1. Cell Biology

EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD

  1. Ginto George
  2. Satoshi Ninagawa
  3. Hirokazu Yagi
  4. Taiki Saito
  5. Tokiro Ishikawa
  6. Tetsushi Sakuma
  7. Takashi Yamamoto
  8. Koshi Imami
  9. Yasushi Ishihama
  10. Koichi Kato
  11. Tetsuya Okada  Is a corresponding author
  12. Kazutoshi Mori  Is a corresponding author
  1. Kyoto University, Japan
  2. Nagoya City University, Japan
  3. Hiroshima University, Japan
https://doi.org/10.7554/eLife.53455
Share this article
Cite this article
  1. Ginto George
  2. Satoshi Ninagawa
  3. Hirokazu Yagi
  4. Taiki Saito
  5. Tokiro Ishikawa
  6. Tetsushi Sakuma
  7. Takashi Yamamoto
  8. Koshi Imami
  9. Yasushi Ishihama
  10. Koichi Kato
  11. Tetsuya Okada
  12. Kazutoshi Mori
(2020)
EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD
eLife 9:e53455.
https://doi.org/10.7554/eLife.53455
  2. Download BibTeX
  3. Download .RIS

Abstract

Sequential mannose trimming of N-glycan (Man9GlcNAc2 -> Man8GlcNAc2 -> Man7GlcNAc2) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no a1,2-mannosidase activity toward Man9GlcNAc2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man9GlcNAc2 to Man8GlcNAc2(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.

Data availability

All data generated or analysed during this study are included in the manuscript.

Article and author information

Author details

  1. Ginto George

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.

  2. Satoshi Ninagawa

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.

  3. Hirokazu Yagi

    Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9296-0225

  4. Taiki Saito

    Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
    Competing interests
    The authors declare that no competing interests exist.

  5. Tokiro Ishikawa

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1718-6764

  6. Tetsushi Sakuma

    Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0396-1563

  7. Takashi Yamamoto

    Department of Mathematical and Life Sciences, Hiroshima University, Hiroshima, Japan
    Competing interests
    The authors declare that no competing interests exist.

  8. Koshi Imami

    Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.

  9. Yasushi Ishihama

    Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.

  10. Koichi Kato

    Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
    Competing interests
    The authors declare that no competing interests exist.

  11. Tetsuya Okada

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    For correspondence
    tokada@upr.biophys.kyoto-u.ac.jp
    Competing interests
    The authors declare that no competing interests exist.

  12. Kazutoshi Mori

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    For correspondence
    mori@upr.biophys.kyoto-u.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7378-4019

Funding

Ministry of Education, Culture, Sports, Science, and Technology (18K06216)

  • Satoshi Ninagawa

Ministry of Education, Culture, Sports, Science, and Technology (17H06414)

  • Hirokazu Yagi

Ministry of Education, Culture, Sports, Science, and Technology (19K06658)

  • Tokiro Ishikawa

Ministry of Education, Culture, Sports, Science, and Technology (18K06110)

  • Tetsuya Okada

Ministry of Education, Culture, Sports, Science, and Technology (17H01432)

  • Kazutoshi Mori

Ministry of Education, Culture, Sports, Science, and Technology (17H06419)

  • Kazutoshi Mori

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Adam Linstedt, Carnegie Mellon University, United States

Version history

  1. Received: November 8, 2019
  2. Accepted: February 7, 2020
  3. Accepted Manuscript published: February 17, 2020 (version 1)
  4. Version of Record published: February 24, 2020 (version 2)

Copyright

© 2020, George et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,721
    views
  • 378
    downloads
  • 35
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Ginto George
  2. Satoshi Ninagawa
  3. Hirokazu Yagi
  4. Taiki Saito
  5. Tokiro Ishikawa
  6. Tetsushi Sakuma
  7. Takashi Yamamoto
  8. Koshi Imami
  9. Yasushi Ishihama
  10. Koichi Kato
  11. Tetsuya Okada
  12. Kazutoshi Mori
(2020)
EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD
eLife 9:e53455.
https://doi.org/10.7554/eLife.53455

Share this article

https://doi.org/10.7554/eLife.53455

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD

    Ginto George, Satoshi Ninagawa ... Kazutoshi Mori
    Research Advance Updated

    Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

    1. Cancer Biology
    2. Cell Biology

    Exosomes: How tumors escape the immune system

    Stefanie Schmieder
    Insight

    Mutations in the gene for β-catenin cause liver cancer cells to release fewer exosomes, which reduces the number of immune cells infiltrating the tumor.

    1. Cell Biology
    2. Neuroscience

    Unraveling the link between neuropathy target esterase NTE/SWS, lysosomal storage diseases, inflammation, abnormal fatty acid metabolism, and leaky brain barrier

    Mariana I Tsap, Andriy S Yatsenko ... Halyna R Shcherbata
    Research Article Updated

    Mutations in Drosophila Swiss cheese (SWS) gene or its vertebrate orthologue neuropathy target esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.