Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD
- Kyoto University, Japan
- Nagoya City University, Japan
- Hokkaido University, Japan
- National Institute of Health Sciences, Japan
- Osaka University, Japan
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
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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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
Further reading
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- Cell Biology
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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- Biochemistry and Chemical Biology
- Genetics and Genomics
Yerba mate (YM, Ilex paraguariensis) is an economically important crop marketed for the elaboration of mate, the third-most widely consumed caffeine-containing infusion worldwide. Here, we report the first genome assembly of this species, which has a total length of 1.06 Gb and contains 53,390 protein-coding genes. Comparative analyses revealed that the large YM genome size is partly due to a whole-genome duplication (Ip-α) during the early evolutionary history of Ilex, in addition to the hexaploidization event (γ) shared by core eudicots. Characterization of the genome allowed us to clone the genes encoding methyltransferase enzymes that catalyse multiple reactions required for caffeine production. To our surprise, this species has converged upon a different biochemical pathway compared to that of coffee and tea. In order to gain insight into the structural basis for the convergent enzyme activities, we obtained a crystal structure for the terminal enzyme in the pathway that forms caffeine. The structure reveals that convergent solutions have evolved for substrate positioning because different amino acid residues facilitate a different substrate orientation such that efficient methylation occurs in the independently evolved enzymes in YM and coffee. While our results show phylogenomic constraint limits the genes coopted for convergence of caffeine biosynthesis, the X-ray diffraction data suggest structural constraints are minimal for the convergent evolution of individual reactions.
