Potent Glycan Inhibitors of Myelin-associated Glycoprotein Enhance Axon Outgrowth in Vitro

Alka A Vyas,Ola Blixt,James C Paulson,Ronald L Schnaar

doi:10.1074/jbc.m500250200

Alka A Vyas, Ola Blixt + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m500250200

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Abstract

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

Highlights

Myelin-associated glycoprotein (MAG, Siglec-4) is one of several endogenous axon regeneration inhibitors that limit recovery from central nervous system injury and disease
The injured adult mammalian central nervous system (CNS)1 is a highly inhibitory environment for axon regeneration, in large part due to specific axon regeneration inhibitors expressed on residual myelin that persists at sites of CNS injury and on astrocytes recruited to injury sites [1,2,3]
Inhibitors include three myelin proteins, myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp), and chondroitin sulfate proteoglycans, which are found on the astrocytic scar and on residual myelin

Summary

EXPERIMENTAL PROCEDURES

Materials—Glycosides of 3-sialyl T, 6-sialyl T, and disialyl T, each bearing an ␣-linked N-acetyl carboxymethyl threonine as aglycone, were prepared as described previously [20]. The proteins were transferred onto polyvinylidene difluoride membranes using a semidry transfer cell (Bio-Rad) and blotted with biotinylated peanut agglutinin as described previously [26], except that membranes were blocked for 2 h at ambient temperature, incubated with peanut agglutinin (10 ␮g/ml) for 16 h at 4 °C and with streptavidin-conjugated alkaline phosphatase (1:1500; Jackson ImmunoResearch Laboratories) for 1 h at ambient temperature, and developed using nitro blue tetrazoliumbromochloroindolyl phosphate (Sigma). To test the specificity of benzyl-␣-GalNAc treatment, gangliosides were extracted from inhibitor-treated and control cells in chloroformmethanol-water (4:8:3), resolved by silica gel TLC using chloroformmethanol-0.25% aqueous KCl (60:35:8) as developing solvent, and stained with resorcinol reagent as described previously [27, 28]

RESULTS

MAG bindingb

Axon outgrowthc

DISCUSSION