The UDP-glucose:Glycoprotein Glucosyltransferase Is Organized in at Least Two Tightly Bound Domains from Yeast to Mammals

Abstract

The endoplasmic reticulum UDP-Glc:glycoprotein glucosyltransferase (GT) exclusively glucosylates nonnative glycoprotein conformers. GT sequence analysis suggests that it is composed of at least two domains: the N-terminal domain, which composes 80% of the molecule, has no significant similarity to other known proteins and was proposed to be involved in the recognition of non-native conformers and the C-terminal or catalytic domain, which displays a similar size and significant similarity to members of glycosyltransferase family 8. Here, we show that N-and C-terminal domains from Rattus norvegicus and Schizosaccharomyces pombe GTs remained tightly but not covalently bound upon a mild proteolytic treatment and could not be separated without loss of enzymatic activity. The notion of a two-domain protein was reinforced by the synthesis of an active enzyme upon transfection of S. pombe GT null mutants with two expression vectors, each of them encoding one of both domains. Transfection with the Cterminal domain-encoding vector alone yielded an inactive, rapidly degraded protein, thus indicating that the N-terminal domain is required for proper folding of the C-terminal catalytic portion. If, indeed, the N-terminal domain is, as proposed, also involved in glycoprotein conformation recognition, the tight association between N-and C-terminal domains may explain why only Nglycans in close proximity to protein structural perturbations are glucosylated by the enzyme. Although S. pombe and Drosophila melanogaster GT N-terminal domains display an extremely poor similarity (16.3%), chimeras containing either yeast N-terminal and fly Cterminal domains or the inverse construction were enzymatically and functionally active in vivo, thus indicating that the N-terminal domains of both GTs shared three-dimensional features.

The Endoplasmic Reticulum Glucosyltransferase Recognizes Nearly Native Glycoprotein Folding Intermediates

“…We have previously determined that GT has at least two independent folding domains located at the N-and C-terminal ends, comprising 80 and 20% of the molecule, respectively. Both domains remained tightly but not covalently bound upon a mild proteolytic treatment, after which they could not be separated by common biochemical procedures without loss of enzymatic activity (21). The C-terminal portion is the catalytic domain as it shares sequence similarity and the ability to bind UDP-Glc analogs with glycosyltransferase family 8 enzymes (22).…”

“…The C-terminal portion is the catalytic domain as it shares sequence similarity and the ability to bind UDP-Glc analogs with glycosyltransferase family 8 enzymes (22). On the other hand, there is strong evidence suggesting that the Nterminal domain is the portion recognizing the folding status of glycoprotein substrates in addition to being required for the C-terminal domain stability (21,23). It may be speculated that on GT recognition of molten globule-like structures, the contact surface between N-and C-terminal domains becomes somehow partially distorted, thus, allowing binding of the substrate glycoprotein hydrophobic surface.…”

Protein Folding in the Endoplasmic Reticulum

“…UGT1 contains an amino-terminal folding sensor domain and a carboxy-terminal transferase domain (Arnold and Kaufman 2003;Guerin and Parodi 2003). UGT1 modifies glycans based on the structural integrity of the glycoprotein substrate (Caramelo and Parodi 2008).…”

Single-particle electron microscopy structure of UDP-glucose:glycoprotein glucosyltransferase suggests a selectivity mechanism for misfolded proteins

“…By glucosylating misfolded proteins, UGGT plays an important role in preventing misfolded proteins from exiting the ER. UGGT is conserved and essential (8,9). It is a large monomeric protein of more than 1500 residues and found in almost all eukaryotes.…”