isolated membranes from N18 cells, double-labelled with [14C]fucose and [3H]fucose, revealed heterogeneous turnover rates for specific plasma-membrane glycoproteins. Comparisons of polyacrylamide gels of isolated plasma membranes from [3H]fucose-labelled control cells and [14C]fucose-labelled tunicamycin-treated cells revealed that both rapidly and slowly metabolized, although not all, membrane glycoproteins became resistant to degradation after glycosylation inhibition.     

A single mutation in the E2 glycoprotein important for neurovirulence influences binding of sindbis virus to neuroblastoma cells

The amino acid at position 55 of the E2 glycoprotein (E2(55)) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E2(55) = histidine) differs only at this position from virus strain 633 (E2(55)= glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50 to 500 and by 633 only at an MOI of 5,000, while both viruses infected essentially 100% of BHK cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15 to 20%) than fusion with BHK-derived liposomes ( approximately 50%). Binding of TE and 633 to N18, but not BHK, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco's modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0, while that of TE did not change. The same pattern was observed with RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH dependent. Growth of N18 and BHK cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E2(55) impairs SV binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells, contributing to greater neurovirulence.     

Structure of mouse myelin-associated glycoprotein gene.

The mouse myelin-associated glycoprotein gene was isolated from a mouse gene library. This gene was split into 13 exons distributed about 15 kb in length. Each extracellular immunoglobulin-related domain was encoded by a single exon, and RNA splicing between those exons occurred between the first and second nucleotides of the junctional codon, the features of which are conserved in most of the genes of the immunoglobulin superfamily. The sequence of the 5'-flanking region appeared to have some regions homologous to other myelin proteins, which suggested that they were possible cis-elements for specific expression of oligodendrocytes.     

Preparation and characterization of antisera to the myelin-associated glycoprotein

Rabbits were immunized with the myelin-associated glycoprotein (MAG) that had been purified from isolated rat brain myelin by selective extraction with lithium diiodosacicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. Antibodies to MAG were detected qualitatively by immunodiffusion and quantitatively by a double antibody assay utilizing [³H]fucose-labeled MAG as antigen. The antisera were capable of precipitating between 300 and 500 g of MAG/ml of serum under the conditions of the assay. Preincubation of the anti-MAG serum with other glycoproteins or glycolipids did not inhibit the precipitation of labeled MAG. Similarly, preincubation of the antiserum with LIS-phenol extracts of non-neural tissues did not inhibit the immune precipitation of MAG. The specificity of the antiserum was also indicated by the selective double antibody precipitation of MAG from solubilized whole myelin that contained a heterogeneous mixture of [³H]fucose-labeled glycoproteins. The antibodies to MAG were not effectively absorbed by whole brain homogenate or purified myelin, indicating that the antigenic site(s) is not accessible in the intact membranes, but can be exposed by treatment with detergent or partial purification. Low levels of antibodies reacting with MAG were detected in three rabbits with experimental allergic encephalomyelitis induced by injection of purified myelin in complete Freund's adjuvant.     

Binding partners for curcumin in human schwannoma cells: Biologic Implications

Curcumin (diferuloylmethane) is a potent anti-inflammatory and anti-tumorigenic agent that has shown preclinical activity in diverse cancers. Curcumin up-regulates heat shock protein 70 (hsp70) mRNA in several different cancer cell lines. Hsp70 contributes to an escape from the apoptotic effects of curcumin by several different mechanisms including prevention of the release of apoptosis inducing factor from the mitochondria and inhibition of caspases 3 and 9. Previously we showed that the combination of curcumin plus a heat shock protein inhibitor was synergistic in its down-regulation of the proliferation of a human schwannoma cell line (HEI-193) harboring an NF2 mutation, possibly because curcumin up-regulated hsp70, which also binds merlin, the NF2 gene product. In order to determine if curcumin also interacts directly with hsp70 and to discover other binding partners of curcumin, we synthesized biotinylated curcumin (bio-curcumin) and treated HEI-193 schwannoma cells. Cell lysates were prepared and incubated with avidin-coated beads. Peptides pulled down from this reaction were sequenced and it was determined that biotinylated curcumin bound hsp70, hsp90, 3-phosphoglycerate dehydrogenase, and a β-actin variant. These binding partners may serve to further elucidate the underlying mechanisms of curcumin’s actions.     

cDNA cloning and amino acid sequence for human myelin-associated glycoprotein

cDNA clones of human myelin-associated glycoprotein were isolated and analyzed. The combination of the two overlapping cDNA clones covered the full coding region and the complete amino acid sequence was deduced. In rat and mouse, expression of the two forms of mRNA is developmentally regulated; the mRNA without exon 12 portion is expressed mainly in the actively myelinating stage of development. Although the cDNA library used here was prepared from adult human brain poly(A)+ RNA, all five clones obtained corresponded to the mRNA without exon 12 portion.     

The expression of recombinant large myelin-associated glycoprotein cytoplasmic domain and the purification of native myelin-associated glycoprotein from rat brain and peripheral nerve

The myelin-associated glycoprotein (MAG) is a transmembrane protein of the immunoglobulin superfamily existing as two isoforms (L-MAG and S-MAG) that are differentially expressed by myelinating glial cells of the central and peripheral nervous systems, where MAG represents 1 and 0.1% of the total myelin proteins, respectively. The polypeptide chains of the two isoforms differ only by the carboxy terminus of their respective cytoplasmic domains, which most probably determine the isoform-specific functions. Here, we describe the expression of the L-MAG cytoplasmic domain as a GST fusion protein. The recombinant protein was used to raise polyclonal antibodies against the L-MAG-specific carboxy terminus and against the region of the MAG cytoplasmic domain common to both S-MAG and L-MAG. These antibodies, which function in dot blotting, Western blotting, and immunoprecipitation, were used to immunopurify native MAG from both rat brain and peripheral nerves in quantities and purity sufficient for the realization of most biochemical and functional studies. The antibodies and the recombinant and native MAG proteins provide much needed tools for the study of the common and isoform-specific properties and functions of L-MAG and S-MAG.     

Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells

The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 2 · 10}{}^{\mathrm{?7}}\text{M}$). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.     

A monoclonal anti-idiotypic antibody against a human monoclonal IgM with specificity for myelin-associated glycoprotein

A human monoclonal IgM lambda antibody, directed against MAG, obtained from a patient with polyneuropathy associated with a gammopathy, was used as an immunogen to generate mouse monoclonal anti-idiotype antibodies. One hybridoma antibody, designated A8F2, reacts uniquely with the M-IgM of the patient, shows high affinity binding to the patient's M-IgM, and dose-dependently inhibits binding of the patient's M-IgM to its specific antigen MAG. Thus, A8F2 is a monoclonal anti-idiotype antibody that recognizes a region of the MAG binding site of the patient's IgM. Use of this anti-idiotype antibody in a competition RIA revealed the presence of naturally occurring anti-idiotype in the patient's serum. Because anti-idiotype antibodies may be part of a mechanism for down-regulation of antibody production, the use of A8F2 to induce a specific immunosuppression should be considered.     

Abnormal glycosylation of myelin-associated glycoprotein in quaking mouse brain

Brain slices from actively myelinating (26–28 days) quaking and normal littermates were dual-labeled with radioactive mannose and fucose for 2 h. Following the incubation myelin was isolated by sucrose density gradient centrifugation and the incorporation of sugars into the major myelinassociated glycoprotein (MAG) determined. The incorporation of mannose (an internal monosaccharide) and fucose (a terminal monosaccharide) was impaired in quaking by approximately 70 and 83% respectively as compared to control. The mannose/fucose ratio in quaking myelin was approximately 70% higher than in control. The results indicate an abnormal processing of the N-linked oligosaccharide moiety of MAG in quaking oligodendrocytes.     

Biochemical demonstration of the myelin-associated glycoprotein in the peripheral nervous system

Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [3H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, W1, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [3H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [14C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and W1 fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole LIS-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

Binding of soluble myelin-associated glycoprotein to specific gangliosides induces the association of p75NTR to lipid rafts and signal transduction

Myelin-associated glycoprotein (MAG) is a potent inhibitor of neurite outgrowth from a variety of neurons. Here we show that gangliosides, GT1b and GD1a, as well as the Nogo receptor, are functional binding partners for soluble MAG-Fc. Postnatal cerebellar neurons from mice deficient in the GalNAcT gene are insensitive to MAG with regard to neurite outgrowth and lack in the activation of RhoA. MAG-Fc or the antibody to GT1b and GD1a elicits recruitment of p75(NTR.) to lipid rafts, specialized microdomain for signal transduction. Disruption of lipid rafts results in abolishment of inhibitory effects of MAG-Fc and the Nogo peptide. These findings establish gangliosides as functional binding partners for soluble MAG. Gangliosides may play a role in translocation of p75(NTR.) to lipid rafts for initiation of the signal transduction.     

Rap1 is involved in the signal transduction of myelin-associated glycoprotein

Myelin-associated glycoprotein (MAG) is a well-characterized axon growth inhibitor in the adult vertebrate nervous system. Several signals that play roles in inhibiting axon growth have been identified. Here, we report that soluble MAG induces activation of Rap1 in postnatal cerebellar granule neurons (CGNs) and dorsal root ganglion (DRG) neurons. The p75 receptor associates with activated Rap1 and is internalized in response to MAG. After MAG is applied to the distal axons of the sciatic nerves, the activated Rap1, internalized p75 receptor, and MAG are retrogradely trafficked via axons to the cell bodies of the DRG neurons. Rap1 activity is required for survival of the DRG neurons as well as CGNs when treated with MAG. The transport of the signaling complex containing the p75 receptor and Rap1 may play a role in the effect of MAG.     

Synthesis of sialic acid derivatives as ligands for the myelin-associated glycoprotein (MAG)

The trisaccharide substructure 13 of the ganglioside GQ1balpha shows a remarkable affinity for the myelin-associated glycoprotein (MAG). In the search for structurally simplified and pharmacokinetically improved mimics of 13, sialosides with modifications at the reducing and non-reducing end were synthesized. The biological evaluation of mimics 12a-o was performed in a competitive target-based assay. It was found that the relative inhibitory potency (rIP) of antagonist 12h was enhanced by more than 1000-fold in comparison to the reference trisaccharide 13, despite the former having a much simpler structure. In addition, the sialic acid derivatives, for example, 12h, have clearly improved pharmacokinetic properties due to the presence of aromatic moieties, a lower molecular weight, and a reduced number of polar hydroxy functions compared to the reference compound 13.     

The myelin-associated glycoprotein is phosphorylated in the peripheral nervous system.

Phosphorylation of the myelin-associated glycoprotein (MAG) in the peripheral nervous system is demonstrated by immunoprecipitation from myelin proteins radiolabeled in vivo, in nerve slices and in a cell-free system. Phosphoamino acid analysis of immunoprecipitated MAG revealed the presence of radioactivity in phosphoserine, but not in phosphothreonine or phosphotyrosine. Only the shorter isoform of MAG (S-MAG) was detected by immunostaining of nitrocellulose sheets with anti-MAG anti-serum after enzymatic deglycosylation of immunoprecipitated MAG labeled in nerve slices. Autoradiography of the same Western blots revealed that most of the radioactive phosphate was in S-MAG, demonstrating that the polypeptide backbone of S-MAG is phosphorylated in the PNS.