Gigaxonin glycosylation regulates intermediate filament turnover and may impact giant axonal neuropathy etiology or treatment
Po-Han Chen, Jimin Hu, Jianli Wu, Duc T. Huynh, Timothy J. Smith, Samuel Pan, Brittany J. Bisnett, Alexander B. Smith, Annie Lu, Brett M. Condon, Jen-Tsan Chi, Michael Boyce
Po-Han Chen, Jimin Hu, Jianli Wu, Duc T. Huynh, Timothy J. Smith, Samuel Pan, Brittany J. Bisnett, Alexander B. Smith, Annie Lu, Brett M. Condon, Jen-Tsan Chi, Michael Boyce
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Research Article Cell biology

Gigaxonin glycosylation regulates intermediate filament turnover and may impact giant axonal neuropathy etiology or treatment

Abstract

Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor–dependent manner. MS analyses of human gigaxonin revealed 9 candidate sites of O-GlcNAcylation, 2 of which — serine 272 and threonine 277 — are required for its ability to mediate IF turnover in gigaxonin-deficient human cell models that we created. Taken together, the results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the nongenetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.

Authors

Po-Han Chen, Jimin Hu, Jianli Wu, Duc T. Huynh, Timothy J. Smith, Samuel Pan, Brittany J. Bisnett, Alexander B. Smith, Annie Lu, Brett M. Condon, Jen-Tsan Chi, Michael Boyce

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Figure 9

Nutrient-sensitive regulation of gigaxonin O-GlcNAcylation.

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Nutrient-sensitive regulation of gigaxonin O-GlcNAcylation. (A) SH-SY5Y ...
(A) SH-SY5Y cells were treated with 10% or 0.1% serum for 72 hours, as indicated, and lysed. Endogenous gigaxonin was immunoprecipitated and analyzed by WB. Gigaxonin O-GlcNAcylation is reduced after serum starvation, as indicated by 2 different anti–O-GlcNAc monoclonal antibodies (representative results from 3 biological replicates). Arrows indicate gigaxonin bands. (B) 293T cells were transfected with WT HA-gigaxonin for 24 hours, treated with control (4 mM) or glutamine-free medium for an additional 48 hours under various glucose concentrations, as indicated, and then lysed. HA-gigaxonin was immunoprecipitated and analyzed by WB. Gigaxonin O-GlcNAcylation is reduced after glutamine starvation under low-glucose conditions, as indicated by anti–O-GlcNAc (RL2) WB. Arrows indicate gigaxonin bands. (C) Quantification of gigaxonin glycosylation under glutamine (gln) starvation and low-glucose conditions (0.4 g/L) in B (n = 3; black dots represent individual biological replicates; mean ± SD; Student’s t test, **P < 0.01).