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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 128–134 (2004)Cite this article

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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Figure 1: β-Glc activity and recognition by UGGT.
Figure 2: Crystal structure of exo-(1,3)-β-glucanase at 1.75-Å resolution.
Figure 3: β-Glc activity and trypsin sensitivity under crystal growth conditions.
Figure 4: Probing the β-Glc structure with UGGT, trypsin and N-glycanases.
Figure 5: Mutagenesis of β-Glc produces an enzymatically active substrate that is recognized by UGGT.
Figure 6: pH-induced structural defects of β-Glc F280S mutants are recognized by UGGT.
Figure 7: Probing the integrity of the structure of F280S mutants with UGGT and trypsin.

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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

  1. Sean C Taylor

    Present address: Montreal Proteomics Network, Genome Quebec Innovation Centre, McGill University, 740 Dr. Penfield, Montreal, Quebec, Canada, H3A 2B2

  2. Andrew D Ferguson

    Present address: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, 75390-9050, USA

  3. Sean C Taylor and Andrew D Ferguson: These authors contributed equally to this work.

Authors and Affiliations

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

    Sean C Taylor & David Y Thomas

  2. Department of Anatomy and Cell Biology, McGill University, Montreal, H3A 2B2, Quebec, Canada

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

  3. Montreal Proteomics Network, Genome Quebec Innovation Centre, McGill University, 740 Dr. Penfield, Montreal, Quebec, Canada, H3A 2B2

    Andrew D Ferguson

  4. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, 75390-9050, USA

    Sean C Taylor

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Correspondence to David Y Thomas.

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Taylor, S., Ferguson, A., Bergeron, J. et al. The ER protein folding sensor UDP-glucose glycoprotein–glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation. Nat Struct Mol Biol 11, 128–134 (2004). https://doi.org/10.1038/nsmb715

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