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

  • 2,692
    Page views
  • 377
    Downloads
  • 28
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Cell Biology

    Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes

    Fabian Link, Alyssa Borges ... Markus Engstler
    Research Article

    Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

    1. Cell Biology
    2. Neuroscience

    Tissue-specific O-GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning

    Haibin Yu, Dandan Liu ... Kai Yuan
    Research Article

    O-GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O-GlcNAc binding activity derived from Clostridium perfringens OGA (CpOGA) with TurboID proximity labeling in Drosophila, we developed an O-GlcNAcylation profiling tool that translates O-GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O-GlcNAc interactome in major brain regions of Drosophila and found that components of the translational machinery, particularly ribosomal subunits, were abundantly O-GlcNAcylated in the mushroom body of Drosophila brain. Hypo-O-GlcNAcylation induced by ectopic expression of active CpOGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of O-GlcNAcylation in Drosophila, and suggests a possibility that O-GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.