Publications

• The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.

  Sousa M., Parodi A.J.

  The UDP-Glc:glycoprotein glucosyltransferase is a soluble enzyme of the endoplasmic reticulum that glucosylates protein-linked Man7-9GlcNAc2 to form the monoglucosylated derivatives. In vivo the reaction products are immediately deglucosylated by glucosidase II. The glucosyltransferase has a uniqu ... >> More

  The UDP-Glc:glycoprotein glucosyltransferase is a soluble enzyme of the endoplasmic reticulum that glucosylates protein-linked Man7-9GlcNAc2 to form the monoglucosylated derivatives. In vivo the reaction products are immediately deglucosylated by glucosidase II. The glucosyltransferase has a unique property: it glucosylates misfolded, but not native, glycoproteins. It has been proposed that the glucosyltransferase participates, together with calnexin, in the control mechanism by which only properly folded glycoproteins can exit from the endoplasmic reticulum. In this paper it is demonstrated that the glucosyltransferase recognizes two elements in the acceptor substrates: the innermost N-acetylglucosamine unit of the oligosaccharide and protein domains exposed in denatured, but not in native, conformations. Both determinants have to be covalently linked. In many cases the first element is not accessible to macromolecular probes in native conformations. Concerning the protein domains, it is demonstrated here that the glucosyltransferase interacts with hydrophobic amino acids exposed in denatured conformations. More disordered conformations, i.e. those exposing more hydrophobic amino acids, were found to be those having higher glucose acceptor capacity. It is suggested that both accessibility of the innermost N-acetylglucosamine unit and binding to hydrophobic patches determine the exclusive glucosylation of misfolded conformations by the glucosyltransferase. << Less

  EMBO J 14:4196-4203(1995) [PubMed] [EuropePMC]

• Biophysical properties of UDP-glucose:glycoprotein glucosyltransferase, a folding sensor enzyme in the ER, delineated by synthetic probes.

  Sakono M., Seko A., Takeda Y., Hachisu M., Ito Y.

  UDP-glucose:glycoprotein glucosyltransferase plays a key role in glycoprotein quality control in the endoplasmic reticulum, by virtue of its ability to discriminate folding states. Although lines of evidence have clarified the ability of UGGT to recognize a partially unfolded protein, its mechanis ... >> More

  UDP-glucose:glycoprotein glucosyltransferase plays a key role in glycoprotein quality control in the endoplasmic reticulum, by virtue of its ability to discriminate folding states. Although lines of evidence have clarified the ability of UGGT to recognize a partially unfolded protein, its mechanistic rationale has been obscure. In this study, the substrate recognition mechanism of UGGT was studied using synthetic substrate of UGGT. Although UGGT has high extent of surface hydrophobicity, it clearly lacks property of typical molecular chaperones. Furthermore, it was revealed that the addition of the substrate caused secondary structure change of UGGT in a dose-dependent manner, resulting that the K(d) value of the UGGT-substrate interaction was estimated from theoretical formula based on 1:1 complexation between UGGT and the acceptor substrate. Moreover, the kinetic analysis of glucosyltransferase activity of UGGT elucidated Michaelis constant K(m) correctly. << Less

  Biochem. Biophys. Res. Commun. 426:504-510(2012) [PubMed] [EuropePMC]

• The recognition motif of the glycoprotein-folding sensor enzyme UDP-Glc:glycoprotein glucosyltransferase.

  Totani K., Ihara Y., Tsujimoto T., Matsuo I., Ito Y.

  The folding of glycoproteins is primarily mediated by a quality control system in the ER, in which UDP-Glc:glycoprotein glucosyltransferase (UGGT) serves as a "folding sensor". In this system, client glycoproteins are delivered to UGGT after the trimming of their innermost glucose residue by gluco ... >> More

  The folding of glycoproteins is primarily mediated by a quality control system in the ER, in which UDP-Glc:glycoprotein glucosyltransferase (UGGT) serves as a "folding sensor". In this system, client glycoproteins are delivered to UGGT after the trimming of their innermost glucose residue by glucosidase II, which releases them from the lectin chaperones calnexin (CNX) and calreticulin (CRT). UGGT is inactive against folded proteins, allowing them to proceed to the Golgi apparatus for further processing to complex- or hybrid-type glycoforms. On the other hand, this enzyme efficiently glucosylates incompletely folded glycoproteins to monoglucosylated structures, providing them with an opportunity to interact with CNX/CRT. In order to clarify the mode of this enzyme's substrate recognition, we conducted a structure-activity relationship study using a series of synthetic probes. The inhibitory activities of various glycans suggest that UGGT has a strong affinity for the core pentasaccharide (Man3GlcNAc2) of high-mannose-type glycans. Our comparison of the reactivity of acceptors that have been modified by various aglycons supports the hypothesis that UGGT recognizes the hydrophobic region of client glycoproteins. Moreover, we discovered fluorescently labeled substrates that will be valuable for highly sensitive detection of UGGT activity. << Less

  Biochemistry 48:2933-2940(2009) [PubMed] [EuropePMC]

• Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.

  Sousa M.C., Ferrero-Garcia M.A., Parodi A.J.

  It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endo ... >> More

  It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endoplasmic reticulum mannosidases decreases the extent of glycoprotein glucosylation. All five different glycoproteins tested (bovine and porcine thyroglobulins, phytohemagglutinin, soybean agglutinin, and bovine pancreas ribonuclease B) were found to be poorly glucosylated or not glucosylated unless they were subjected to treatments that modified their native conformations. The effect of denaturation was not to expose the oligosaccharides but to make protein determinants, required for enzymatic activity, accessible to the glucosyltransferase because (a) cleavage of denatured glycoproteins by unspecific (Pronase) or specific (trypsin) proteases abolished their glucose acceptor capacities almost completely except when the tryptic peptides were held together by disulfide bonds and (b) high mannose oligosaccharides in native glycoproteins, although poorly glucosylated or not glucosylated, were accessible to macromolecular probes as concanavalin A-Sepharose, endo-beta-N-acetylglucosaminidase H, and jack bean alpha-mannosidase. In addition, denatured, endo-beta-N-acetylglucosaminidase H deglycosylated glycoproteins were found to be potent inhibitors of the glucosylation of denatured glycoproteins. It is suggested that in vivo only unfolded, partially folded, and malfolded glycoproteins are glucosylated and that glucosylation stops upon adoption of the correct conformation, a process that hides the protein determinants (possibly hydrophobic amino acids) from the glucosyltransferase. << Less

  Biochemistry 31:97-105(1992) [PubMed] [EuropePMC]

• Glycoprotein folding and quality-control mechanisms in protein-folding diseases.

  Ferris S.P., Kodali V.K., Kaufman R.J.

  Biosynthesis of proteins--from translation to folding to export--encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to pri ... >> More

  Biosynthesis of proteins--from translation to folding to export--encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC) system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER). A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes. << Less

  Dis Model Mech 7:331-341(2014) [PubMed] [EuropePMC]