Myelin-associated Glycoprotein Isoforms Research Articles

High-affinity heterotetramer formation between the large myelin-associated glycoprotein and the dynein light chain DYNLL1.

The close association of myelinated axons and their myelin sheaths involves numerous intercellular molecular interactions. For example, myelin-associated glycoprotein (MAG) mediates myelin-to-axon adhesion and signalling via molecules on the axonal surface. However, knowledge about intracellular binding partners of myelin proteins, including MAG, has remained limited. The two splice isoforms of MAG, S- and L-MAG, display distinct cytoplasmic domains and spatiotemporal expression profiles. We used yeast two-hybrid screening to identify interaction partners of L-MAG and found the dynein light chain DYNLL1 (also termed dynein light chain 8). DYNLL1 homodimers are known to facilitate dimerization of target proteins. L-MAG and DYNLL1 associate with high affinity, as confirmed with recombinant proteins invitro. Structural analyses of the purified complex indicate that the DYNLL1-binding segment is localized close to the L-MAG C terminus, next to the Fyn kinase Tyr phosphorylation site. The crystal structure of the complex between DYNLL1 and its binding segment on L-MAG shows 2:2 binding in a parallel arrangement, indicating a heterotetrameric complex. The homology between L-MAG and previously characterized DYNLL1-ligands is limited, and some details of binding site interactions are unique for L-MAG. The structure of the complex between the entire L-MAG cytoplasmic domain and DYNLL1, as well as that of the extracellular domain of MAG, were modelled based on small-angle X-ray scattering data, allowing structural insights into L-MAG interactions on both membrane surfaces. Our data imply that DYNLL1 dimerizes L-MAG, but not S-MAG, through the formation of a specific 2:2 heterotetramer. This arrangement is likely to affect, in an isoform-specific manner, the functions of MAG in adhesion and myelin-to-axon signalling. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page712.

N‐CAM Dysfunction and Unexpected Accumulation of PSA‐NCAM in Brain of Adult‐Onset Autosomal‐Dominant Leukodystrophy

Previously, myelin from cerebral white matter (CWM) of two subjects of a family with orthochromatic adult-onset autosomal-dominant leukodystrophy (ADLD) was disclosed to exhibit defective large isoform of myelin-associated glycoprotein (L-MAG) and patchy distribution only in the elder subject. L-MAG and neural cell adhesion molecule (N-CAM) (N-CAM 180, 140, and 120) are structurally related and concur to myelin/axon interaction. In early developmental stages, in neurons and glia N-CAM is converted into polysialylated (PSA)-NCAM by two sialyltransferases sialyltransferase-X (STX) and polysialyltransferase-1 (PST). Notably, PSA-NCAM disrupts N-CAM adhesive properties and is nearly absent in the adult brain. Here, CWM extracts and myelin of the two subjects were searched for the expression pattern of the N-CAM isoforms and PSA-NCAM, and their CWM was evaluated for N-CAM, STX and PST gene copy number and gene expression as mRNA. Biochemically, we disclosed that in CWM extracts and myelin from both subjects, PSA-NCAM accumulates, N-CAM 180 considerably increases, N-CAM 140 is modestly modified and N-CAM 120 remarkably decreases; duplication of genes encoding N-CAM, STX and PST was not revealed, whereas PST mRNA was clearly increased. Immunohistochemically, in CWM of both subjects, we found an unusually diffuse accumulation of PSA-NCAM without inflammation markers. PSA-NCAM persistence, up-regulated PST mRNA and previously uncovered defective L-MAG may be early pathogenetic events in this ADLD form.

The role of glycosphingolipid metabolism in the developing brain

Glycosphingolipids (GSLs) are amphipathic lipids ubiquitously expressed in all vertebrate cells and body fluids, but they are especially abundant in the nervous system. The synthesis of GSLs generally is initiated in the endoplasmic reticulum and completed in the Golgi apparatus, followed by transportation to the plasma membrane surface as an integral component. The amount and expression patterns of GSLs change drastically in brains during the embryonic to postnatal stages. Recent studies have revealed that GSLs are highly localized in cell surface microdomains and function as important components that mediate signal transduction and cell adhesion. Also in developing brains, GSLs are suggested to play important roles in nervous system formation. Disturbance of GSL expression and metabolism affects brain function, resulting in a variety of diseases, particularly lysosomal storage diseases. In this review, we describe some aspects of the roles of GSLs, especially of gangliosides, in brain development.

Unraveling the differential expression of the two isoforms of myelin-associated glycoprotein in a mouse expressing GFP-tagged S-MAG specifically regulated and targeted into the different myelin compartments

The two myelin-associated glycoprotein (MAG) isoforms are cell adhesion molecules that differ only in their cytoplasmic domains, but their specific roles are not well understood. In this study, we present a transgenic mouse line that specifically expresses GFP-tagged S-MAG correctly regulated and targeted into the myelin sheath allowing the specific discrimination of L- and S-MAG on the subcellular level. Here, we describe the differential expression pattern and spatial distribution of L- and S-MAG during development as well as in the adult central and peripheral nervous system. In peripheral nerves, where S-MAG is the sole isoform, we observed S-MAG concentrated in different ring-like structures such as periaxonal and abaxonal rings, and discs spanning through the compact myelin sheath perpendicular to the axon. In summary, our data provide new insight in the subcellular distribution of the two isoforms fundamental for the understanding of their specific functions in myelin formation and maintenance.

Myelin proteolipid protein, basic protein, the small isoform of myelin-associated glycoprotein, and p42MAPK are associated in the Triton X-100 extract of central nervous system myelin.

To further our understanding of the functions of the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), and other myelin proteins, such as 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG), bovine brain myelin was extracted with Triton X-100, and protein complexes in the detergent-soluble fraction were isolated by coimmunoprecipitation and sucrose density gradient sedimentation. MBP, PLP, and the small isoform of MAG (S-MAG) were coimmunoprecipitated from the detergent-soluble fraction by anti-PLP, anti-MBP or anti-MAG monoclonal antibodies. Additionally, a 30 kDa phosphoserine-containing protein and two phosphotyrosine-containing proteins (M(r) 30 and 42 kDa) were found in the coimmunoprecipitates. The 42 kDa protein is probably p42MAPK, in that MAPK was shown also to be present in the immunoprecipitated complex. CNP, the small PLP isoform DM20, the large MAG isoform L-MAG, MOG, CD44, MEK, p44MAPK, and actin were not present in the immunoprecipitates, although they were present in the detergent-soluble fraction. Lipid analysis revealed that the PLP-MBP-S-MAG coimmunoprecipitated with some phospholipids and sulfatide but not cholesterol or galactosylceramide. However, the complex had a high density, indicating that the lipid/protein ratio is low, and it was retained on a Sepharose CL6B column, indicating that it is not a large membrane fragment. Given that MAG is localized mainly in the periaxonal region of myelin, where it interacts with axonal ligands, the PLP-MBP-S-MAG complex may come from these regions, where it could participate in dynamic functions in the myelin sheath and myelin-axonal interactions.

L-isoform but not S-isoform of myelin associated glycoprotein promotes neurite outgrowth of mouse cerebellar neurons

Myelin associated glycoprotein (MAG) has growth promoting effect on mouse cerebellar neurons. In the present study, we examined which isoform of MAG has the effect. cDNA for L-MAG and S-MAG was stably transfected into BALB/c 3T3 cells, on which cerebellar neurons were cultured. The neurons were stained with antibody against microtubule-associated protein-2. Neurites of the neurons cultured on cells expressing L-MAG extended significantly further than those cultured on cells expressing S-MAG or on control cells. Therefore, intracellular domain of MAG may have the potential to affect MAG-neurite interaction.

Selective myelin defects in the anterior medullary velum of thetaiep mutant rat

The taiep rat is a myelin mutant in which initial hypomyelination is followed by progressive demyelination of the CNS. An in vitro study suggests that accumulation of microtubules within oligodendrocytes is the cause of the taiep myelin defects (Song et al., 1999). In this article, we analyze microtubule accumulation in relation to taiep myelin defects in vivo in the anterior medullary velum (AMV), a CNS tissue that enables entire oligodendrocyte units to be resolved. Immunohistochemical analysis demonstrated notably high levels of beta-tubulin and the microtubule associated protein tau in the somata and processes of taiep oligodendrocytes. This was correlated with markedly reduced expression of the myelin proteins, proteolipid protein (PLP), myelin basic protein (MBP), 2',3 -cyclic nucleotide 3'-phosphodiesterase, and both large (L) and small (S) isoforms of myelin-associated glycoprotein (MAG). Moreover, PLP and L-MAG, which are dependent on the microtubule system for intracellular transport, accumulated in the perinuclear cytoplasm of the taiep oligodendrocyte. The myelin deficit was most marked in the area of the AMV populated by the small somata oligodendrocytes that have fine long processes that support numerous myelin sheaths of small diameter axons. Type III/IV oligodendrocytes, which have large somata and short processes that support a small number of myelin sheaths of large diameter axons, were also affected to a certain degree in compact myelin sheath formation. These results support the hypothesis that myelin loss and oligodendrocyte disruption in the taiep mutant result from a defect in the microtubule system that transports myelin components from the somata to the myelin sheath.

Calcium-dependent interaction between the large myelin-associated glycoprotein and S100beta.

The myelin-associated glycoprotein is a transmembrane cell adhesion molecule expressed by myelinating glial cells of the nervous system. So far, only protein kinases have been reported to interact with the cytoplasmic domains of the two isoforms of the myelin-associated glycoprotein. We report here the identification of the first nonkinase intracellular ligand for the large isoform of the myelin-associated glycoprotein as the S100beta protein. The interaction is dependent on the presence of calcium. We have also localized the S100beta-binding site in the cytoplasmic domain specific to the large myelin-associated glycoprotein isoform to a putative basic amphipathic alpha-helix. A synthetic peptide corresponding to this region bound to S100beta in a calcium-dependent manner with a stoichiometric ratio of 1:1 (K(D) approximately 7 microM). We suggest that the observed interaction may play a role in the regulation of the myelinating glial cell cytoskeleton and the divalent cation-dependent signal transduction events during myelin formation and maintenance.

Demyelination and altered expression of myelin-associated glycoprotein isoforms in the central nervous system of galactolipid-deficient mice.

Vertebrate myelin is enriched in the lipid galactocerebroside (GalC) and its sulfated derivated sulfatide. To understand the in vivo function of these lipids, we analyzed myelination in mice that contain a null mutation in the gene encoding UDP-galactose:ceramide galactosyltransferase, the enzyme responsible for catalyzing the final step in GalC synthesis. Galactolipid-deficient myelin is regionally unstable and progressively degenerates. At postnatal day 30, demyelination is restricted to the midbrain and hindbrain, but by postnatal day 90, it spreads throughout the central nervous system. Activated microglial cells and reactive astrocytes appear with the loss of myelin in older animals. Nonetheless, major myelin protein gene mRNA levels are normal throughout the life of these animals, suggesting that widespread oligodendrocyte death is not the primary cause of demyelination. The developmental switch in myelin-associated glycoprotein isoform expression, however, does not occur normally in these mice, suggesting an alteration in oligodendrocyte maturation. Taken together, these findings indicate that GalC and sulfatide are required for the long-term maintenance of myelin and that their absence may have subtle effects on the development of oligodendrocytes.

Evidence against altered forms of MAG in the dysmyelinated mouse mutant claw paw.

Evidence against altered forms of MAG in the dysmyelinated mouse mutant claw paw.

The Cytoplasmic Domain of the Large Myelin-Associated Glycoprotein Isoform Is Needed for Proper CNS But Not Peripheral Nervous System Myelination

The myelin-associated glycoprotein (MAG) is a member of the immunoglobulin gene superfamily and is thought to play a critical role in the interaction of myelinating glial cells with the axon. Myelin from mutant mice incapable of expressing MAG displays various subtle abnormalities in the CNS and degenerates with age in the peripheral nervous system (PNS). Two distinct isoforms, large MAG (L-MAG) and small MAG (S-MAG), are produced through the alternative splicing of the primary MAG transcript. The cytoplasmic domain of L-MAG contains a unique phosphorylation site and has been shown to associate with the fyn tyrosine kinase. Moreover, L-MAG is expressed abundantly early in the myelination process, possibly indicating an important role in the initial stages of myelination. We have adapted the gene-targeting approach in embryonic stem cells to generate mutant mice that express a truncated form of the L-MAG isoform, eliminating the unique portion of its cytoplasmic domain, but that continue to express S-MAG. Similar to the total MAG knockouts, these animals do not express an overt clinical phenotype. CNS myelin of the L-MAG mutant mice displays most of the pathological abnormalities reported for the total MAG knockouts. In contrast to the null MAG mutants, however, PNS axons and myelin of older L-MAG mutant animals do not degenerate, indicating that S-MAG is sufficient to maintain PNS integrity. These observations demonstrate a differential role of the L-MAG isoform in CNS and PNS myelin.

The Cytoplasmic Domain of the Large Myelin-Associated Glycoprotein Isoform Is Needed for Proper CNS But Not Peripheral Nervous System Myelination

The Cytoplasmic Domain of the Large Myelin-Associated Glycoprotein Isoform Is Needed for Proper CNS But Not Peripheral Nervous System Myelination

128 Amino acid dimorphism of the small myelin-associated glycoprotein isoform in the peripheral nervous system

International Journal of Developmental NeuroscienceVolume 14, Issue S1 p. 81-81 Article 128 Amino acid dimorphism of the small myelin-associated glycoprotein isoform in the peripheral nervous system Petri Kursula, Petri KursulaSearch for more papers by this authorVeli-Pekka Lehto, Veli-Pekka LehtoSearch for more papers by this authorAnthony M. Heape, Anthony M. HeapeSearch for more papers by this author Petri Kursula, Petri KursulaSearch for more papers by this authorVeli-Pekka Lehto, Veli-Pekka LehtoSearch for more papers by this authorAnthony M. Heape, Anthony M. HeapeSearch for more papers by this author First published: July-August 1996 https://doi.org/10.1016/0736-5748(96)80318-2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume14, IssueS111th Biennial Meeting of the International Society for Developmental NeuroscienceJuly-August 1996Pages 81-81 RelatedInformation

Differential intracellular sorting of the myelin-associated glycoprotein isoforms.

Myelin-associated glycoprotein (MAG), a myelin-specific protein, is expressed as two isoforms, designated as L-MAG and S-MAG. Both share identical extracellular and transmembrane domains but differ in their cytoplasmic domains. L-MAG is expressed earlier during myelination than S-MAG. These features, as well as others, suggest that the isoforms have different functions. To confirm this hypothesis, both isoforms were expressed transiently and stably in Madin-Darby canine kidney (MDCK) epithelial cells, and the localization of the isoforms was studied. In both transiently and stably transfected cells, L-MAG sorted primarily to the basolateral membrane. In single transfected cells, S-MAG sorted primarily to the apical membrane. When groups of adjacent cells became transiently transfected, S-MAG accumulated at areas of cell-cell contact within the basolateral membrane. In stably transfected cells S-MAG sorted to the basolateral membrane. The data suggest that L-MAG contains an invariable basolateral sorting signal, but that the sorting of S-MAG is dependent upon extrinsic factors, such as coexpression by adjacent (contacting) cells. As MDCK cells sort the MAG isoforms differently, these data support the hypothesis that the MAG isoforms do perform different functions.

The myelin-associated glycoprotein of the peripheral nervous system in trembler mutants contains increased alpha 2-3 sialic acid and galactose.

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 myelin‐associated glycoprotein of the peripheral nervous system in trembler mutants contains increased α2–3 sialic acid and galactose

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 α2–3 linked sialic acid, most glycoproteins of both control and trembler nerve contained primarily α2–6 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 α2–3 linked sialic acid and galactose. The abnormal glycosylation of MAG in trembler mutants may contribute to the myelin pathology. © 1996 Wiley-Liss, Inc.

Limited postnatal ethanol exposure permanently alters the expression of mRNAS encoding myelin basic protein and myelin-associated glycoprotein in cerebellum.

Experiments were designed to test the hypothesis that ethanol exposure during development can selectively affect the expression of specific isoforms of myelin protein gene expression in the rat cerebellum. We focused on myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) gene expression. Both of these genes are alternatively spliced to yield 4 (MBP) or 2 (MAG) mRNA isoforms. Prenatal ethanol exposure, delivered to the dams in a liquid diet, did not significantly alter the expression of MBP or MAG gene expression in the cerebellums of 15-day-old pups, as measured by quantitative in situ hybridization using specific oligodeoxynucleotide probes. In contrast, postnatal ethanol exposure delivered directly to the pups over a 6-day period by gastrostomy tube (PN days 4-10) reduced the expression of specific MBP and MAG isoforms in the cerebellum of animals in adulthood. These data demonstrate that ethanol exposure, especially during the period of rapid myelination, has selective effects on mRNA isoforms encoding specific MBPs and MAG.

Expression of myelin-associated glycoprotein isoforms after sciatic nerve crush injury in mice.

The large myelin-associated glycoprotein isoform (L-MAG) protein and small myelin-associated glycoprotein isoform (S-MAG) protein were demonstrated after sciatic nerve crush injury in mice by an immunoblotting technique using specific antibodies to the L-MAG protein and the S-MAG protein, respectively. Immunoblots indicated a rapid decrease in expression of both isoform proteins in the crushed sciatic nerves to < 10% of the control side. By 13 d after injury, L-MAG protein expression had quickly recovered to 100% of the control level. Following the increase in L-MAG protein expression, S-MAG protein expression recovered to 100% by 20 d after injury. It has been reported that the developmental maximum expression of L-MAG protein precedes that of S-MAG protein in both central and peripheral nervous system (CNS and PNS). Our previous work demonstrated that L-MAG mRNA was characteristically induced at the time of most active myelination, including remyelination in the CNS. We here have shown the expression of L-MAG protein precedes that of S-MAG protein during active remyelination in the PNS. This suggests that it plays an important role in the early stage of myelin formation.

Myelin-associated glycoprotein is phosphorylated by protein kinase C.

The myelin-associated glycoprotein (MAG) is a neural recognition molecule involved in heterophilic interactions between myelin-forming cells and neurons. To characterize the molecular mechanisms underlying post-translational modifications which may be instrumental in signal transduction following the recognition event, we have studied the stimuli leading to modification of 32P-orthophosphate incorporation into MAG in cultures of oligodendrocytes or transformed differentiated Schwann cells. Here we show that in oligodendrocytes both the 67 and 72 kD isoforms of MAG were phosphorylated exclusively on serine, while in the transformed Schwann cells only the 67 kD isoform was found to be present and phosphorylated. The phorbol ester phorbol-12-myristoyl-13-acetate (PMA) did not affect biosynthesis of the protein backbone, but enhanced incorporation of phosphate by a factor of 2-3, indicating the involvement of protein kinase C. Exclusive phosphorylation of serine residues was also observed, when purified MAG was incubated with protein kinase C in the presence of [gamma-32P]ATP. In searching for the physiological stimuli which may trigger phosphorylation of MAG, cultures of oligodendrocytes were exposed to extracellular signals, such as coculture with dorsal root ganglion and spinal cord neurons carrying the MAG receptor, to membrane fractions of these neurons, monoclonal MAG antibody 513 binding to the recognition site of MAG, or platelet-derived growth factor. None of these additives modified the phosphorylation of MAG. These observations point to the possibility that phosphorylation of MAG is controlled by yet unknown intracellular cues rather than by extracellular signals interacting with cell surface receptors of oligodendrocytes.

Expression of the large myelin-associated glycoprotein isoform in rat oligodendrocytes around cerebral infarcts.

An immunohistochemical method that uses two specific antisera-distinguishable myelin-associated glycoprotein (MAG) isoforms showed an expression of large MAG isoform (L-MAG) protein in the oligodendrocyte cytoplasms in the white matter around experimental cerebral infarcts produced by occlusion of the left middle cerebral artery in the rat. L-MAG protein also was detected in the white matter on the contralateral side. This protein appears prior to S-MAG protein in myelination and remyelination in the central and peripheral nervous systems, and is believed to function in myelin formation. Because L-MAG protein has been found in the oligodendrocyte cytoplasm only in the early development period, its appearance in this cytoplasm after ischemic insult is evidence of MAG regeneration.