The UDP-Glc:glycoprotein glucosyltrasferase is a soluble protein of the endoplasmic reticulum

Abstract

N-linked oligosaccharides devoid of glucose residues are transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum. The reaction products have been identified, depending on the organisms, as protein-linked Glc1Man5-9GlcNAc2. Incubation of right side-sealed vesicles from rat liver with UDP-[14C]Glc, Ca2+ ions and denatured thyroglobulin led to the glucosylation of the macromolecule only when the vesicles had been disrupted previously by sonication or by the addition of detergents to the glucosylation mixture. Similarly, maximal glucosylation of denatured thyroglobulin required disruption of microsomal vesicles isolated from the protozoan Crithidia fasciculata. Treatment of the rat liver vesicles with trypsin led to the inactivation of the UDP-Glc:glycoprotein glucosyltransferase only when proteolysis was performed in the presence of detergents. The glycoprotein glucosylating activity could be solubilized upon sonication of right side-sealed vesicles in an isotonic medium, upon passage of them through a French press or by suspending the vesicles in an hypotonic medium. Moreover, the enzyme appeared in the aqueous phase when the vesicles were submitted to a Triton X-114/water partition. Solubilization was not due to proteolysis of a membrane-bound enzyme. The enzyme could also be solubilized from C. fasciculata microsomal vesicles by procedures not involving membrane disassembly. About 30% of endogenous glycoproteins glucosylated upon incubation of intact rat liver microsomal vesicles with UDP-[14C]GLc could be solubilized by sonication or by suspending the vesicles in 0.1 M Na2CO3. These and previous results show that the UDP-Glc:glycoprotein glucosyltransferase is a soluble protein present in the lumen of the endoplasmic reticulum. In addition, both soluble and membrane-bound glycoproteins may be glucosylated by the glycoprotein glucosylating activity.

Association between the 15-kDa Selenoprotein and UDP-glucose:Glycoprotein Glucosyltransferase in the Endoplasmic Reticulum of Mammalian Cells

“…Previously characterized eukaryotic selenoproteins were involved in redox processes (2), and redox function has been anticipated for Sep15, but the quality control of protein folding has not been linked to a redox process. (ii) UGTR is located in the ER (17), and no selenoprotein has yet been found to occur in this cellular compartment. In addition to Sep15, two other known mammalian selenoproteins, glutathione peroxidase 3 and selenoprotein P, contain N-terminal signal peptides.…”

The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

“…Moreover, this result provided a convenient method for assaying the transferring enzyme (UDP-Glc:glycoprotein glucosyltransferase, GT), which was detected in microsomal membranes of mammalian, plant, fungal and trypanosomatid cells (27). Further work showed that GT was a soluble protein of the ER lumen and that the reaction products had the glucose unit linked to the same mannose with the same α(1,3) bond as in Glc 1 Man 9 GlcNAc 2 -P-P-dolichol ( Figure 1) (27,28). As observed for thyroglobulin, other glycoproteins (phytohemagglutinin, soybean agglutinin, ribonuclease B) were not glucosylated unless they had been previously denatured (29).…”

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

“…Upon release, the terminal glucose is cleaved by glucosidase II, producing a shortened glycan, Man 9 GlcNAc 2 , which can no longer bind the lectin chaperones. Incompletely folded or misfolded proteins after the first cycle of folding are recognized by UDP-glucose:glycoprotein glucosyltransferase (UGGT), which adds back a glucose residue to regenerate the monoglucosylated form for additional rounds of lectin chaperone-assisted refolding (6,7). By glucosylating misfolded proteins, UGGT plays an important role in preventing misfolded proteins from exiting the ER.…”