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GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion

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Abstract

Synthesis of glycosaminoglycans, such as heparan sulfate (HS) and chondroitin sulfate (CS), occurs in the lumen of the Golgi, but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNAcT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans.

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Acknowledgements

We thank Ge Yu, Judith Meyers Opp and the University of Michigan Advanced Genomics Core for their contribution in the RNA-Seq experiment. We thank Drs. Venkatesha Basrur, Felipe da Veiga Leprevost, Alexey Nesvizhskii and the Mass Spectrometry-Based Proteomics Resource Facility in the Department of Pathology at the University of Michigan for their contribution in Proteomics analysis. We thank the members of the Wang lab and Linhardt lab for stimulating discussions and technical support.

Funding

Y. W. was supported by the National Institutes of Health (Grant R35GM130331), Mizutani Foundation for Glycoscience, MCubed, and the Fast Forward Protein Folding Disease Initiative of the University of Michigan; E.A. was supported by a University of Michigan Rackham Predoctoral fellowship. R.J.L, F.Z. and Y.S. was supported by GlycoMIP a National Science Foundation Materials Innovation Platform funded through Cooperative Agreement DMR-1933525.GlycoMIP.

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Author notes

  1. Erpan Ahat and Yuefan Song authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, 1105 North University Avenue, Ann Arbor, MI, 48109-1085, USA

    Erpan Ahat, Whitney Reid, Jie Li, Sarah Bui & Yanzhuang Wang

  2. Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Yuefan Song, Ke Xia, Fuming Zhang & Robert J. Linhardt

  3. Department of Neurology, University of Michigan School of Medicine, Ann Arbor, MI, USA

    Yanzhuang Wang

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  1. Erpan Ahat
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  2. Yuefan Song
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  3. Ke Xia
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Contributions

E.A., Y.S., F.Z., R.L. and Y.W. designed the experiments; E.A. prepared the samples and Y.S., K.X. performed labeling and LC–MS experiments. E.A. performed most of the other experiments. W.R. and S.B. performed data search and analysis from the RNA-seq and Proteomics experiments. J.L. and E.A. performed the flow cytometry experiment. E.A., Y.S., R.L. and Y.W. analyzed the data. E.A. and Y.W. made the figures and wrote the first draft; Y.S. K.X., F.Z. and R.L. edited the manuscript. All authors discussed the results and contributed to the final manuscript.

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Correspondence to Robert J. Linhardt or Yanzhuang Wang.

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Ahat, E., Song, Y., Xia, K. et al. GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion. Cell. Mol. Life Sci. 79, 199 (2022). https://doi.org/10.1007/s00018-022-04223-3

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