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The ER protein folding sensor UDP-glucose glycoprotein–glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation

Nature Structural & Molecular Biology volume 11, pages 128134 (2004) | Download Citation

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Abstract

We present in vitro data that explain the recognition mechanism of misfolded glycoproteins by UDP-glucose glycoprotein–glucosyltransferase (UGGT). The glycoprotein exo-(1,3)-β-glucanase (β-Glc) bearing two glycans unfolds in a pH-dependent manner to become a misfolded substrate for UGGT. In the crystal structure of this glycoprotein, the local hydrophobicity surrounding each glycosylation site coincides with the differential recognition of N-linked glycans by UGGT. We introduced a single F280S point mutation, producing a β-Glc protein with full enzymatic activity that was both recognized as misfolded and monoglucosylated by UGGT. Contrary to current views, these data show that UGGT can modify N-linked glycans positioned at least 40 Å from localized regions of disorder and sense subtle conformational changes within structurally compact, enzymatically active glycoprotein substrates.

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References

  1. 1.

    & Intracellular functions of N-linked glycans. Science 291, 2364–2369 (2001).

  2. 2.

    , & Molecular chaperone systems in the endoplasmic reticulum. In Molecular Chaperones in the Cell (ed. Lund, P.) 180–200 (Oxford Univ. Press, Oxford, UK, 2000).

  3. 3.

    Importance of glycosidases in mammalian glycoprotein biosynthesis. Biochim. Biophys. Acta. 1473, 96–107 (1999).

  4. 4.

    & Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631–664 (1985).

  5. 5.

    & Substrate specificities of rat liver microsomal glucosidases which process glycoproteins. J. Biol. Chem. 255, 2255–2258 (1980).

  6. 6.

    et al. The heterodimeric structure of glucosidase II is required for its activity, solubility and localization in vivo. Glycobiology 10, 815–827 (2000).

  7. 7.

    , , , & Lectin control of protein folding and sorting in the secretory pathway. Trends Biochem. Sci. 28, 49–57 (2003).

  8. 8.

    et al. Conformation independent binding of monoglucosylated ribonuclease B to calnexin. Cell 88, 29–38 (1997).

  9. 9.

    et al. Enhanced catalysis of ribonuclease B folding by the interaction of calnexin or calreticulin with ERp57. J. Biol. Chem. 273, 6009–6012 (1998).

  10. 10.

    , & Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. Biochemistry 31, 97–105 (1992).

  11. 11.

    & The noncatalytic portion of human UDP-glucose:glycoprotein glucosytransferase I confers UDP-glucose binding and transferase function to the catalytic domain. J. Biol. Chem. 278, 43320–43328 (2003).

  12. 12.

    & The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. EMBO J. 14, 4196–4203 (1995).

  13. 13.

    , , , & UDP-Glc:glycoprotein glucosyltransferase recognizes structures and solvent accessible hydrophobic patches in molten globule-like folding intermediates. Proc. Natl. Acad. Sci. USA 100, 86–91 (2003).

  14. 14.

    , , , & Glycopeptide specificity of the secretory protein folding sensor UDP-glucose glycoprotein:glucosyltransferase. EMBO Rep. 4, 405–411 (2003).

  15. 15.

    & Recognition of local glycoprotein misfolding by the ER folding sensor UDP-glucose:glycoprotein glucosyltransferase. Nat. Struct. Biol. 7, 278–280 (2000).

  16. 16.

    et al. Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme. Glycobiology 10, 403–412 (2000).

  17. 17.

    , , , & Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose. Biochemistry 35, 10648–10660 (1996).

  18. 18.

    et al. Selective elongation of the oligosaccharide attached to the second potential glycosylation site of yeast exoglucanase: effects on the activity and properties of the enzyme. Biochem. J. 304, 917–922 (1994).

  19. 19.

    , & A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae. Arch. Microbiol. 151, 391–398 (1989).

  20. 20.

    et al. Destabilizing effects of replacing a surface lysine of cytochrome c with aromatic amino acids: implications for the denatured state. Biochemistry 32, 183–190 (1993).

  21. 21.

    et al. The magnitude of changes in guanidine-HCl unfolding m-values in the protein, iso-1-cytochrome c, depends upon the substructure containing the mutation. Protein Sci. 7, 1789–1795 (1998).

  22. 22.

    , & The N-glycans of jack bean α-mannosidase. Structure, topology and function. Eur. J. Biochem. 264, 168–75 (1999).

  23. 23.

    , & A family of yeast expression vectors. Gene 52, 225–233 (1987).

  24. 24.

    , , & New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994).

  25. 25.

    , & One-step transformation of yeast in stationary phase. Curr. Genet. 21, 83–84 (1992).

  26. 26.

    & Preparative elution of proteins blotted to Immobilon membranes. Anal. Biochem. 168, 48–53 (1988).

  27. 27.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  28. 28.

    , , & Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

  29. 29.

    et al. The structure of the exo-β-(1,3)-glucanase from Candida albicans in native and bound forms: relationship between a pocket and groove in family 5 glycosyl hydrolases. J. Mol. Biol. 294, 771–783 (1999).

  30. 30.

    et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

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Acknowledgements

We thank M. Cygler, A. Matte and J. Schrag for their assistance and G. Larriba for antibodies. Operating grants from the Canadian Institutes of Health Research (to J.J.M.B. and D.Y.T) financially supported this work. A.D.F. was the recipient of a Canadian Institutes of Health Research postdoctoral fellowship, and is currently a fellow of the Human Frontier Science Program.

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Author notes

    • Sean C Taylor
    •  & Andrew D Ferguson

    Present addresses: Montreal Proteomics Network, Genome Quebec Innovation Centre, 740 Dr. Penfield, McGill University, Montreal, Quebec, Canada H3A 2B2 (S.C.T.) and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9050, USA (A.D.F.).

    • Sean C Taylor
    •  & Andrew D Ferguson

    These authors contributed equally to this work.

Affiliations

  1. Biochemistry Department, Faculty of Medicine, McGill University, McIntyre Medical Sciences Building, 3655 Boulevard Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6.

    • Sean C Taylor
    •  & David Y Thomas
  2. Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 2B2.

    • Sean C Taylor
    • , John J M Bergeron
    •  & David Y Thomas

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The authors declare no competing financial interests.

Corresponding author

Correspondence to David Y Thomas.

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DOI

https://doi.org/10.1038/nsmb715

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