A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein

Abstract

The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.

Functional cooperation between BiP and calreticulin in the folding maturation of a glycoprotein in Trypanosoma cruzi

“…Given the particular features of protein N -glycosylation in T. cruzi mentioned above, this is the first in vivo confirmation that UGGT recognizes preferentially advanced folding stages of an endogenous glycoprotein. This agrees with previous in vitro experiments or in vivo ones but using heterologously expressed glycoproteins [8,9,11,35]. In uggt −/− cells TcrCATL was unable to finish its folding process and was retained in the ER by BiP in a mostly extended conformation.…”

Oncogenic deletion mutants of gp130 signal from intracellular compartments

“…We therefore hypothesize that the observed increase in association is due to local folding defects in domain D2 of the extracellular domain of gp130 DYY, evoked by the deletion mutation. It has been indeed shown that local folding defects in transmembrane proteins are recognized by the UDP-glucose:glycoprotein glucosyltransferase (UGGT) leading to reglucosylation of the glycan and, in consequence, a prolonged association of the misfolded protein with calnexin (Pearse et al, 2008;Taylor et al, 2004). Biophysical analysis of the recombinant gp130 domains D2-D3 containing a deletion mutation will reveal whether the mutations result in local structural instability.…”

Protein Folding in the Endoplasmic Reticulum

“…The released substrate is now free to fold. If after a single round of lectin chaperone binding a nonnative conformation persists, the quality-control sensor UGT1 (UDP-glucose: glycoprotein glucosyltransferase 1) will transfer a glucose back onto the unglucosylated glycoprotein, regenerating monoglucosylated glycans (Labriola et al 1995;Sousa and Parodi 1995;Pearse et al 2008). The reglucosylated substrate can then reassociate with the lectin chaperones to continue with attempts to fold properly (Hammond et al 1994;Hebert et al 1995;Van Leeuwen and Kearse 1997;Wada et al 1997;Molinari et al 2005;Pearse et al 2008;.…”