Lectin-binding proteins in central-nervous-system myelin. Detection of glycoproteins of purified myelin on polyacrylamide gels by [3h]concanavalin A binding.

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.     

FRACTIONATION OF GLYCOPROTEINS ASSOCIATED TO ADULT RAT BRAIN MYELIN FRACTIONS

Abstract— The chloroform-methanol insoluble residue of adult rat brain myelin fractions (My-CMI) contains only 20% of protein but all myelin-associated glycoproteins (Z anetta et al ., 1977a). After solubilization in sodium dodecyl sulphate, these glycoproteins were separated by sequential affinity chromatography on 4 immobilized lectins. Ten fractions (9 of which contained only glycoproteins) were obtained. Glycoproteins added up to at least 25% of My-CMI proteins. Many minor glycoproteins were detected in the different fractions. However most of them appeared not to be intrinsic to myelin. On the contrary a major glycoprotein electrophoretic band, component A, appeared to be intrinsic to myelin although its presence also on oligodendrocyte plasma membrane cannot be excluded. Component A was tentatively identified with the'major myelin associated glycoprotein'described by QUARLES (1972, 1973 a, b ). It accounted for less than 0.4% of proteins and 8% of glycoproteins of myelin fractions and consisted of at least two'glycopolypeptides'which, both, bind to concanavalin A and to the Ulex europeus lectin. The other major glycoprotein, component B, did not bind to any of the lectins used and, thus, must have N -acetyl neuraminic acid as only terminal sugar. The separation of myelin-associated glycoproteins according to their affinity for lectins allowed a tentative identification of the lectin binding sites of myelin sheath.     

Lectin-binding proteins in central-nervous-system myelin. Binding of glycoproteins in purified myelin to immobilized lectins

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. [(3)H]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 alpha-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 alpha-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 [(3)H]fucose-labelled glycoproteins from adult myelin and [(14)C]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.     

The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin-Deficient (mld) Mutant Mice

The myelin-deficient (mld) mutation is autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mld. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin-associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low-molecular-mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.     

[3 5S]Sulfate incorporation into myelin glycoproteins I. Central nervous system

The in vivo incorporation of [^{3 5}S]sulfate and [³H]fucose into rat brain myelin was investigated. Most of the ^{3 5}S in the myelin was in sulfatide, but about 4% was associated with the residual proteins after chloroform/methanol extraction. Polyacrylamide gel electrophoresis of these proteins indicated that the major ^{3 5}S-labeled component corresponded to the major fucose-labeled glycoprotein. The labeling of this predominant glycoprotein with sulfate was more selective than with fucose, since there was relatively little incorporation of sulfate into some of the minor fucose-labeled glycoproteins. There was little or no ^{3 5}S associated with proteolipid or basic protein on polyacrylamide gels. The fucose-labeled glycoproteins were converted to glycopeptides by pronase digestion and separated into two major classes by gel filtration on Sephadex-G50. Only the higher molecular weight class contained significant amounts of ^{3 5}S. The association of ^{3 5}S with the glycopeptides was not due to binding of sulfatide or free inorganic sulfate. The results indicate that the predominant myelin-associated glycoprotein in rat brain is sulfated.     

High Expression of the HNK-1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

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.     

ANOMALIES OF MYELIN-ASSOCIATED GLYCOPROTEINS IN''QUAKING MICE

Abstract— 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.     

Demyelination Induced in Aggregating Brain Cell Cultures by a Monoclonal Antibody Against Myelin/Oligodendrocyte Glycoprotein

A monoclonal antibody (8-18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti-MOG antibody in the presence of complement, myelin basic protein (MBP) concentration decreased in a dose-related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase. In the absence of complement, anti-MOG antibody did not induce detectable demyelination. In contrast to the effect of anti-MOG antibody and as expected, anti-MBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side of the myelin membrane, is a good target antigen for antibody-induced demyelination. Indeed, they show that a purified anti-MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin-associated glycoprotein. These observations may have important implications regarding the possible role of anti-MOG antibodies in demyelinating diseases.     

Hypotheses regarding myelination derived from comparisons of myelin subfractions.

Patterns of myelin-associated proteins and glycoproteins in subfractions of central nervous system myelin and changes in these during development have been reviewed. Several hypotheses are put forward regarding classification of these proteins as structural components of myelin, as components of the membranes from which myelin is derived or as molecules present to play a role in myelin formation rather than as true components of compact myelin. 2′, 3′-Cyclic nucleotide 3′-phosphohydrolase is considered to fit into this last category and two different hypothesis are proposed for its role. The major myelin glycoprotein is postulated to serve a function in recognition of axons by oligodendrocytes, a function not needed in the peripheral nervous system where Schwann cells and axons develop in close contact.     

Demonstration of five major glycoproteins in myelin and myelin subfractions.

Myelin was found to contain five major glycoproteins with molecular weights of 120000, 95000, 88000, 43000 and 38000. Light myelin contained only 5-7% of the amount of these glycoproteins in whole myelin, whereas heavy myelin and the membrane fraction contained amounts nearly identical with whole myelin. Since all the major and minor glycoproteins, with the exception of 120000-mol-wt. glycoprotein, were detected only after treating the myelin membrane with neuraminidase, N-acetylneuraminic acid is a terminal sugar residue in these glycoproteins.     

External labeling of galactose in surface membrane glycoproteins of the intact myelin sheath.

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.     

Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain.

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— 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.     

Appearance of Myelin proteins during vertebrate evolution

Myelin, defined as an arrangement of spirally fused unit membranes, is an acquisition of vertebrates and first appeared during evolution in Gnathostomata. In all species studied PNS and CNS myelins contain the myelin-associated glycoprotein (MAG) and the myelin basic protein (MBP). Throughout phylogeny PNS myelin is characterized by the major P₀ glycoprotein which is called IP in fishes. The PNS myelin proteins did not evolve further except for the addition of P₂ protein from reptiles onward. In Elasmobranchii and Chondrostei, PNS and CNS myelin proteins are similar. CNS myelin of actinopterygian fishes possesses a 36,000 Da protein (36K) in addition to P₀-like IP glycoproteins. In tetrapod CNS myelin, P₀ is replaced by the proteolipid protein (PLP) and the Wolfgram protein (WP). Of particular interest in a transitional phylogenetic sense are the lungfish Protopterus, carrying glycosylated PLP (g-PLP) but no P₀, 36K or WP, and the bichir Polypterus, showing simultaneous presence of P₀, 36K and PLP.

These results indicate that myelin proteins could be valuable molecular markers in establishing vertebrate phylogenetic relationships and in reconstructing the fish-tetrapod transition.     

Nogo on the go

Growth inhibition in the central nervous system (CNS) is a major barrier to axon regeneration. Recent findings indicate that three distinct myelin proteins, myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp), inhibit axon growth by binding a common receptor, the Nogo66 receptor (NgR), and likely converge on a common signaling cascade.     

Myelin-Associated Glycoprotein and Related Glycoconjugates in Developing Cat Peripheral Nerve: A Correlative Biochemical and Morphometric Study

The expression and accumulation of the myelin-associated glycoprotein (MAG) and other glycoconjugates have been studied during myelination in the developing cat peripheral nervous system. The glycoconjugates studied have in common a similar carbohydrate determinant which is bound by many antibodies, including the mouse monoclonal antibody HNK-1, and human IgM paraproteins from patients with neuropathy. In addition to MAG, the reactive glycoconjugates include a 60-kilodalton (kD) glycoprotein and a group of 20-26 kD glycoproteins, as well as a group of recently identified acidic glycolipids, the major one of which is sulfate-3-glucuronyl paragloboside (SGPG). The accumulation of these glycoproteins and glycolipids is compared with the established myelin proteins P0, P1, and P2 and with morphometric indices of myelin volume and axonal perimeter. The study demonstrates that MAG appears and accumulates very early during myelination, being present at 15% of the maximum level prior to the appearance of P0, and at 80% of the maximum level when P0 is at 30% of its maximum level. In the adult, the level of MAG falls to 60% maximum. The 60 kD and 20-26 kD glycoproteins accumulate at the same time as or later than P0, suggesting that they are either compact myelin proteins or in membranes closely associated with compact myelin. SGPG accumulates with P0 early in myelination, but falls to 60% of maximum in the adult. By comparing biochemical and morphometric data, we demonstrate that P0 and other compact myelin proteins accumulate synchronously with the increase in myelin area. MAG accumulation, however, is closely related to changes in axonal perimeter, consistent with a predominant localization of MAG to the periaxonal membranes in the peripheral nervous system.     

GLIAL VERSUS NEURONAL ORIGIN OF MYELIN PROTEINS AND GLYCOPROTEINS STUDIED BY COMBINED INTRAOCULAR AND INTRACRANIAL LABELLING

Abstract— —The contribution of axonal transport to the production of myelin proteins and glycoproteins was investigated using the double labelling technique of combined intraocular and intracerebral injections in the same animal. Myelin and an axolemma-enriched fraction were isolated from pooled optic nerves, chiasma and optic tracts. Separation by gel electrophoresis showed that typical myelin proteins and glycoproteins were only significantly labelled by intracerebral injection. Intraocular injection labelled high molecular weight proteins other than the major Wolfgram protein and the major myelin glycoprotein. Fifteen days after intraocular injection the label was concentrated in a high molecular weight protein which migrated slightly more slowly than the major Wolfgram protein. The pattern of proteins and glycoproteins in myelin labelled by intraocular injection was very similar to that obtained in the axolemma-enriched fraction by the same route. These results indicate that neuronal metabolism and axonal transport do not contribute significantly to the synthesis of specific myelin proteins and glycoproteins, but suggest that the components of myelin fractions which are labelled by intraocular injection are contaminants of axolemmal origin. One of these glycoproteins may prove a useful marker of axolemma membranes.     

Developmental regulation of the carbohydrate composition of glycoproteins associated with central nervous system myelin

Abstract: Glycoproteins from central nervous system myelin were evaluated for developmental alterations in their carbohydrate composition by autoradiographic analysis of radioiodinated lectin binding after separation by high-resolution sodium dodecyl sulfate-pore gradient slab gel electrophoresis (SDS-PGE). Sixteen lectin-binding components were assessed in highly purified myelin preparations from 15-day, 18-day, and adult rat brains, using the lectins Triticum vulgaris (wheat germ agglutinin) and Ulex europeus (gorse agglutinin I). Developmental changes in lectin binding for individual glycoproteins were evaluated semiquantitatively by comparing densitometric scans of the auto radiographs. Both increases and decreases in lectin binding for individual components were observed as a consequence of development, as well as the appearance and disappearance of lectin binding to three low-molecular-weight components. No changes in electrophoretic mobility and hence glycoprotein molecular weight were observed in any components when using these lectins. These developmental changes in lectin binding suggest that increases in glycoprotein (receptor) density occur, as well as an elaboration of oligosaccharide branching for individual glycoproteins. In addition, the appearance of a new glycoprotein in the adult myelin membrane could imply a new functional role not present in the immature membrane. These observations suggest that dynamic alterations of myelin-associated glycoproteins occur during development. Such developmental regulation of membrane glycoproteins increases the significance of their potential role in myelination and myelin maintenance.     

Distribution of the myelin-associated glycoprotein and P0 protein during myelin compaction in quaking mouse peripheral nerve

Ultrastructural studies have shown that during early stages of Schwann cell myelination mesaxon membranes are converted to compact myelin lamellae. The distinct changes that occur in the spacing of these Schwann cell membranes are likely to be mediated by the redistribution of (a) the myelin-associated glycoprotein, a major structural protein of mesaxon membranes; and (b) P0 protein, the major structural protein of compact myelin. To test this hypothesis, the immunocytochemical distribution of these two proteins was determined in serial 1-micron-thick Epon sections of ventral roots from quaking mice and compared to the ultrastructure of identical areas in an adjacent thin section. Ventral roots of this hypomyelinating mouse mutant were studied because many fibers have a deficit in converting mesaxon membranes to compact myelin. The results indicated that conversion of mesaxon membranes to compact myelin involves the insertion of P0 protein into and the removal of the myelin-associated glycoprotein from mesaxon membranes. The failure of some quaking mouse Schwann cells to form compact myelin appears to result from an inability to remove the myelin-associated glycoprotein from their mesaxon membranes.     

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.