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 8

S272A and T277A gigaxonin mutants have reduced interaction with vimentin and NF-L.

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S272A and T277A gigaxonin mutants have reduced interaction with vimentin...
(A) 293T cells were cotransfected with constructs encoding GFP or HA-WT-, HA-S272A-, or HA-T277A-gigaxonin and MYC-CUL3, as indicated, for 46 hours. Lysates were subjected to myc IP and analyzed by WB. The interaction between S272A or T277A gigaxonin and CUL3 is not reduced relative to WT (n = 2). (B) 293T cells were cotransfected with constructs encoding HA-WT-, HA-S272A-, or HA-T277A-gigaxonin and EGFP-vimentin (EGFP-vim), as indicated, for 30 hours. Lysates were subjected to HA or GFP IP and analyzed by WB. The S272A and T277A gigaxonin mutants show reduced interaction with vimentin and NF-L. (C and D) Quantification of the relative binding efficiency between vimentin and the indicated gigaxonin variants from IP-WB in B. The vimentin–WT gigaxonin interaction was normalized to 100%. n = 3; black dots represent individual biological replicates. The mean values from the 3 replicates were analyzed first by 1-way ANOVA and then using Tukey’s HSD post hoc test. Error bars represent SD; **P < 0.01. (E) 293T cells were cotransfected with constructs encoding EGFP-vimentin and HA-WT-, HA-S272A-, or HA-T277A-gigaxonin, as indicated, for 30 hours. Lysates were subjected to GFP IP and analyzed by ubiquitin (Ub) WB. Polyubiquitination of vimentin is reduced in S272A or T277A gigaxonin–expressing cells compared with WT (n = 3).