Myelin-associated glycoprotein (MAG, Siglec-4) is unique among the siglecs in that it is expressed exclusively on neuronal glial cells. It is the most highly conserved among the siglecs in mammalian species. This siglec paradigm is unique in its activity of stabilizing axon-myelin interactions. MAG has a cytoplasmic domain that is devoid of ITIMs, but contains a tyrosine-based motif associated with binding the FYN tyrosine kinase, believed to play a role in its activity in myelin-axon interactions. MAG recognizes as ligands sialoside sequences found on gangliosides that are abundant in axonal membranes. It is one of several proteins in myelin that negatively regulate axon outgrowth following tissue injury, an activity that involves MAG-ligand interactions. Evidence suggests that inhibition of MAG-ligand interactions may enhance neurite outgrowth and repair of injured neurons.
CFG Participating Investigators contributing to the understanding of this paradigm
Several CFG Participating Investigators (PIs) have contributed to identification of MAG as a siglec and to understanding the functions of MAG, including: Paul Crocker, Sørge Kelm, James Paulson, Ronald Schnaar
Progress toward understanding this GBP paradigm
This section documents what is currently known about MAG, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Pages for human and mouse MAG (a.k.a. Siglec-4a) in the CFG database.
on glycolipids and/or glycoproteins
Specificity for linkage of sialic acid to underlying Gal:
about 10-fold better binding to Neu5Acα2,3Gal-R than Neu5Acα2,6Gal-R
Underlying glycan structures can enhance binding:
Enhanced binding through additional internal sialic acids:
higher binding to
Mouse knockout experiments have implicated MAG-binding brain gangliosides GD1a and/or GT1b as MAG ligands (see "Biosynthesis of Ligands" below).
Cellular expression of GBP and ligands
MAG (Siglec-4) is expressed exclusively on myelin, which is produced by oligodendrocytes (the myelinating cells of the central nervous system) and Schwann cells (the myelinating cells of the peripheral nervous system). In both central and peripheral nervous systems, MAG is enriched on the innermost wrap of myelin, directly apposing the axon surface.
MAG recognizes as ligands sialoside sequences found on gangliosides that are abundant in axonal membranes.
Biosynthesis of ligands
Mice null for the ganglioside-specific N-acetylgalactosaminyltransferase gene B4galnt1 (GM2/GD2 synthase) have similar nervous system phenotypic deficits as MAG-null mice (see "Biological roles of GBP-ligand interaction" below). These data implicate MAG-binding brain gangliosides GD1a and/or GT1b as MAG ligands..
Siglec-4 is a heavily glycosylated protein of about 100kDa with 30% of its mass being made up by carbohydrates distributed over eight glycosylation sites. The extracellular part of Siglec-4 consists of five Ig-like domains (one V-set domain and four C2-set domains). Two splice variants for Siglec-4 are found in mammals, L-MAG (72kDa) and S-MAG (67kDa), which differ in their cytoplasmic domain. L-MAG contains a tyrosine phosphorylation site that is missing in S-MAG.
Biological roles of GBP-ligand interaction
MAG is expressed on the innermost myelin membrane wrap, directly apposed to the axon surface. Although it is not required for myelination, MAG enhances long-term axon survival, helps structure myelin gaps (nodes of Ranvier) essential for rapid nerve conduction, regulates the axon cytoskeleton and protects axons from acute toxic insults. In addition to its role in axon-myelin stabilization, MAG inhibits axon regeneration after injury; MAG on residual myelin membranes at injury sites actively signals axons to halt elongation. Whether MAG's stabilizing effects and its inhibition of axon regeneration are part of the same signaling system is under investigation.
MAG has multiple receptors on the axon surface, including gangliosides GD1a/GT1b, the GPI-anchored Nogo receptors (NgR1 and NgR2), and transmembrane proteins PirB and β-integrin. Some of these interactions involve MAG's glycan binding capability, while others may not. The following biological roles of MAG have been experimentally linked to its glycan binding activity using genetic, biochemical, and/or pharmacological criteria:
1. Long term axon stabilization: B4galnt1-null mice, which lack the termini of complex gangliosides, display the same progressive axon degeneration phenotype as Mag-null mice. Double null mice (B4galnt1, Mag) are similar. 
2. Nodes of Ranvier: B4galnt1-null and Mag-null mice have similar deficits in the structures of Nodes of Ranvier
3. Cytoskeletal organization: B4galnt1-null, Mag-null and double-null mice have similarly reduced neurofilament spacing and reduced axon diameter.
4. Axon protection: MAG-mediated protection of axons from toxic insults is diminished in B4galnt1-null mice or after treatment of axons with sialidase.
5. Regulating axon regeneration: MAG-mediated inhibition of axon regeneration is diminished in B4galnt1-null mice, after treatment with sialidase, or by addition of MAG-binding soluble glycans.
MAG signaling is bidirectional, into the myelinating cells and into myelin-ensheathed axons. Signaling into myelinating cells may involve tyrosine phosphorylation of the MAG intracellular domain downstream of ligand engagement, whereas signals into the axon are likely to involve activation of the small non-receptor GTPase RhoA.
CFG resources used in investigations
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for Siglec-4.
Probes for mouse and human MAG (under the name Siglec-4) have been included on all four versions of the CFG glycogene microarray.
Knockout mouse lines
The CFG has phenotyped the MAG-deficient mouse.
Investigators have used CFG carbohydrate compounds to study MAG ligand specificity.
Compared to other Siglecs, Siglec-4 is most conserved. Based on sequence similarity orthologous proteins can be identified in all vertebrate genomes available so far (several mammals, chicken, Xenopus, zebrafish and fugu). Sialic acid binding activity selective for 2,3-linked Sia has been shown for the avian ortholog (SMP and fish Siglec-4 from zebrafish (Danio rerio) and fugu (Takifugu rubripes)). Whereas the primary sequences of the Sia-binding N-terminal domains is 97 % identical between rodents and man and share over 50 % sequence identity between fish and mammals, the cytoplasmic tail is much less conserved (20% identical amino acids between fish and mammals).
- ↑ Crocker, P. R., Paulson, J. C. & Varki, A. Siglecs and their roles in the immune system. Nat Rev Immunol 7, 255-266 (2007).
- ↑ 2.0 2.1 2.2 Schnaar, R. L. Brain gangliosides in axon-myelin stability and axon regeneration. FEBS Lett (2009).
- ↑ Yang, L. J. et al. Sialidase enhances spinal axon outgrowth in vivo. Proc Natl Acad Sci U S A 103, 11057-11062 (2006).
- ↑ Mountney A., Zahner M.R., Lorenzini I., Oudega M., Schramm L.P., Schnaar R.L. Sialidase enhances recovery from spinal cord contusion injury. Proc Natl Acad Sci U S A 107, 11561-11566, 2010
- ↑ 5.0 5.1 Vyas, A. A., Blixt, O., Paulson, J. C. & Schnaar, R. L. Potent glycan inhibitors of myelin-associated glycoprotein enhance axon outgrowth in vitro. J Biol Chem 280, 16305-16310 (2005).
- ↑ Kelm, S. et al. Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily. Curr Biol. 4, 965-972 (1994)
- ↑ Collins, B. E., Kiso, M., Hasegawa, A., Tropak, M. B., Roder, J. C., Crocker, P. R., Schnaar, R. L. Binding specificities of the sialoadhesin family of I-type lectins. Sialic acid linkage and substructure requirements for binding of myelin-associated glycoprotein, Schwann cell myelin protein, and sialoadhesin. J Biol Chem. 272, 16889-16895 (1997)
- ↑ Collins, B. E., Yang, L. J., Mukhopadhyay, G., Filbin, M. T., Kiso, M., Hasegawa, A., Schnaar, R.L. Sialic acid specificity of myelin-associated glycoprotein binding. J Biol Chem. 272, 1248-1255 (1997)
- ↑ 9.0 9.1 Strenge, K., Schauer, R., Bovin, N., Hasegawa, A., Ishida, H., Kiso, M., Kelm, S. Glycan specificity of myelin-associated glycoprotein and sialoadhesin deduced from interactions with synthetic oligosaccharides. Eur J Biochem. 258, 677-685 (1998)
- ↑ Blixt, O., Collins, B. E., van den Nieuwenhof, I. M., Crocker, P. R., Paulson, J. C. Sialoside specificity of the siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelin-associated glycoprotein. J Biol Chem. 278, 31007-31019 (2003)
- ↑ Yang, L. J., Zeller, C.B., Shaper, N.L., Kiso, M., Hasegawa, A., Shapiro, R.E., Schnaar, R.L. Gangliosides are neuronal ligands for myelin-associated glycoprotein. Proc. Natl. Acad. Sci. USA 93, 814-818 (1996).
- ↑ 12.0 12.1 Collins, B.E., Ito, H., Sawada, N., Ishida, H., Kiso, M., Schnaar, R.L. Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs. J Biol Chem. 274, 37637-37643 (1999)
- ↑ 13.0 13.1 Quarles RH. Myelin-associated glycoprotein (MAG): past, present and beyond. J Neurochem. 100, 1431-1448 (2007).
- ↑ 14.0 14.1 14.2 Pan, B., Fromholt, S.E., Hess, E.J., Crawford, T.O., Griffin, J.W., Sheikh, K.A., Schnaar, R.L. Myelin-associated glycoprotein and gangliosides mediate axon-myelin stability: Neuropathology and behavioral deficits in single- and double-null mice. Exp. Neurol. 195, 208-217 (2005)
- ↑ 15.0 15.1 Umemori, H., Sato, S., Yagi, T., Aizawa, S., Yamamoto, T. Initial events of myelination involve Fyn tyrosine kinase signalling. Nature 367, 572-576 (1994)
- ↑ 16.0 16.1 Jaramillo, M. L., Afar, D. E., Almazan, G., Bell, J. C. Identification of tyrosine 620 as the major phosphorylation site of myelin-associated glycoprotein and its implication in interacting with signaling molecules. J Biol Chem. 269, 27240-27245 (1994)
- ↑ Pernet V., Joly S., Christ F., Dimou L., Schwab M.E. Nogo-A and myelin-associated glycoprotein differently regulate oligodendrocyte maturation and myelin formation. J Neurosci. 16, 7435-44 (2008).
- ↑ Susuki K., Baba H., Tohyama K., Kanai K., Kuwabara S., Hirata K., Furukawa K., Furukawa K., Rasband M.N., Yuki N. Gangliosides contribute to stability of paranodal junctions and ion channel clusters in myelinated nerve fibers. Glia 55, 746-757. 2007.
- ↑ Nguyen T., Mehta N.R., Conant K., Kim K.J., Jones M., Calabresi P.A., Melli G., Hoke A., Schnaar R.L., Ming G.L., Song H., Keswani S.C., Griffin J.W. Axonal protective effects of the myelin-associated glycoprotein. J Neurosci. 21, 630-637 (2009).
- ↑ Mehta N.R., Nguyen T., Bullen J.W., Griffin J.W., Schnaar R.L. Myelin-associated glycoprotein (MAG) protects neurons from acute toxicity using a ganglioside-dependent mechanism. ACS Chem Neurosci. 1, 215-222, 2010.
- ↑ Vyas A.A., Patel H.V., Fromholt S.E., Heffer-Lauc M., Vyas K.A., Dang J., Schachner M., Schnaar R.L. Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration. Proc Natl Acad Sci U S A 99, 8412-8417, 2002.
- ↑ Yiu G., He Z. Glial inhibition of CNS axon regeneration. Nat. Rev. Neurosci. 7, 617–627, 2006.
- ↑ 23.0 23.1 Lehmann, F., Gäthje, H., Kelm, S., Dietz, F. Evolution of sialic acid-binding proteins: molecular cloning and expression of fish siglec-4. Glycobiology 14, 959-968 (2004)
The CFG is grateful to the following PIs for their contributions to this wiki page: Paul Crocker, Sorge Kelm, James Paulson, Ron Schnaar