UDP-Glc:glycoprotein glucosyltransferase recognizes structured and solvent accessible hydrophobic patches in molten globule-like folding intermediates

Abstract

Protein folding in the cell involves the action of different molecular chaperones and folding-facilitating enzymes. In the endoplasmic reticulum (ER), the folding status of glycoproteins is stringently controlled by a glucosyltranferase enzyme (GT) that creates monoglucosylated structures recognized by ER resident lectins (calnexin͞ calreticulin, CNX͞CRT). GT serves as a folding sensor because it only glucosylates misfolded or partly folded glycoproteins. Nevertheless, the molecular mechanism behind this recognition process remains largely unknown. In this paper we explore the structural determinants for GT recognition by using a single domain model protein. For this purpose we used a family of chemically glycosylated proteins derived from chymotrypsin inhibitor-2 as GT substrates. T he folding status of glycoproteins is strictly censored in the endoplasmic reticulum (ER), before they attain their final destination to membranes, organelles, or the extracellular environment (1). Proteins that fail to fold properly are initially retained in the ER and eventually degraded in the proteasomes. The quality control system involves the action of UDPGlc:glycoprotein glucosyltranferase (GT) and glucosidase II (GII) enzymes and the ER resident lectins calnexin (CNX) and calreticulin (CRT). Proteins that are incompletely folded are glucosylated by GT, thus generating Glc 1 Man 9 GlcNAc 2 structures. On binding those structures, CNX and͞or CNR retain the monoglucosylated glycoproteins in the ER. Eventually, the Glc units added by GT are removed by GII, causing the release of glycoproteins from the lectin anchors. Glucosylationdeglucosylation cycles catalyzed by the opposing activities of GT and GII continue until native conformations are attained. Glycoproteins then become substrates of GII but not of GT and are thus free to pursue their travel through the secretory pathway.The CNX͞CRT-monoglucosylated glycan interaction is one of the mechanism by which cells retain incompletely folded glycoproteins in the ER and, in addition, it enhances folding efficiency by preventing protein aggregation and allowing intervention of additional ER chaperones and folding accessory proteins. The key element in this mechanism is GT as it is the only component exclusively acting on incompletely folded glycoproteins. This feature is based on the recognition of two structural determinants not exposed in native conformations. One of them is the innermost GlcNAc unit, which is buried within the protein scaffold in native but solvent exposed in nonnative conformations (2). In addition, there is an ill-defined determinant in incompletely folded polypeptides that is also recognized by GT. This protein determinant has eluded identification so far, mainly because of the difficulties intrinsic to the study of partly folded conformations of possible intermediates along the folding pathway of nascent polypeptides. It has been suggested that hydrophobic residues exposed in incompletely folded conformers could be the protein elements recognized by GT as exp...

The Endoplasmic Reticulum Glucosyltransferase Recognizes Nearly Native Glycoprotein Folding Intermediates

“…CI2-(1-25), CI2-(1-40), and CI2-(1-64) were obtained as described previously (8). The remaining fragments were produced by introducing a stop codon at the appropriate positions by the inverse PCR method using the pTZ18U-based vector (11).…”

“…Proteins were purified by reverse phase high performance liquid chromatography using a Vydac C8 column with a linear gradient of acetonitrile/water in 0.1% trifluoroacetic acid and dried in a SpeedVac. Because GT substrates are high mannose-type glycoproteins, thyroglobulin/Pronasederived high mannose-type glycopeptides were covalently linked to the new-engineered C7 compounds by using sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate as described previously (8). Glycosylated CI2s will be referred to as GCI2s.…”

“…CI2 is an extremely useful model to study chaperone and GT recognition of nascent chains (8). However, because GT substrates are glycoproteins, the original E7 in CI2 was replaced by a cysteine (E7C) to which a high mannose-type glycopeptide derived from bovine thyroglobulin was attached through a bifunctional chemical cross-linker (glycosylated CI2s are to be referred to as GCI2s).…”

“…Current evidence points to exposure of hydrophobic residues as the common determinant (6,7). In a recent work we presented evidence showing that GT recognized local stretches of hydrophobic residues in small disordered fragments rather poorly but actively glucosylated glycoproteins showing long range interactions in molten globule-like conformations, thus, exposing anilinonaphthalene sulfonic acid (ANS) binding hydrophobic patches (8).…”

Protein Folding in the Endoplasmic Reticulum

“…UGT1 modifies glycans based on the structural integrity of the glycoprotein substrate (Caramelo and Parodi 2008). Studies using purified UGT1 and engineered substrates have showed that UGT1 recognizes near-native molten globule substrates through surface-exposed hydrophobic patches (Sousa and Parodi 1995;Caramelo et al 2003Caramelo et al , 2004. More recent studies using a cell-based reglucosylation assay revealed that the magnitude of substrate misfolding determines the level of reglucosylation and that reglucosylation occurs posttranslationally .…”