The lumen of the endoplasmic reticulum (ER) provides a specialized environment to assure the folding and oligomerization of secretory proteins to their native conformations. UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a biosensor in the ER that detects the folding state of glycoproteins. UGGT-catalyzed monoglucosylation of incompletely folded glycoproteins leads to their continued retention in the ER through their association with the lectins calnexin and calreticulin for further folding or for degradation. Purified recombinant UGGT from rat liver and glycoprotein substrates from a mutant strain of Saccharomyces cerevisiae were used in an in vitro system to examine the peptide components recognized by UGGT in unfolded glycoproteins and glycopeptides. Mass spectrometry was used to measure and quantitate the levels of glucose incorporation into these substrates that was directly related to their level of recognition by UGGT. To assess the capacity of UGGT for sensing non-native structures in glycoprotein substrates, Exo-1,3-beta-glucanase (beta-Glc) from S. cerevisiae was crystallized and its structure determined. A mutagenesis strategy was used to mutate solvent-exposed residues to yield the beta-Glc F280S point mutant that retained enzymatic activity while still being recognized by UGGT. These data suggest that UGGT recognizes solvent-exposed hydrophobic patches in the primary and tertiary structure of glycoproteins even in near-native conformations.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84438 |
| Date | January 2002 |
| Creators | Taylor, Sean Caldwell |
| Contributors | Bergeron, John (advisor), Thomas, David (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001986177, proquestno: AAINQ88584, Theses scanned by UMI/ProQuest. |
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