Folding Sensor Research Articles

The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation.

We present in vitro data that explain the recognition mechanism of misfolded glycoproteins by UDP-glucose glycoprotein-glucosyltransferase (UGGT). The glycoprotein exo-(1,3)-beta-glucanase (beta-Glc) bearing two glycans unfolds in a pH-dependent manner to become a misfolded substrate for UGGT. In the crystal structure of this glycoprotein, the local hydrophobicity surrounding each glycosylation site coincides with the differential recognition of N-linked glycans by UGGT. We introduced a single F280S point mutation, producing a beta-Glc protein with full enzymatic activity that was both recognized as misfolded and monoglucosylated by UGGT. Contrary to current views, these data show that UGGT can modify N-linked glycans positioned at least 40 A from localized regions of disorder and sense subtle conformational changes within structurally compact, enzymatically active glycoprotein substrates.

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

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 (calnexincalreticulin, CNXCRT). 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. Structural characterization of species showing higher glucose acceptor capacity suggests that GT recognizes solvent accessible hydrophobic patches in molten globule-like conformers mimicking intermediate folding stages of nascent glycoproteins. It was further confirmed that BiP (binding protein, a chaperone of the heat shock protein 70 family) preferentially recognized neoglycoproteins displaying extended conformations, thus providing a molecular rationale for the sequential BiP-CNXCRT interaction with folding glycoproteins observed in vivo.

Organizational diversity among distinct glycoprotein endoplasmic reticulum-associated degradation programs.

Protein folding and quality control in the early secretory pathway function as posttranslational checkpoints in eukaryote gene expression. Herein, an aberrant form of the hepatic secretory protein alpha1-antitrypsin was stably expressed in a human embryonic kidney cell line to elucidate the mechanisms by which glycoprotein endoplasmic reticulum-associated degradation (GERAD) is administered in cells from higher eukaryotes. After biosynthesis, genetic variant PI Z underwent alternative phases of secretion and degradation, the latter of which was mediated by the proteasome. Degradation required release from calnexin- and asparagine-linked oligosaccharide modification by endoplasmic reticulum mannosidase I, the latter of which occurred as PI Z was bound to the molecular chaperone grp78/BiP. That a distinct GERAD program operates in human embryonic kidney cells was supported by the extent of PI Z secretion, apparent lack of polymerization, inability of calnexin to participate in the degradation process, and sequestration of the glycoprotein folding sensor UDP-glucose:glycoprotein glucosyltransferase in the Golgi complex. Because UDP-glucose:glycoprotein glucosyltransferase sustains calnexin binding, its altered distribution is consistent with a GERAD program that hinders the reentry of substrates into the calnexin cycle, allowing grp78/BiP to partner with a lectin, other than calnexin, in the recognition of a two-component GERAD signal to facilitate substrate recruitment. How the processing of a mutant protein, rather than the mutation itself, can contribute to disease pathogenesis, is discussed.

Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control.

The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.

Monoglucosylated Oligomannosides Are Released during the Degradation Process of Newly Synthesized Glycoproteins

The Chinese hamster ovary mutant MI8-5 is known to synthesize Man(9)GlcNAc(2)-P-P-dolichol rather than the fully glucosylated lipid intermediate Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This nonglucosylated oligosaccharide lipid precursor is used as donor for N-glycosylation. In this paper we demonstrate that a significant part of the glycans bound to the newly synthesized glycoproteins in MI8-5 cells are monoglucosylated. The presence of monoglucosylated glycans on glycoproteins determines their binding to calnexin as part of the quality control machinery. Furthermore, we point out the presence of Glc(1)Man(5)GlcNAc(1) in the cytosol of MI8-5 cells. This indicates that part of the monoglucosylated glycoproteins can be directed toward a deglycosylation process that occurs in the cytosol. Besides studies on glycoprotein degradation based on the disappearance of protein moieties, MI8-5 cells can be used as a tool to elucidate the various step leading to glycoprotein degradation by studying the fate of the glycan moieties.

Quality control in the secretory assembly line.

As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway.

Recognition of local glycoprotein misfolding by the ER folding sensor UDP-glucose:glycoprotein glucosyltransferase.

The endoplasmic reticulum (ER) contains a stringent quality control system that ensures the correct folding of newly synthesized proteins to be exported via the secretory pathway. In this system UDP-Glc:glycoprotein glucosyltransferase (GT) serves as a glycoprotein specific folding sensor by specifically glucosylating N-linked glycans in misfolded glycoproteins thus retaining them in the calnexin/calreticulin chaperone cycle. To investigate how GT senses the folding status of glycoproteins, we generated RNase B heterodimers consisting of a folded and a misfolded domain. Only glycans linked to the misfolded domain were found to be glucosylated, indicating that the enzyme recognizes folding defects at the level of individual domains and only reglucosylates glycans directly attached to a misfolded domain. The result was confirmed with complexes of soybean agglutinin and misfolded thyroglobulin.

Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum.

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.

Modification of the T Cell Antigen Receptor (TCR) Complex by UDP-glucose:Glycoprotein Glucosyltransferase: TCR FOLDING IS FINALIZED CONVERGENT WITH FORMATION OF αβδεγε COMPLEXES

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing alpha, beta, gamma, delta, epsilon, and zeta molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3alpha beta chains and formation of CD3-associated, disulfide-linked alpha beta heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

小胞体におけるフォールディング検知器，ＵＤＰ‐グルコース： 糖タンパク質グルコース転移酵素

小胞体におけるフォールディング検知器，ＵＤＰ‐グルコース： 糖タンパク質グルコース転移酵素

Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control.

Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor.