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The myelin-associated glycoprotein (MAG) is selectively localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths suggesting that it functions in glia-axon interactions in the PNS and CNS, and this is supported by much experimental evidence. In addition, MAG is now well known as one of several white matter inhibitors of neurite outgrowth in vitro and axonal regeneration in vivo, and this latter area of research has provided a substantial amount of information about neuronal receptors or receptor complexes for MAG. This article makes the hypothesis that the capacity of MAG to inhibit outgrowth of immature developing or regenerating neurites is an aberration of its normal physiological function to promote the maturation, maintenance, and survival of myelinated axons. The overview summarizes the literature on the function of MAG in PNS and CNS myelin sheaths and its role as an inhibitor of neurite outgrowth to put this hypothesis into perspective. Additional research is needed to determine if receptors and signaling systems similar to those responsible for MAG inhibition of neurite outgrowth also promote the maturation, maintenance, and survival of myelinated axons as hypothesized here, or if substantially different MAG-mediated signaling mechanisms are operative at the glia-axon junction.

Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with [35S] sulfate. Separation of the ganglioside fraction by two-dimensional TLC showed that, except for galactosylceramide-sulfate, none of the sulfate-labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components. At least eight sulfate-labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate. Six of the sulfate-labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A. ureafaciens neuraminidase. In vivo labeling of lipids from 14-day-old rat brain with [35S]-sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue. These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis.

Treatment of cultured rat oligodendroglial progenitors with either platelet-derived growth factor (PDGF) or fibroblast growth factor-2 (FGF-2) activated extracellular signal regulated kinase 2 (ERK2). Activation was transient in response to PDGF, whereas it was greater and more prolonged in response to FGF-2. ERK2 activation by PDGF was preceded by a very rapid, robust and transient tyrosine phosphorylation of the PDGF receptor. Although there was consistently more activation of ERK2 in response to FGF-2 than to PDGF, immunostaining of FGF receptors 1 (FGFR1) and 2 (FGFR2) and their tyrosine phosphorylation in progenitors was very weak, and both receptors were up-regulated during differentiation to oligodendrocytes. Tyrosine phosphorylation of the FGF receptors was maximal from 15 to 60 min of treatment and was sustained for many hours. Binding of radioiodinated FGF-2 to FGFR1 was predominant in progenitors, whereas binding to FGFR2 was predominant in oligodendrocytes. ERK2 activation by PDGF was more sensitive to inhibition of tyrosine kinases, whereas ERK2 activation by FGF-2 was relatively more sensitive to inhibitors of protein kinase C. These differences in signal transduction pathways probably contribute to the different cellular responses of oligodendroglial lineage cells to PDGF and FGF-2, respectively.

Although MAG-null mice myelinate relatively normally except for subtle structural abnormalities in the periaxonal region of myelin sheaths, they develop more severe pathological changes as they age. The purpose of this study was to further define the biochemical aspects of CNS pathology caused by an absence of MAG. Proteins associated with myelin and oligodendrocytes were quantified by densitometry of western blots in whole brain homogenates, as well as in isolated myelinated axons and myelin. Neither myelin yields, nor levels of myelin basic protein and proteolipid protein, were decreased in comparison to control levels in 14-month-old MAG-null mice. On the other hand, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and the 120 kD neural cell adhesion molecule (N-CAM) were substantially reduced in whole brain, myelinated axons, and myelin. Tubulin, Na(+)K(+)ATPase and Fyn tyrosine kinase were also reduced significantly in myelin-related fractions, but not in whole brain homogenate. The decreased levels of these proteins suggest pathological abnormalities in oligodendrocytes. Furthermore, significant reductions of CNPase and 120 kD NCAM were also present at 2 months, indicating that the oligodendroglial abnormalities begin at a relatively early age. Neither TUNEL assays nor multiplex RT-PCR for mRNAs of apoptosis-related proteins in the aging MAG-null mice provided evidence for apoptotic oligodendrocytes. These biochemical findings suggest oligodendroglial damage in MAG-null mice and support the morphological observations pointing to a progressive "dying-back oligodendrogliopathy" as a consequence of MAG deficiency.

High titers of serum antibodies to neural antigens occur in several forms of neuropathy. These include neuropathies associated with monoclonal gammopathy, inflammatory polyneuropathies, and paraneoplastic neuropathies. The antibodies frequently react with glycosylated cell surface molecules, including glycolipids, glycoproteins, and glycosaminoglycans, but antibodies to intracellular proteins have also been described. There are several correlations between antibody specificity and clinical symptoms, such as anti-MAG antibodies with demyelinating sensory or sensorimotor neuropathy, anti-GM1 ganglioside antibodies with motor nerve disorders, antibodies to gangliosides containing disialosyl moieties with sensory ataxic neuropathy and Miller-Fisher syndrome, and antibodies to the neuronal nuclear Hu antigens with paraneoplastic sensory neuronopathy. These correlations suggest that the neuropathies may be caused by the antibodies, but evidence for a causal relationship is stronger in some examples than others. In this review, we discuss the origins of the antibodies, evidence for and against their involvement in pathogenic mechanisms, and the implications of these findings for therapy.

A growing number of glycoproteins have been identified and characterized in myelin and myelin-forming cells. In addition to the major P0 glycoprotein of compact PNS myelin and the myelin-associated glycoprotein (MAG) in the periaxonal membranes of myelin-forming oligodendrocytes and Schwann cells, the list now includes peripheral myelin protein-22 (PMP-22), a 170 kDa glycoprotein associated with PNS myelin and Schwann cells (P170k/SAG), Schwann cell myelin protein (SMP), myelin/oligodendrocyte glycoprotein (MOG), and oligodendrocyte-myelin glycoprotein (OMgp). Many of these glycoproteins are members of the immunoglobulin superfamily and express the adhesion-related HNK-1 carbohydrate epitope. This review summarizes recent findings concerning the structure and function of these glycoproteins of myelin sheaths with emphasis on the physiological roles of oligosaccharide moieties.

The myelin-associated glycoprotein (MAG) exhibits an abnormally high apparent molecular weight in sciatic nerve, but not in brain, of dysmyelinating trembler mutants (Inuzuka et al.: J Neurochem 44:793-797, 1985). Antibodies to the large and small isoforms of MAG (L- and S-MAG) and probes for oligosaccharide structure were used to determine if this was due to overexpression of L-MAG or increased glycosylation. Nerves from both control and trembler 36-day-old mice contained primarily S-MAG with only traces of L-MAG. The distribution of the two isoforms appeared normal in trembler mice, and both isoforms exhibited the higher apparent molecular weight. Lectin binding showed that, in contrast to brain in which most glycoproteins contain primarily alpha 2-3 linked sialic acid, most glycoproteins of both control and trembler nerve contained primarily alpha 2-6 linked sialic acid. Lectin binding and glycosidase treatments demonstrated that the higher molecular weight of MAG in trembler nerves was due to an increased content of alpha 2-3 linked sialic acid and galactose. The abnormal glycosylation of MAG in trembler mutants may contribute to the myelin pathology.

The relative expression of large (L) and small (S) iso-forms of the myelin-associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using anti-sera specific for L- and S-MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetyineuraminic acid residues linked α2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oli-gosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants. © 1995 Wiley-Liss, Inc.

Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12-14 days in vitro. Addition of 50 micrograms/ml exogenous Neu-NAc alpha 2-3Gal beta 1-4Glc beta 1-1'ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [14C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [14C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [14C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O-2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3.

An autoantibody occurring in the serum of an apparently normal rabbit that immunocytochemically stains myelin sheaths and oligodendrocytes in rat brain was shown to react specifically with the 46-kDa isoform of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) (EC 3.1.4.37) in a number of species. Identification of the shorter isoform of the enzyme (CNP1) as the antigen was achieved by comparing the immunostaining of Western blots by the autoantibody with that of a well-characterized anti-CNP antiserum. The 46-kDa antigen reacting with the autoantibody exhibited the same Mr and pI as the small isoform of CNP on two-dimensional gels and showed a similar enrichment in purified CNS myelin. The autoantibody has very high affinity for CNP1 and is capable of detecting the very low amounts of this enzyme in peripheral nerve, spleen, adrenal gland, pancreas, testis, and intestine. Testing the reactivity of the autoantibody with synthetic peptides by enzyme-linked immunosorbent assay revealed that it reacted with the N-acetylated decapeptide corresponding to the N-terminus of CNP1, but did not react if the peptide was not acetylated or if the acetyl group was replaced with a palmityl group. The lack of reactivity with CNP2, which differs from CNP1 by a 20-amino acid extension at the N-terminus of the protein as a result of alternative splicing, may be due to the absence of the N-acetyl moiety that is part of the epitope and/or blocking of antibody binding to the decapeptide by extension of the polypeptide chain. This highly specific antibody for the shorter isoform of CNP may be useful in studies to establish possible differences in the location or function of the two isoforms of this enzyme.

Isolated adult bovine oligodendrocytes maintained in vitro for 10 days were treated for 1 day with 50 micrograms/ml of GM3 ganglioside (NeuNac alpha 2-3Gal beta 1-4Glc beta 1-1'ceramide) in serum-free culture medium. The treated oligodendrocytes had significantly longer processes with more branching than control cells in the same medium without GM3. The treatment also stimulated the release of a series of 22-100-kDa, [3H]glucosamine-labeled glycoproteins into the culture medium. Treatment of oligodendrocytes maintained in vitro for 50 days with GM3 for 1 day resulted in a thickening of the processes and the appearance of many fine branches on existing processes as well as a similar stimulation of glycoprotein release into the medium.

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.

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 immuno-precipitated 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.

Members of the immunoglobulin superfamily include a large number of cell adhesion molecules and receptors that are important for mediating interactions between cells in the nervous system. Keywords: cell adhesion molecule (CAM); glia; neural cell adhesion molecule (N-CAM); neuron; signal transduction

The spontaneously immortalized S16 Schwann cell line expresses higher levels of myelin-associated glycoprotein (MAG) and PO glycoprotein and their messenger RNAs when grown at high density than at low density [Sasagasako et al: J Neurochem 1996;66:1432–1439]. This up-regulation of myelin protein expression at high density is not associated with decreased cellular proliferation and may be caused by direct cell-to-cell contact. To investigate the hypothesis that increased mRNA levels for myelin proteins are caused by contact between Schwann cells, sparse S16 cell cultures were treated for 48 h with plasma-membrane-enriched fractions isolated from dense S16 cells. The treatment had no effect on the proliferation of the cells, but MAG and PO mRNAs were elevated 2- and 1.3-fold, respectively, in comparison to untreated cells. These effects on levels of myelin protein mRNAs were eliminated by pretreatment of the membrane fraction with heat or trypsin and were not caused by plasma membrane fractions from NIH 3T3 cells. These data support the hypothesis that homotypic contact between Schwann cells up-regulates expression of myelin proteins and suggest the possibility of autotypic contact-mediated regulation of myelinogenesis by adjacent spiraled membranes of individual Schwann cells.

The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85-kDa band. GNA identifies mannose-rich oligosaccharides because it is highly specific for terminal alpha-mannose residues. After purification of this 85-kDa glycoprotein band by chromatography on GNA-agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N-Glycanase or endoglycosidase H produced a 65-kDa band, indicating that it consisted of about one-fourth N-linked oligomannosidic carbohydrate moieties. High-performance anion-exchange chromatography and fluorescence-assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85-kDa mannose-rich glycoprotein component contained the SHP substrate-1 for phosphotyrosine phosphatases and at least one other member of the signal-regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor-like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.

Mammalian peripheral nervous system (PNS) myelin contains several glycoproteins with molecular weights of 19 to 28 kDa, including the major 28 kDa P0 glycoprotein and a recently cloned protein called PMP22. Some glycoproteins in this Mr range in humans, cats and some other mammals react with HNK1, a mouse monoclonal antibody that identifies a carbohydrate epitope shared between the immune system and a number of adhesion proteins in the nervous system. A variety of antibodies to PO, PMP-22, and the carbohydrate determinants reacting with HNK1 were used to characterize immunochemically these 19 to 28 kDa glycoproteins of cat PNS myelin. The HNK1-reactive components include P0 and two slightly smaller 23 to 26 kDa proteins that are immunologically related to PO. However, HNK1 reacts most strongly with a lower molecular weight glycoprotein that does not react with the antibodies to P0 and was identified as PMP-22. Since the carbohydrate structure reacting with HNK1 is generally expressed on adhesion molecules, this result suggests that PMP-22 may function in cell-cell or membranemembrane interactions. Furthermore, the related human anti-MAG monoclonal lgM antibodies from patients with neuropathy also react strongly with PMP-22, suggesting that it may be a target antigen in the pathogenesis of this disease. Purified PNS and CNS myelin from bony fish (toadfish and trout) were also shown to contain major glycoproteins, in the same 19 to 28 kDa Mr range, that react very strongly with HNK1. It is known that fish myelin has major proteins of this size that are immunologically and structurally related to mammalian P0, and it is demonstrated here that one of the strongly HNK1-positive proteins reacted well with an antiserum raised to bovine P0. The presence of high levels of the adhesion-related HNK1 epitope on these major myelin proteins of fish suggests that this carbohydrate structure may have played a role in the molecular evolution of myelin. © 1993 Wiley-Liss, Inc.1

Although the myelin-associated glycoprotein (MAG) cannot be detected in primary cultures of rat Schwann cells in the absence of neurons, MAG expression was demonstrated in some lines of cultured Schwann cells that had been immortalized by repetitive passaging. Radioimmunoassay of one such Schwann cell line, S-16, showed a remarkably high MAG concentration of about 1 ng/microgram of total protein, a level that is comparable to the MAG concentration in adult sciatic nerve. The S-16 cells divide very rapidly, are rounder than normal Schwann cells, and elaborate many processes after reaching high density. The cells are galactocerebroside positive, but express little or no P0 glycoprotein or myelin basic protein. As in nerve, the MAG synthesized by the cultured cells is primarily the shorter isoform (S-MAG). Furthermore, the posttranslational processing resembles that occurring in vivo including a similar degree of glycosylation, sulfation of oligosaccharides, and phosphorylation of the polypeptide. The sensitivity of MAG to treatment of the intact cells with trypsin or neuraminidase, as well as surface labeling with [3H]borohydride reduction after periodate oxidation, demonstrated that most of the MAG expressed by the S-16 cells is located on the cell surface. This line of immortalized Schwann cells expressing a remarkably high level of MAG should be useful for investigating the cell biology and function of this glycoprotein.

A high proportion of patients with neuropathy have immunoglobulin M (IgM) paraproteins that react with carbohydrate determinants on the myelin-associated glycoprotein (MAG) and two sphingoglycolipids, 3-sulfoglucuronyl paragloboside (SGPG) and 3-sulfoglucuronyl lactosaminyl paragloboside. In order to characterize the fine specificities of these human antibodies further, the binding of 10 anti-MAG paraproteins to several chemically modified derivatives of SGPG was compared with the binding to intact SGPG by both TLC-overlay and enzyme-linked immunosorbent assay. The following derivatives were tested: the desulfated lipid, glucuronyl paragloboside (GPG); the methyl ester of GPG (MeGPG); the methyl ester of SGPG, 3-sulfomethylglucuronyl paragloboside (SMeGPG); and 3-sulfoglucosyl paragloboside (SGlcPG) produced by reduction of the carboxyl group of the glucuronic acid with sodium borohydride. All 10 IgM paraproteins and the related mouse IgM antibody, HNK-1, reacted most strongly with intact SGPG, but variations in the reactivity with the derivatives revealed striking differences in the structural requirements for binding between the antibodies. Five distinct patterns of reactivity were observed: (a) three of the human antibodies and HNK-1 exhibited partial reactivity with the sulfated derivatives, SMeGPG and SGlcPG, but not with GPG or MeGPG, indicating an absolute requirement for the sulfate group; (b) two of the human antibodies reacted only with GPG, demonstrating a requirement for the free carboxyl group on the glucuronic acid; (c) three of the antibodies bound to SMeGPG, SGlcPG, and GPG, but not to MeGPG, suggesting that at least one negative charge was needed for binding; (d) one antibody bound to SMeGPG and GPG, but not to SGlcPG or MeGPG, suggesting that both the carbonyl group and at least one negative charge were required; and (e) another antibody bound to MeGPG, SMeGPG, and SGlcPG, but not to GPG, a pattern that is difficult to explain simply based on the chemical structures. Interestingly, only those antibodies that exhibited reactivity with GPG bound to a third, minor, unidentified glycolipid of normal peripheral nerve that chromatographs faster than SGPG. The results clearly demonstrate heterogeneity in the fine specificities of the anti-MAG antibodies that may affect their pathogenic properties.

An earlier investigation from our laboratory (Ilyas AA, Wilson HJ, Quarles RH, et al. Serum antibodies to gangliosides in Guillain-Barre syndrome. Ann Neurol 1988;23:440-447) demonstrating the presence of high levels of antiganglioside ganglioside antibodies in the sera of 5 of 26 patients with Guillain-Barre Syndrome (GBS) but not in control sera is summarized. The ganglioside antigens varied among the 5 patients with positive findings, and the antiganglioside antibodies decreased concurrently with clinical improvement in those patients for whom longitudinal samples were available for analysis. The results are discussed in the context of antibodies to acidic glycolipids in other types of neuropathy and other studies on antiglycolipid antibodies in GBS. Data showing the occurrence of lower levels of antibodies to P2 protein, P0 glycoprotein, and myelin-associated glycoprotein in some of the GBS patients are also summarized. The findings from our laboratory combined with the results of others make it unlikely that antiganglioside antibodies have a notable pathogenic effect in most patients with GBS, but the possibility remains that they are of pathogenic importance in some patients with the highest antibody titers.

The myelin-associated glycoprotein (MAG) is a type I transmembrane glycoprotein localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths where it functions in glia-axon interactions. It contains five immunoglobulin (Ig)-like domains and is in the sialic acid-binding subgroup of the Ig superfamily. It appears to function both as a ligand for an axonal receptor that is needed for the maintenance of myelinated axons and as a receptor for an axonal signal that promotes the differentiation, maintenance and survival of oligodendrocytes. Its function in the maintenance of myelinated axons may be related to its role as one of the white matter inhibitors of neurite outgrowth acting through a receptor complex involving the Nogo receptor and/or gangliosides containing 2,3-linked sialic acid. MAG is expressed as two developmentally regulated isoforms with different cytoplasmic domains that may activate different signal transduction pathways in myelin-forming cells. MAG contains a carbohydrate epitope shared with other glycoconjugates that is a target antigen in autoimmune peripheral neuropathy associated with IgM gammopathy and has been implicated in a dying back oligodendrogliopathy in multiple sclerosis.

The monoclonal HNK-1 antibody, a marker for human natural killer cells, strongly reacted with human peripheral nerve gangliosides in the enzyme-linked immunosorbent assay. Autoradiography after the binding of HNK-1 to thin-layer chromatograms of peripheral nerve gangliosides followed by radioiodinated goat anti-mouse IgM revealed that HNK-1 was reacting with a minor ganglioside that chromatographed between GM1 and GD1a. The antigen was insensitive to digestion with neuraminidase and pronase.

Concanavalin A strongly agglutinates purified fragments of immature and mature rat brain myelin, but only weakly agglutinates mature bovine and human myelin fragments. A sensitive method involving [3H]concanavalin binding to sodium dodecyl sulphate/polyacrylamide gels was used to detect the concanavalin A-binding proteins in purified myelin. When applied to mature rat brain myelin proteins that had been labelled in vivo with [14C]fucose, the distribution of the [3H]concanavalin A on the gel was very similar to that of [14C]fucose with the major peak corresponding to the major myelin-associated glycoprotein. The technique revealed that the immature form of the myelin-associated glycoprotein with a slightly larger apparent molecular weight also bound concanavalin A, and that in purified immature rat myelin the quantitative importance of some of the other glycoproteins in binding concanavalin A was increased relative to the myelin-associated glycoprotein. The separated proteins of bovine and human myelin bound more [3H]-concanavalin A than those of rat myelin. In these species, the myelin-associated glycoprotein was a major concanavalin A-binding protein, although two higher-molecular-weight glycoproteins also bound significant quantities of [3H]concanavalin A. The results indicate that there are receptors for concanavalin A on the surface of rat, bovine and human myelin membranes and suggest that the myelin-associated glycoprotein is one of the principal receptors.

This paper evaluates the effects of two premixed commercial buffer systems on baseline changes, artifacts, and contaiminants in amino acid analysis using o -phthalaldehyde detection. The effects of additional components such as isopropanol are discussed. An alternative combination of premixed commercial buffers that minimizes artifacts and contaminants is proposed.

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance.

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.

Small cell lung cancer (SCLC) is a bronchogenic carcinoma of neuroectodermal origin that expresses a variety of nervous system markers characteristic of neuroendocrine cells. In addition, SCLC cell lines and biopsies have been shown immunocytochemically to express an antigen recognized by HNK-1, a mouse monoclonal antibody which recognizes a surface antigen on natural killer cells and on the myelin-associated glycoprotein (MAG) and other nervous system glycoconjugates. Immunoblot data are presented which identify 2 groups of HNK-1-reactive plasma membrane glycoproteins with Mrs of about 80 000 and 130 000, respectively, from several SCLC cell lines. Using antibodies to MAG carbohydrate and protein determinants as probes, it is shown that the SCLC glycoproteins reacting with HNK-1 do not appear to share structural similarity with MAG apart from carbohydrate determinants. Using similar techniques with a panel of polyclonal antibodies, data are shown indicating that there is no cross-reactivity of SCLC proteins with other myelin proteins including P0, P1, P2, proteolipid protein and myelin basic protein. A possible role of the carbohydrate antigen in mediating nervous system disease associated with SCLC is suggested.

— Proteins and glycoproteins in a myelin fraction isolated from Quaking mutant mice were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and stained with Fast Green or with periodic acid-Schiff reagents. Double labelling experiments with [3H]fucose and [14C]fucose were also used to compare glycoproteins in myelin from the mutant mice with those from control mice. In the myelin fraction from the Quaking mice the basic proteins and proteolipid protein were decreased relative to the high molecular weight proteins. Some glycoproteins which are present in small amounts in myelin from normal mice were increased relative to the major glycoprotein in the myelin fraction of the Quaking mice. Furthermore, the major myelin-associated glycoprotein was shifted toward higher apparent molecular weight in comparison with controls of the same age or even with 9-day-old controls. The abnormal glycoproteins in the mutant myelin fraction could be a factor in the impairment of myelination.

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.

The capacities of immature and mature rat brain myelin, bovine myelin and human myelin to be agglutinated by soya-bean agglutinin, Ricinus communis agglutinin, wheatgerm agglutinin, and Lotus tetragonolobus agglutinin were examined. The first two lectins, which are specific for galactose and N-acetylgalactosamine, strongly agglutinated immature and mature rat myelin, weakly agglutinated bovine myelin, but did not affect human myelin. The other myelin and lectin combinations resulted in very weak or no agglutination. [3H]Fucose-labelled glycoproteins of purified adult rat brain myelin were solubilized with sodium dodecyl sulphate and allowed to bind to concanavalin A–Sepharose and each of the other lectins mentioned above, which had been immobilized on agarose. About 60% of the radioactive fucose was in glycoproteins that bound to concanavalin A–Sepharose and these glycoproteins could be eluted with solutions containing methyl α-d-mannoside and sodium dodecyl sulphate. Periodate/Schiff staining or radioactive counting of analytical gels showed that most of the major myelin-associated glycoprotein (apparent mol.wt. approx. 100000) bound to the concanavalin A, whereas the glycoproteins that did not bind were mostly of lower molecular weight. Preparative polyacrylamide-gel electrophoresis of the glycoprotein fraction that was eluted with methyl α-d-mannoside yielded a relatively pure preparation of the myelin-associated glycoprotein. Similar results were obtained with each of the other lectins, i.e. the myelin-associated glycoprotein was in the fraction that bound to the immobilized lectin. Double-labelling experiments utilizing [3H]fucose-labelled glycoproteins from adult myelin and [14C]fucose-labelled glycoproteins from 14-day-old rat brain myelin did not reveal any difference in the binding of the mature and immature glycoproteins to any of the immobilized lectins. The results in this and the preceding paper [McIntyre, Quarles & Brady (1979) Biochem. J.183, 205–212] suggest that the myelin-associated glycoprotein is one of the principal receptors for concanavalin A and other lectins in myelin, and that this property can be utilized for the purification of this glycoprotein.

Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin-associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower-molecular-weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin-related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.

Immunoglobulins from patients with paraproteinemic polyneuropathy were screened for reactivity with nerve and brain glycolipids by ELISA and/or a thin-layer-chromatogram-overlay technique. The myelin-associated glycoprotein (MAG) has been shown to be an antigen in many neuropathy patients with IgM gammopathy, but this study focused on seven neuropathy patients in which the IgM paraproteins had been shown not to react with this glycoprotein. Five of these seven had IgM that reacted with components in the acidic glycolipid fraction of human sciatic nerve, and three of these IgMs also reacted with components in the acidic glycolipid fraction of human brain. Little or no reactivity with glycolipids was detected for two patients with neuropathy and IgG gammopathy or for two with neuropathy and IgA gammopathy. The results suggest that neuropathy-patient IgM paraproteins not reactive with MAG often react with acidic glycolipids and thus define a subset of paraproteinemic neuropathies. Since the IgM paraproteins that react with MAG also react with acidic glycolipids of nerve, glycolipid antigens appear to be quite common among the IgM paraproteinemic neuropathies.

The myelin-associated glycoprotein (MAG) is a 100 K-dalton, integral membrane glycoprotein containing 30% carbohydrate that is in central and peripheral myelin sheaths. Recent research on its function during normal myelinogenesis and possible roles in the pathology of demyelinating diseases is reviewed. Emphasis is placed on studies supporting a role for MAG in glia-axon interactions. Also, a highly immunogenic carbohydrate antigen on MAG, that is shared with natural killer cells and binds IgM paraproteins associated with neuropathy, is considered in detail.

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.

A sulfated 100K-dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK-1 bind to this glycoprotein. It is shown here that mouse anti-myelin-associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti-MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti-melanoma antibodies (10C5 and 11B5) resemble HNK-1 in binding to MAG and to some 19-28K-dalton glycoproteins and sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti-melanoma antibodies cross-react with neural cell adhesion molecule (N-CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti-melanoma antibodies fall into a class of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, anti-human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N-CAM, and several protein and lipid glycoconjugates of the PNS.

Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base.

It was recently established that anti-myelin associated glycoprotein (MAG) IgM paraproteins associated with neuropathy and a substantial number of experimentally produced rat and mouse monoclonal antibodies that react with MAG (e.g. HNK-1) also bind to some sulfated glucuronic acid-containing sphingoglycolipids of human peripheral nerve. A species study revealed that these glycolipids could be detected readily by TLC overlay experiments in the acidic glycolipid fractions from human, monkey, bovine, cat and dog peripheral nerve. The glycolipids were also present in the nerves of rat, mouse, rabbit, guinea pig and chicken, but their concentration was about an order of magnitude lower. These antigenic glycolipids were present in the purified myelin fraction from cat nerve, but their level was not enriched over that in whole homogenate. Partial characterization of the epitopes in the glycolipids was accomplished by comparing binding of the human and experimental monoclonal antibodies to sulfated glucuronyl paragloboside (SGPG), to the desulfated lipid (GPG), and to the methyl ester of the desulfated lipid (MeGPG). All of the human, mouse and rat antibodies reacted with the intact SGPG, but none exhibited binding to MeGPG indicating that either the sulfate or the free car☐yl group on SGPG was required for reactivity. Five out of 11 human IgM paraproteins retained partial and variable reactivity with GPG showing that the sulfate was not absolutely required for binding, while the other 6 did not react with GPG. These results demonstrate idiotypic heterogeneity among the IgM paraproteins. Only 1 of 14 monoclonal antibodies produced experimentally in mice or rats retained reactivity with GPG. The data indicate that the sulfated glucuronic acid moiety of the glycolipid is a critical part of the antigenic determinant for all of the human, mouse and rat monoclonal antibodies tested.

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.

Antibodies to complex glycolipids occur in patients with a variety of diseases of the peripheral nervous system. Many patients with demyelinating neuropathy occurring in association with IgM paraproteinemia have a monoclonal antibody that reacts with a carbohydrate determinant shared between sulfate-3-glucuronyl paragloboside (SGPG), the myelin-associated glycoprotein and other glycoproteins of peripheral nerve. Other patients with neuropathy in association with IgM paraproteinemia have monoclonal antibodies reacting with carbohydrate determinants on various gangliosides. More than 80% of the IgM monoclonal antibodies from patients of this type that have been screened in our laboratory react with SGPG or ganglioside antigens. High levels of antibodies reacting with ganglioside antigens are also found in some patients with inflammatory neuropathies such as Guillain-Barré Syndrome and chronic relapsing inflammatory polyneuropathy. The pathogenetic significance of these antibodies reacting with acidic sphingoglycolipids remains to be established.

The myelin-associated glycoprotein (MAG) was quantitated in the CNS and PNS of quaking mice and the levels compared to the levels of myelin basic protein (MBP) and 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) activity. In the brainstems of 36-day-old quaking mice, MBP, MAG, and CNPase were reduced to 12, 16, and 29% of control levels, respectively. In the sciatic nerves of the 36-day-old quaking mice, MBP and CNPase were 38 and 75% of control levels, respectively, whereas the concentration of MAG was unchanged or slightly increased. Similar quantitative results were obtained for the sciatic nerves and spinal roots of 7-month-old quaking mice. Immunoblots showed that the principal MAG band from the brainstems, sciatic nerves, and spinal roots of the quaking mice had a higher than normal apparent Mr. In addition, there was a minor component reacting with anti-MAG antiserum in the brain-stems of the quaking mice that had a slightly lower Mr than control MAG and was not detected in the normal mice. The results for the quaking mice are compared with those from similar studies on other mutants with dysmyelination of the CNS and PNS.

Specific antibodies have been developed against P1, P2, and P0 myelin proteins and were used to study the localization of these proteins in the rat peripheral nervous system. Both peripheral and central nervous system myelin sheaths contain P1 protein. P0 and P2 proteins are found exclusively in peripheral nervous system myelin sheaths. Antisera to P1 and P0 proteins stain all peripheral nervous system myelin sheaths uniformly. P2 protein is not a component of all peripheral nervous system myelin sheaths. In sheaths that do contain P2 protein, it is concentrated in the area of the Schmidt-Lanterman incisures.

A novel method is described for the biotinylation of immunoglobulins. The procedure relies on the generation of reactive aldehydes on the carbohydrate moieties of the immunoglobulin by oxidation with sodium periodate and subsequent reaction with biotin hydrazide. The method is simple and specific and results in stable conjugates retaining full immunologic activity. It has been applied successfully to a number of mouse monoclonal antibodies of both IgG and IgM classes, and to human IgM preparations. The procedure may also be applied to conjugation of immunoglobulins with fluorescent dyes.

In developing optic nerve and medulla of the rat, the concentration of the myelin-associated glycoprotein (MAG) increases rapidly between 10 and 30 days of age to reach adult levels of 4.4 and 4.1 ng per μg total protein, respectively. The general shapes of the developmental curves for MAG and basic protein (BP) are similar, but the rapid accumulation of MAG progresses earlier than that of BP by about 3–6 days.

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 [ 3 H]fucose of [ 14 C]fucose, and purified myelin fractions were prepared. Proteins were extracted with SDS and separated by polyacrylamide gel electrophoresis. Using double-labeling techniques, the 3 H- and 14 C-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.

The myelin-associated glycoprotein (MAG) and the brain 1B236 protein are 100-kDa glycoproteins containing 30% carbohydrate that exist in two developmentally regulated forms and are specific to the nervous system. Recent cDNA cloning experiments in several laboratories using primarily immunological means of identification have determined the complete primary sequence of a rat brain glycoprotein that seems to correspond to both MAG and 1B236, suggesting that these proteins are identical. However, MAG was previously considered to be an oligodendrocyte/myelin specific component in the CNS at all ages, whereas 1B236 was thought to be primarily a neuronal component in adult rats but synthesized by oligodendrocytes at the time of active myelination. The composite term 1B236/MAG was proposed to describe the molecule identified by the cDNAs. In order to explore further the relationship between MAG and 1B236, as well as their developmentally regulated forms, experiments were carried out on rat samples utilizing synthetic peptides corresponding to sequences throughout the 1B236 molecule, antisera raised to synthetic peptides in the C-terminus of 1B236 that distinguish between the two developmentally regulated forms, and well-characterized polyclonal and monoclonal antibodies raised to purified MAG. Epitope mapping demonstrated that reactive sites were distributed throughout the extracellular and intracellular domains of 1B236/MAG. Only antibodies reacting with the smaller of the two forms of 1B236/MAG detected the glycoprotein in the peripheral nervous system. Both anti-MAG and anti-1B236 antibodies revealed a drastic reduction of the level of 1B236/MAG in 25-day-old myelin-deficient rats and in adult quaking mice, and both types of antibodies revealed a slight shift of 1B236/MAG toward higher apparent Mr in quaking mice as had previously been reported for MAG. The results indicate that MAG and 1B236 are almost certainly identical since they cannot be distinguished immunologically by the reagents available and that quantitatively most of the glycoprotein is associated with oligodendrocytes and myelin rather than neurons at all ages.

A method for the detection and quantitation of glycoproteins on nitrocellulose electroblots is described. Protein mixtures may be solubilized in sodium dodecyl sulfate prior to labeling, which is especially useful when dealing with membrane proteins. Mild periodate oxidation produces aldehydes on the oligosaccharide moieties which are then specifically condensed with biotin aminocaproyl hydrazide. After polyacrylamide gel electrophoresis and transfer of proteins onto nitrocellulose membranes, biotinylated glycoproteins are detected with enzyme-linked streptavidin and quantitated by densitometric scanning. As little as 1 ng of α1-acid glycoprotein can be detected by this method. The use of mild oxidation conditions renders the method highly selective for the detection of sialic acid-containing glycoproteins.

— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [3H]glucosamine or N-[3H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [3H]fucose and [14C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.

1. 1. In vitro biosynthesis of glycoproteins in neuroendocrine tissue was studied by incubating rat pineal glands with L -[3H]fucose and determining trichloroacetic acid-insoluble radioactivity. After 20 h of incubation 67% of the total trichloroacetic acid-insoluble radioactivity was in the gland and 33% had been released into the medium. Essentially all of the 3H-labelled trichloroacetic acid-insoluble material consisted of [3H]fucose-containing glycoproteins. 2. 2. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate showed a heterogeneous pattern of [3H]glycoproteins in the gland (apparent mol. wts 30 000–200 000) with several characteristic peaks. In contrast, 50% of the [3H]glycoproteins in the medium were in a single broad peak (apparent mol. wts. 30 000–70 000). 3. 3. In experiments in which glands were incubated in the presence of [3H]fucose and [14C]leucine, the ratio of 3H to 14C in macromolecules was 7 times higher in the medium than in the gland.

A discontinuous sucrose gradient was used to separate adult rat brain myelin into light, medium and heavy subfractions. Basic proteins decreased sharply, proteolipid potein changed very little, and high molecular weight proteins increased from the light to the heavy fraction. The concentration of monosialoganglioside GM1 was the highest in the middle fraction. The amount of carbohydrate in the major myelin-associated glycoprotein per mg total myelin protein increased 3.5-fold from the light to the heavy fraction. 2′,3′-Cyclic nucleotide 3′-phosphohydrolase, which is related to myelin or the oligodendroglial membrane, and acetylcholinesterase, which is in neural membranes such as the axolemma, both increased between the light and the heavy fraction, although their relative distributions among the three fractions were different. The glycoprotein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase had similar distributions suggesting that they were concentrated in similar locations, possibly in the loose myelin and oligodendroglial plasma membrane. Electron microscopic examination of the subfractions was consistent with this interpretation.