1. Biochemistry and Chemical Biology
  2. Cell Biology

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

  1. Ginto George
  2. Satoshi Ninagawa
  3. Hirokazu Yagi
  4. Jun-ichi Furukawa
  5. Noritaka Hashii
  6. Akiko Ishii-Watabe
  7. Ying Deng
  8. Kazutoshi Matsushita
  9. Tokiro Ishikawa
  10. Yugoviandi P Mamahit
  11. Yuta Maki
  12. Yasuhiro Kajihara
  13. Koichi Kato
  14. Tetsuya Okada  Is a corresponding author
  15. Kazutoshi Mori  Is a corresponding author
  1. Kyoto University, Japan
  2. Nagoya City University, Japan
  3. Hokkaido University, Japan
  4. National Institute of Health Sciences, Japan
  5. Osaka University, Japan
https://doi.org/10.7554/eLife.70357
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Cite this article
  1. Ginto George
  2. Satoshi Ninagawa
  3. Hirokazu Yagi
  4. Jun-ichi Furukawa
  5. Noritaka Hashii
  6. Akiko Ishii-Watabe
  7. Ying Deng
  8. Kazutoshi Matsushita
  9. Tokiro Ishikawa
  10. Yugoviandi P Mamahit
  11. Yuta Maki
  12. Yasuhiro Kajihara
  13. Koichi Kato
  14. Tetsuya Okada
  15. Kazutoshi Mori
(2021)
Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD
eLife 10:e70357.
https://doi.org/10.7554/eLife.70357
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Abstract

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the a1,6-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 a1,2-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 a1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 1S1, 2, 2S1, 4, 5, 6 and 7.

Article and author information

Author details

  1. Ginto George

    Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.

  2. Satoshi Ninagawa

    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:" 0000-0002-8005-4716

  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. Jun-ichi Furukawa

    Hokkaido University, Sappora, Japan
    Competing interests
    The authors declare that no competing interests exist.

  5. Noritaka Hashii

    National Institute of Health Sciences, Kawasaki, Japan
    Competing interests
    The authors declare that no competing interests exist.

  6. Akiko Ishii-Watabe

    National Institute of Health Sciences, Kawasaki, Japan
    Competing interests
    The authors declare that no competing interests exist.

  7. Ying Deng

    Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.

  8. Kazutoshi Matsushita

    Kyoto University, Kyoto, Japan
    Competing interests
    The authors declare that no competing interests exist.

  9. Tokiro Ishikawa

    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

  10. Yugoviandi P Mamahit

    Osaka University, Toyonaka, Japan
    Competing interests
    The authors declare that no competing interests exist.

  11. Yuta Maki

    Osaka University, Toyonaka, Japan
    Competing interests
    The authors declare that no competing interests exist.

  12. Yasuhiro Kajihara

    Osaka University, Toyonaka, Japan
    Competing interests
    The authors declare that no competing interests exist.

  13. Koichi Kato

    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:" 0000-0001-7187-9612

  14. Tetsuya Okada

    Kyoto University, Kyoto, Japan
    For correspondence
    tokada@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:" 0000-0002-2513-1301

  15. Kazutoshi Mori

    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

MEXT, Japan (18K06216)

  • Satoshi Ninagawa

Kobayashi Foundation

  • Satoshi Ninagawa

MEXT, Japa (17H06414)

  • Hirokazu Yagi

MEXT, Japan (21H02625)

  • Hirokazu Yagi

MEXT, Japan (19K06658)

  • Tokiro Ishikawa

MEXT, Japan (20K21495)

  • Koichi Kato

MEXT, Japan (18K06110)

  • Tetsuya Okada

MEXT, Japan (17H01432)

  • Kazutoshi Mori

MEXT, Japan (17H06419)

  • Kazutoshi Mori

Takeda Science Foundation

  • Satoshi Ninagawa

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

Copyright

© 2021, 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.

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  1. Ginto George
  2. Satoshi Ninagawa
  3. Hirokazu Yagi
  4. Jun-ichi Furukawa
  5. Noritaka Hashii
  6. Akiko Ishii-Watabe
  7. Ying Deng
  8. Kazutoshi Matsushita
  9. Tokiro Ishikawa
  10. Yugoviandi P Mamahit
  11. Yuta Maki
  12. Yasuhiro Kajihara
  13. Koichi Kato
  14. Tetsuya Okada
  15. Kazutoshi Mori
(2021)
Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD
eLife 10:e70357.
https://doi.org/10.7554/eLife.70357

Share this article

https://doi.org/10.7554/eLife.70357

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Further reading

    1. Cell Biology

    UGGT1-mediated reglucosylation of N-glycan competes with ER-associated degradation of unstable and misfolded glycoproteins

    Satoshi Ninagawa, Masaki Matsuo ... Kazutoshi Mori
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    How the fate (folding versus degradation) of glycoproteins is determined in the endoplasmic reticulum (ER) is an intriguing question. Monoglucosylated glycoproteins are recognized by lectin chaperones to facilitate their folding, whereas glycoproteins exposing well-trimmed mannoses are subjected to glycoprotein ER-associated degradation (gpERAD); we have elucidated how mannoses are sequentially trimmed by EDEM family members (George et al., 2020; 2021 eLife). Although reglucosylation by UGGT was previously reported to have no effect on substrate degradation, here we directly tested this notion using cells with genetically disrupted UGGT1/2. Strikingly, the results showed that UGGT1 delayed the degradation of misfolded substrates and unstable glycoproteins including ATF6α. An experiment with a point mutant of UGGT1 indicated that the glucosylation activity of UGGT1 was required for the inhibition of early glycoprotein degradation. These and overexpression-based competition experiments suggested that the fate of glycoproteins is determined by a tug-of-war between structure formation by UGGT1 and degradation by EDEMs. We further demonstrated the physiological importance of UGGT1, since ATF6α cannot function properly without UGGT1. Thus, our work strongly suggests that UGGT1 is a central factor in ER protein quality control via the regulation of both glycoprotein folding and degradation.

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    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 α1,2-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.

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