The Endoplasmic Reticulum Glucosyltransferase Recognizes Nearly Native Glycoprotein Folding Intermediates

Abstract

The UDP-Glc:glycoprotein glucosyltransferase (GT), a key player in the endoplasmic reticulum (ER) quality control of glycoprotein folding, only glucosylates glycoproteins displaying non-native conformations. To determine whether GT recognizes folding intermediates or irreparably misfolded species with nearly native structures, we generated and tested as GT substrates neoglycoprotein fragments derived from chymotrypsin inhibitor 2 (GCI2) bearing from 53 to 64 (full-length) amino acids. Fragment conformations mimicked the last stagefolding structures adopted by a glycoprotein entering the ER lumen. GT catalytic efficiency (V max /K m ) remained constant from GCI2-(1-53) to GCI2-(1-58) and then steadily declined to reach a minimal value with GCI2-(1-64). The same parameter showed a direct hyperbolic relationship with solvent accessibility of the single Trp residue but only in fragments exposing hydrophobic amino acid patches. Mutations introduced (GCI2-(1-63)V63S and GCI2-(1-64)V63S) produced slight structural destabilizations but increased GT catalytic efficiency. This parameter presented an inverse exponential relationship with the free energy of unfolding of canonical and mutant fragments. Moreover, the catalytic efficiency showed a linear relationship with the fraction of unfolded species in water. It was concluded that the GT-derived quality control may be operative with nearly native conformers and that no alternative ER-retaining mechanisms are required when glycoproteins approach their proper folding.A quality control mechanism in the endoplasmic reticulum (ER) 1 is in charge of sensing the folding state of glycoproteins before their transport to the Golgi apparatus (1). Proteins incorrectly folded are initially retained in the ER and eventually degraded by the proteasome, a process known as ER-associated degradation (2, 3). Recent findings on glycoprotein processing in the ER showed that sugar moieties could be exploited to encode information on glycoprotein folding status. About 65% of Asn-XSer/Thr consensus sequences in proteins entering the ER lumen are N-glycosylated (4). The Glc 3 Man 9 GlcNAc 2 glycan transferred is deglucosylated immediately by the sequential action of glucosidases I and II. At this stage the proteins that are not correctly folded are reglucosylated by UDP-Glc:glycoprotein glucosyltransferase (GT), generating Glc 1 Man 9 GlcNAc 2 . This enzyme has a unique property as it glucosylates glycoproteins displaying native-close, molten globule-like but not random coil or compactnative conformations. Monoglucosylated glycans are specifically recognized by two ER resident lectins, calnexin (CNX) and calreticulin (CRT), in charge of retaining folding intermediates and irreparably misfolded glycoproteins in the ER. In addition, lectin binding facilitates conformational maturation of glycoproteins by preventing aggregation and allowing the labor of lectin-associated proteins such as ERp57 (endoplasmic reticulum protein 57), a protein of the protein disulfide isomerase family (5).The single gluc...

Glycoprotein Tertiary and Quaternary Structures Are Monitored by the Same Quality Control Mechanism

“…The glycoprotein quality control system could have evolved, among other reasons, to avoid these situations. If the definitive tertiary structure of the subunits is acquired upon oligomer assembly, individual subunits are expected to be glucosylated by GT until they form the final complex, as GT appeared to be able to glucosylate glycoprotein monomers displaying structures minimally differing from native ones (18). The work presented here suggests that, on the other hand, if the tertiary structure is attained before assembly, GT will recognize the subunit interface of the partially assembled species.…”

Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum

“…Folding and subunit assembly occur at this stage and, if completed, the glycoprotein may be exported from the ER. However, if incompletely folded, the glycoprotein becomes a substrate for UDP-glucose: glycoprotein glucosyltransferase (UGGT), which re-attaches a single glucose only to non-native glycoprotein conformers (Caramelo et al, 2004;Ritter et al, 2005;Taylor et al, 2004). This signals re-binding to Cnx/Crt and the cycle continues until a native conformation is achieved or until degradative processes engage.…”

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

“…Misfolded proteins are kinetically favored through their higher rate of occupancy in the cavity. The size of the pocket favors the binding of intact, partially folded proteins over small hydrophobic glycopeptides (43), and flexibility allows UGGT to act on different misfolded proteins. This simple mechanism explains why the acceptor glycan and exposed hydrophobic segment need to be in the same molecule and why misfolded, deglycosylated proteins inhibit UGGT activity.…”