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Unglycosylation at Asn-633 made extracellular domain of E-cadherin folded incorrectly and arrested in endoplasmic reticulum, then sequentially degraded by ERAD

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Abstract

The human E-cadherin is a single transmembrane domain protein involved in Ca2+-dependent cell–cell adhesion. In a previous study, we demonstrated that all of four potential N-glycosylation sites in E-cadherin are occupied by N-glycans in human breast carcinoma cells in vivo and the elimination of N-glycan at Asn-633 dramatically affected E-cadherin expression and made it degraded. In this study we investigated the molecular mechanism of E-cadherin, which lacks N-glycosylation at Asn-633 (M4), degradation and the role of the N-glycan at Asn-633 in E-cadherin folding. We treated cells stably expressed M4 E-cadherin with MG123, DMM, respectively. Either MG132 or DMM could efficiently block degradation of M4 E-cadherin. M4 E-cadherin was recognized as the substrate of ERAD and was retro-translocated from ER lumen to cytoplasm by p97. It was observed that the ration of M4 E-cadherin binding to calnexin was significantly increased compared with that of other variants, suggesting that it was a misfolded protein, though cytoplasmic domain of M4 E-cadherin could associate with β-catenin. Furthermore, we found that N-glycans of M4 E-cadherin were modified in immature high mannose type, suggesting that it could not depart to Golgi apparatus. In conclusion, this study revealed that N-glycosylation at Asn-633 is essential for E-cadherin expression, folding and trafficking.

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Abbreviations

EC:

extracellular domain

AJs:

adherence junction complexes

ER:

endoplasmic reticulum

ERAD:

endoplasmic reticulum-associated degradation

WT:

wild type E-cadherin

M1:

E-cadherin lacking N-glycan at Asn-554 site

M2:

E-cadherin lacking N-glycan at Asn-566 site

M3:

E-cadherin lacking N-glycan at Asn-618 site

M4:

E-cadherin lacking N-glycan at Asn-633 site

M123:

E-cadherin has a single N-glycan at Asn-633 site

Endo H:

endoglycosidase

PNGase F:

peptide N-glycosidase F

PBS:

phosphate-buffered saline

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Acknowledgements

This work was supported by Shanghai Leading Academic Disciplines Project, Project Number: B110, National Nature Science Foundation of China, No 30670468 and the Grant of Shanghai Health Bureau, No. 044083.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai, 200032, People’s Republic of China

    Feng Zhou, Jianmin Su, Le Fu, Yong Yang, Lineng Zhang, Liying Wang, Hongbo Zhao, Zengxia Li & Xiliang Zha

  2. Key Laboratory of Glycoconjugate Research, Ministry of Health, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai, 200032, People’s Republic of China

    Feng Zhou, Jianmin Su, Yong Yang, Lineng Zhang, Liying Wang, Hongbo Zhao, Zengxia Li & Xiliang Zha

  3. Department of general surgery, Huashan Hospital, Fudan University, 12 Middle Wu Lu Mu Qi Road, Shanghai, 200040, People’s Republic of China

    Diancai Zhang

  4. Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, 130 Dong An Road, Shanghai, 200032, People’s Republic of China

    Xiliang Zha

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  1. Feng Zhou
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  2. Jianmin Su
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Zhou, F., Su, J., Fu, L. et al. Unglycosylation at Asn-633 made extracellular domain of E-cadherin folded incorrectly and arrested in endoplasmic reticulum, then sequentially degraded by ERAD. Glycoconj J 25, 727–740 (2008). https://doi.org/10.1007/s10719-008-9133-9

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