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Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins

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Abstract

Endoplasmic reticulum-associated degradation (ERAD) is a key cellular process whereby misfolded proteins are removed from the endoplasmic reticulum (ER) for subsequent degradation by the ubiquitin/proteasome system. In the present work, analysis of the released, free oligosaccharides (FOS) derived from all glycoproteins undergoing ERAD, has allowed a global estimation of the mechanisms of this pathway rather than following model proteins through degradative routes. Examining the FOS produced in endomannosidase-compromised cells following α-glucosidase inhibition has revealed a mechanism for clearing Golgi-retrieved glycoproteins that have failed to enter the ER quality control cycle. The Glc3Man7GlcNAc2 FOS species has been shown to be produced in the ER lumen by a mechanism involving a peptide: N-glycanase-like activity, and its production was sensitive to disruption of Golgi-ER trafficking. The detection of this oligosaccharide was unaffected by the overexpression of EDEM1 or cytosolic mannosidase, both of which increased the production of previously characterised cytosolically localised FOS. The lumenal FOS identified are therefore distinct in their production and regulation compared to FOS produced by the conventional route of misfolded glycoproteins directly removed from the ER. The production of such lumenal FOS is indicative of a novel degradative route for cellular glycoproteins that may exist under certain conditions.

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Acknowledgments

The authors would like to thank Prof. Pirkko Heikinheimo and Elina Kuokkanen, as well as Dr. John Christianson, for providing plasmids. Dr. Anthony Marriott is thanked for providing CHPV. Amicus Therapeutics are thanked for providing a chemical reagent. The authors would like to thank Oxford Glycobiology Institute and the Australian Research Council for financial support and Clarendon Fund/New College (Robert Lyns) for support of NVK.

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Authors and Affiliations

  1. Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    Dominic S. Alonzi, Nikolay V. Kukushkin, Sarah A. Allman, Lorna Pierce, Raymond A. Dwek & Terry D. Butters

  2. School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, VIC, 3010, Australia

    Zalihe Hakki & Spencer J. Williams

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  1. Dominic S. Alonzi
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Correspondence to Terry D. Butters.

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D.S. Alonzi and N.V. Kukushkin contributed equally to this work.

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Alonzi, D.S., Kukushkin, N.V., Allman, S.A. et al. Glycoprotein misfolding in the endoplasmic reticulum: identification of released oligosaccharides reveals a second ER-associated degradation pathway for Golgi-retrieved proteins. Cell. Mol. Life Sci. 70, 2799–2814 (2013). https://doi.org/10.1007/s00018-013-1304-6

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