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O-GlcNAcylation of tubulin inhibits its polymerization

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Abstract

The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins is an abundant and reversible modification that involves many cellular processes including transcription, translation, cell proliferation, apoptosis, and signal transduction. Here, we found that the O-GlcNAc modification pattern was altered during all-trans retinoic acid (tRA)-induced neurite outgrowth in the MN9D neuronal cell line. We identified several O-GlcNAcylated proteins using mass spectrometric analysis, including α- and β-tubulin. Further analysis of α- and β-tubulin revealed that O-GlcNAcylated peptides mapped between residues 173 and 185 of α-tubulin and between residues 216 and 238 of β-tubulin, respectively. We found that an increase in α-tubulin O-GlcNAcylation reduced heterodimerization and that O-GlcNAcylated tubulin did not polymerize into microtubules. Consequently, when O-GlcNAcase inhibitors were co-incubated with tRA, the extent of neurite outgrowth was decreased by 20% compared to control. Thus, our data indicate that the O-GlcNAcylation of tubulin negatively regulates microtubule formation.

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Abbreviations

O-GlcNAc:

O-Linked β-N-acetylglucosamine

tRA:

All-trans retinoic acid

PTM:

Post-translational modification

MAP:

Microtubule-associated protein

OGT:

O-GlcNAc transferase

OGA:

O-GlcNAcase

NButGT:

1,2-Dideoxy-2′-propyl-α-d-glucopyranoso[2,1-d]-Δ2-thiazoline

PUGNAc:

O-(2-Acetamido-2-deoxy-d-glycopyranosylidene)amino-N-phenylcarbamate

ESI:

Electrospray ionization

2-DE:

Two-dimensional electrophoresis

sWGA:

Succinylated wheat germ agglutinin

CBB:

Coomassie Brilliant Blue

IgG HC:

Immunoglobulin heavy chain

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Acknowledgments

This work was supported by grants from National Research Foundation funded by the Ministry of Education, Science and Technology Grant (R0A-2007-000-20011-0), and WCU project (R31-2008-000-10086-0). In addition, S.J., S.Y.P. and J.G.K. are fellowship awardees of the Brain Korea 21 program. This work was made possible through the use of research facilities in the Yonsei Center for Biotechnology.

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Correspondence to Jin Won Cho.

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Ji, S., Kang, J.G., Park, S.Y. et al. O-GlcNAcylation of tubulin inhibits its polymerization. Amino Acids 40, 809–818 (2011). https://doi.org/10.1007/s00726-010-0698-9

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