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.     

Membrane-bound neuraminidase in the developing mouse brain

The specific activity of membrane-bound neuraminidase towards exogenously added gangliosides in the developing mouse brain was investigated. While whole brain was examined in fetuses, studies were carried out comparatively in cortex and cerebellum of postnatal stages. Considerable differences were found among brain areas. There was a rapid increase in forebrain neuraminidase activity before birth. This high level of activity was maintained throughout the first postnatal week, followed by a slow leveling-off to adult values on the 30th day. In the cerebellum a rapid increase in neuraminidase activity occurred between the 10th and 16th postnatal day. After having reached the maximum, enzyme activity declined rapidly, with adult values being reached on the 21st day. Neuraminidase activity in the adult cerebellum exceeds that in the cortex by 65.9 %. The results are discussed in reference to developmental changes in ganglioside metabolism and the possible involvement of neuraminidase in its regulation is pointed out.     

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.     

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.     

Developmental changes of myelin-associated glycoprotein in rat brain: study on experimental hyperphenylalaninemia

We examined developmental changes of myelin-associated glycoprotein (MAG), basic protein (BP), abd proteolipid protein (PLP) in central nervous system myelin isolated from experimental hyperphenylalaninemic rats (PKU rats) and controls. Higher amounts of MAG, including high-molecular-weight MAG in myelin, were found in 12- to 21-day-old control rats than in adult rats. MAG in developing myelin was at a maximum in 18-day-old rats and began to decrease in 21-day-old rats, while PLP and BP in developing myelin increased at these developmental stages. The level of high-molecular-weight MAG decreased in myelin prepared from 21-day-old rats. These results suggest that the decreasing high-molecular-weight MAG is important for compaction of myelin in the early stage of myelination. In myelin from 12- to 18-day-old PKU rats, the ratio of each protein such as MAG, PLP, or BP to that of control was about 0.5 at 12 days, and increased to almost 1.0 at 18 days. The myelination seems to be initially delayed but to be close to that of controls in PKU rats about 18 days old.     

Purification and partial characterization of the myelin-associated glycoprotein from adult rat brain

The myelin-associated glycoprotein was purified from rat central nervous system myelin by selective extraction with lithium diiodosalicylate-phenol followed by gel filtration on a column of Sepharose CL-6B. Amino acid analysis of the purified glycoprotein revealed an excess of acidic over basic amino acids and a relatively high content of nonpolar residues. On the basis of weight, the molecule is about one-third carbohydrate consisting of 5% fucose, 23% mannose, 20% galactose, 34% N-acetylglucosamine, and 18% N-acetylneuraminic acid.     

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 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.     

Developmental change in a myelin-associated glycoprotein: a comparative study in rodents

In developing rats, a fucose-labeled glycoprotein (mGP) which is closely associated with myelin has a higher apparent molecular weight in immature myelin in comparison with mature myelin. In order to see whether this developmental difference occurred in other species, we investigated different rodents of the suborder Myomorpha. Glycoproteins were labeled in vivo by injection of [³H]fucose of [¹⁴C]fucose, and purified myelin fractions were prepared. Proteins were extracted with SDS and separated by polyacrylamide gel electrophoresis. Using double-labeling techniques, the ³H- and ¹⁴C-labeled mGP were compared in different samples. We found the shift in the apparent molecular weight of mGP during development in rats, gerbils, and hamsters, but we were unable to demonstrate it in mice and prairie deer mice. In these latter species, the mGP of smaller apparent molecular weight was already present at the earliest ages at which it was possible to isolate myelin. Although some rodents do not exhibit the developmental change, all showed a common feature: the presence of the same small molecular weight mGP in mature myelin. It may be that the glycoprotein of smaller size is a prerequisite for the normal compaction and maturation of myelin.     

Developmentally regulated alternative splicing of brain myelin-associated glycoprotein mRNA is lacking in the quaking mouse

Evidence is presented that expression of the two myelin-associated glycoprotein mRNAs is developmentally regulated in mouse brain. In quaking mouse, the mRNA without a 45-nucleotide exon portion was scarcely expressed throughout development. We conclude that the mechanism of splicing out the 45-nucleotide exon portion is lacking in quaking mouse.     

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.     

Microtubule-associated protein 1B: a neuronal binding partner for myelin-associated glycoprotein.

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia-axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553-562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.     

Glycoprotein biosynthesis in the developing rat brain IV. Effects of guanosine nucleotides on soluble glycoprotein galactosyl- and N-acetylgalactosaminyltransferases

The effects of GTP on soluble cerebral glycoprotein galactosyl- and N-acetylgalactosaminyltransferases were studied. The transfer of galactose from UDP-galactose to both endogenous and exogenous acceptors was stimulated by GTP while the transfer of N-acetylgalactosamine to endogenous and defined exogenous acceptors was inhibited by the nucleotide. Similar results were obtained with β,γ-methylene GTP, a structural analog of GTP. Evidence is presented which suggests that GTP is as an allosteric modulator of these two glycosyltransferases.     

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.     

Effects of pronase on isolated chloroplasts

Subjecting isolated spinach chloroplasts to mild proteolysis (10-minute incubation at 20 C in 500 micrograms per milliliter pronase) caused chloroplast clumping but did not affect their integrity as measured by their ability to carry out light stimulated, glycerate-3-P-dependent O₂ evolution. Transmission electron microscopy revealed no detectable differences between the control and treated plastids. Mild proteolysis inactivated exogenously added pyruvate kinase and should be a useful technique in certain enzyme distribution studies.     

Antagonists of the myelin-associated glycoprotein: a new class of tetrasaccharide mimics

The tetrasaccharide substructure 1 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 1, biphenyl was identified as a feasible replacement for the core disaccharide Galbeta(1-3)GalNAc according to saturation transfer difference (STD) NMR and molecular modeling investigations. Using Suzuki coupling, a convergent synthesis of the mimics was achieved. To optimize the yields of the coupling reactions, the catalytic effects of microwave irradiation and conventional heating were compared. The biological evaluation of mimics 3 and 4 was performed in a competitive target-based assay. It was found that the relative inhibitory potency (rIP) of antagonist 3 was clearly enhanced in comparison to the reference trisaccharide 2, despite the former having a much simpler structure. In addition to the improved synthetic feasibility, an increase of the partition coefficient between octanol and water (logP), and therefore a beneficial change in the pharmacokinetic properties of 3 and 4 was achieved.     

cDNA cloning of mouse myelin-associated glycoprotein: a novel alternative splicing pattern

The structures of three forms of mouse myelin-associated glycoprotein mRNAs were determined from full-length cDNA clones. Two forms of mRNAs have been reported to be different by alternate inclusion of exon 2 and 12 in rat brain. One of the three forms of clones obtained here appeared to be a novel mRNA which lacked both the exon 2 and 12 portions, although others were identical splicing patterns to those of rat. Northern blot analysis using specific probes to mRNAs with or without the exon 2 portion in normal and quaking mouse confirmed that the splicing of exon 2 and 12 occurred independently.     

Characterization of binding properties of the myelin-associated glycoprotein to extracellular matrix constituents.

The myelin-associated glycoprotein (MAG) can be obtained from adult mouse brain from detergent-lysates of a crude membrane fraction as a 96-100 kd form (detergent solubilized MAG), and from 100,000 g supernatants of homogenates as a 90-96 kd form (soluble MAG). The soluble form distributes into the Triton X-114-poor aqueous phase, while detergent-solubilized MAG predominantly enters the Triton X-114-rich phase. Both molecular forms bind to heparin in hypo- and isotonic buffers. Soluble MAG binds to several collagens (type G, I, II, III, IV, V, VI, IX) with a kd of 5.7 X 10(-8) M for collagen type IX and 2.0 X 10(-7) for collagen type IV. Binding of 125I-labeled MAG to collagen G can be completely inhibited by unlabeled MAG and collagen G, but not by heat-denatured collagen. MAG does not bind to itself, laminin, fibronectin, or the neural cell adhesion molecules L1 and N-CAM. Binding of MAG to collagen G is most effectively blocked by a high molecular weight dextran sulfate, heparan sulfate and heparin, with chondroitin sulfate and a low molecular weight dextran sulfate being less potent blockers. These findings are in agreement with previous observations on the localization of MAG in basal lamina and interstitial collagens of the sciatic nerve in situ.     

Potent glycan inhibitors of myelin-associated glycoprotein enhance axon outgrowth in vitro

Myelin-associated glycoprotein (MAG, Siglec-4) is one of several endogenous axon regeneration inhibitors that limit recovery from central nervous system injury and disease. Molecules that block such inhibitors may enhance axon regeneration and functional recovery. MAG, a member of the Siglec family of sialic acid-binding lectins, binds to sialoglycoconjugates on axons and particularly to gangliosides GD1a and GT1b, which may mediate some of the inhibitory effects of MAG. In a prior study, we identified potent monovalent sialoside inhibitors of MAG using a novel screening platform. In the current study, the most potent of these were tested for their ability to reverse MAG-mediated inhibition of axon outgrowth from rat cerebellar granule neurons in vitro. Monovalent sialoglycans enhanced axon regeneration in proportion to their MAG binding affinities. The most potent glycoside was disialyl T antigen (NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc-R), followed by 3-sialyl T antigen (NeuAcalpha2-3Galbeta1-3GalNAc-R), structures expressed on O-linked glycoproteins as well as on gangliosides. Prior studies indicated that blocking gangliosides reversed MAG inhibition. In the current study, blocking O-linked glycoprotein sialylation with benzyl-alpha-GalNAc had no effect. The ability to reverse MAG inhibition with monovalent glycosides encourages further exploration of glycans and glycan mimetics as blockers of MAG-mediated axon outgrowth inhibition.