Glycoprotein Quality Control System Research Articles

Convergent synthesis of oligomannose-type glycans via step-economical construction of branch structures

Oligomannose-type glycans on glycoproteins are important signaling molecules in the glycoprotein quality control system in the endoplasmic reticulum. Recently, free oligomannose-type glycans generated by the hydrolysis of glycoproteins or dolichol pyrophosphate-linked oligosaccharides were recognized as important signals for immunogenicity. Hence, there is a high demand for pure oligomannose-type glycans for biochemical experiments; however, the chemical synthesis of glycans to achieve high-concentration products is laborious. In this study, we demonstrate a simple and efficient synthetic strategy for oligomannose-type glycans. Sequential regioselective α-mannosylation at the C-3 and C-6 positions of 2,3,4,6-unprotected galactose residues in galactosylchitobiose derivatives was demonstrated. Subsequently, the inversion of the configuration of the two hydroxy groups at the C-2 and C-4 positions of the galactose moiety was successfully carried out. This synthetic route reduces the number of the protection–deprotection reactions and is suitable for constructing different branching patterns of oligomannose-type glycans, such as M9, M5A, and M5B.

Analysis of Selenoprotein F Binding to UDP-Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker.

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between βS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.

Isolation and characterization of high-mannose type glycans containing five or six mannose residues from hen egg yolk

High-mannose type glycans play important roles in biosynthesis of glycoproteins including glycoprotein quality control system. In the endoplasmic reticulum (ER), α1,2-mannosidases cleave several mannose (Man) residues to give small high-mannose type glycans, such as glycans containing five or six mannose residues (M5-glycan or M6-glycan). These glycans are reported to act as a signal for degradation processes of glycoproteins in the ER. In this work, we isolated the M5-glycan and the M6-glycan from delipidated egg yolk and confirmed that their structures were identical to human type glycans based on rigorous NMR experiments, suggesting the potential use for semisynthesis of glycoconjugates and glycan analysis.

Chemical-Synthesis-Based Approach to Glycoprotein Functions in the Endoplasmic Reticulum.

The introduction of Asn-linked glycans to nascent polypeptides occurs in the lumen of the endoplasmic reticulum of eukaryotic cells. After the removal of specific sugar residues, glycoproteins acquire signals in the glycoprotein quality control (GPQC) system and enter the folding cycle composed of lectin-chaperones calnexin (CNX) and calreticulin (CRT), glucosidase II (G-II), and UDP-Glc:glycoprotein glucosyltransferase (UGGT). G-II initiates glycoproteins' entry and exit from the cycle, and UGGT serves as the "folding sensor". This account summarizes our effort to analyze the properties of enzymes and lectins that play important roles in GPQC, especially those involved in the CNX/CRT cycle. To commence our study, general methods for the synthesis of high-mannose-type glycans and glycoproteins were established. Based on these, various substrates to analyze components of the GPQC were created, and properties of CRT, G-II, and UGGT have been clarified.

Structural basis of glycoprotein quality control system

Structural basis of glycoprotein quality control system

Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins.

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-β (IFN-β), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.

Effects of domain composition on catalytic activity of human UDP-glucose:glycoprotein glucosyltransferases.

Uridine diphosphate (UDP)-glucose:glycoprotein glucosyltransferase (UGGT) 1 is a soluble protein residing in the endoplasmic reticulum (ER) and partially in ER-Golgi intermediate compartment. Characteristically, it is able to recognize incompletely folded proteins and re-glucosylate their high-mannose-type glycans. By virtue of this, UGGT1 acts as a folding sensor in the glycoprotein quality control system in the ER. On the other hand, human UGGT2 (HUGT2) has been believed to be an inactive homolog of human UGGT1 (HUGT1), whereas our recent study discovered its activity as UGGT. Although the activity of HUGT2 is significantly lower than HUGT1, C-terminal catalytic region, accounting for approximately 20% of the full-length enzyme, shares high amino acid sequence identity (>85%). In this study, we aimed to clarify the contribution of the noncatalytic domains by comparing activities of truncated forms of recombinant HUGT1/HUGT2 and HUGT1/HUGT2 chimeras with full-length enzymes. Our results obtained by using synthetic substrate indicate that the C-terminal catalytic regions of HUGTs are functional as UGGT. While the activity of HUGT1, but not that of HUGT2, was enhanced by the presence of N-terminal domains, activities of catalytic domains are similar between two homologs.

Calreticulin discriminates the proximal region at the N-glycosylation site of Glc1Man9GlcNAc2 ligand

Calreticulin (CRT) is well known as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However, whether CRT can directly interact with the aglycone moiety (protein portion) of the glycoprotein remains controversial. To improve our understanding of CRT interactions, structure-defined G1M9-derivatives with different aglycones (–OH, –Gly–NH2, and –Gly–Glu–tBu) were used as CRT ligands, and their interactions with recombinant CRT were analyzed using thermal shift analysis. The results showed that CRT binds strongly to a G1M9-ligand in the order –Gly–Glu–tBu > –Gly–NH2 > –OH, which is the same as that of the reglucosylation of Man9GlcNAc2 (M9)-derivatives by the folding sensor enzyme UGGT (UDP-glucose: glycoprotein glucosyltransferase). Our results indicate that, similar to UGGT, CRT discriminates the proximal region at the N-glycosylation site, suggesting a similar mechanism mediating the recognition of aglycone moieties in the ER glycoprotein quality control system.

Profiling Aglycon-Recognizing Sites of UDP-glucose:glycoprotein Glucosyltransferase by Means of Squarate-Mediated Labeling.

Because of its ability to selectively glucosylate misfolded glycoproteins, UDP-glucose:glycoprotein glucosyltransferase (UGGT) functions as a folding sensor in the glycoprotein quality control system in the endoplasmic reticulum (ER). The unique property of UGGT derives from its ability to transfer a glucose residue to N-glycan moieties of incompletely folded glycoproteins. We have previously discovered nonproteinic synthetic substrates of this enzyme, allowing us to conduct its high-sensitivity assay in a quantitative manner. In this study, we aimed to conduct site-selective affinity labeling of UGGT using a functionalized oligosaccharide probe to identify domain(s) responsible for recognition of the aglycon moiety of substrates. To this end, a probe 1 was designed to selectively label nucleophilic amino acid residues in the proximity of the canonical aglycon-recognizing site of human UGGT1 (HUGT1) via squaramide formation. As expected, probe 1 was able to label HUGT1 in the presence of UDP. Analysis by nano-LC-ESI/MS(n) identified a unique lysine residue (K1424) that was modified by 1. Kyte-Doolittle analysis as well as homology modeling revealed a cluster of hydrophobic amino acids that may be functional in the folding sensing mechanism of HUGT1.

ChemInform Abstract: Chemical Synthesis of Homogeneous Glycoproteins for the Study of Glycoprotein Quality Control System

AbstractReview: [36 refs.

Chemical Synthesis of Homogeneous Glycoproteins for the Study of Glycoprotein Quality Control System

AbstractThe glycoprotein quality control system exists in the endoplasmic reticulum to maintain protein homeostasis and prevent accumulation of aberrant glycoproteins. Folding sensor enzyme uridine diphosphate (UDP)glucose : glycoprotein glucosyltransferase (UGGT) plays an important role in this system through its ability to discriminate immature or misfolded glycoproteins from native ones. UGGT transfers a glucose residue to a glycoprotein containing Man9GlcNAc2 (M9; Man=mannose, GlcNAc=N‐acetyl‐D‐glucosamine) N‐glycan only when the glycoprotein has not attained a native form. We chemically prepared homogeneous glycoproteins containing M9 N‐glycan in the native form as well as in misfolded forms and examined them as substrates of UGGT. Glucose transfer to misfolded glycoproteins was clearly observed by LC‐MS, but glycoproteins in the native form were barely glucosylated. Furthermore, we constructed an in vitro glycoprotein folding system in the presence of UGGT and found out that all folding intermediates which appeared during folding were also glucosylated. Through these experiments, we demonstrated the usefulness of chemically synthesized homogeneous glycoproteins as probes to gain insights into the molecular basis of the glycoprotein quality control system.

Emerging Structural Insights into Glycoprotein Quality Control Coupled with N-Glycan Processing in the Endoplasmic Reticulum

In the endoplasmic reticulum (ER), the sugar chain is initially introduced onto newly synthesized proteins as a triantennary tetradecasaccharide (Glc3Man9GlcNAc2). The attached oligosaccharide chain is subjected to stepwise trimming by the actions of specific glucosidases and mannosidases. In these processes, the transiently expressed N-glycans, as processing intermediates, function as signals for the determination of glycoprotein fates, i.e., folding, transport, or degradation through interactions of a series of intracellular lectins. The monoglucosylated glycoforms are hallmarks of incompletely folded states of glycoproteins in this system, whereas the outer mannose trimming leads to ER-associated glycoprotein degradation. This review outlines the recently emerging evidence regarding the molecular and structural basis of this glycoprotein quality control system, which is regulated through dynamic interplay among intracellular lectins, glycosidases, and glycosyltransferase. Structural snapshots of carbohydrate-lectin interactions have been provided at the atomic level using X-ray crystallographic analyses. Conformational ensembles of uncomplexed triantennary high-mannose-type oligosaccharides have been characterized in a quantitative manner using molecular dynamics simulation in conjunction with nuclear magnetic resonance spectroscopy. These complementary views provide new insights into glycoprotein recognition in quality control coupled with N-glycan processing.

Reductions of the components of the calreticulin/calnexin quality-control system by proteasome inhibitors and their relevance in a rodent model of Parkinson's disease.

Evidence indicates that the ubiquitin-proteasome system and the endoplasmic retculum (ER) quality-control system work in concert to ensure that proteins are correctly folded in the ER and that misfolded proteins are retrotransported to the cytosol for degradation by proteasomes. Dysfunction of either system results in developmental abnormalities and even death in animals. This study investigates whether and how proteasome inhibition impacts the components of the calreticulin (CRT)/calnexin (CNX) glycoprotein folding machinery, a typical ER protein quality-control system, in the context of early neuronal injury. Here we report that proteasome inhibitor treatments, at nonlethal levels, reduced protein levels of CRT and ERp57 but not of CNX. These treatments increased protein levels of CRT in culture media, an effect blocked by brefeldin A, an inhibitor of protein trafficking; by contrast, ERp57 was not detected in culture media. Knockdown of CRT levels alone increased the vulnerability of SH-SY5Y, a neuronal cell line, to 6-hydroxydopamine (6-OHDA) toxicity. In a rat model of Parkinson's disease, intrastriatal 6-OHDA lesions resulted in decreased levels of CRT and ERp57 in the midbrain. These findings suggest that reduction of the components of CRT/CNX glycoprotein quality-control system may play a role in neuronal injury in Parkinson's disease and other neurodegenerative disorders associated with dysfunction of the ubiquitin-proteasome system.

Folding of Synthetic Homogeneous Glycoproteins in the Presence of a Glycoprotein Folding Sensor Enzyme

AbstractUDP‐glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re‐glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re‐glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

Folding of Synthetic Homogeneous Glycoproteins in the Presence of a Glycoprotein Folding Sensor Enzyme

UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

2SBP-04 糖タンパク質品質管理システムにおける糖鎖修飾メカニズムの構造基盤(シグナル伝達機構における構造細胞生物学的新展開,新学術領域研究「細胞シグナリング複合体によるシグナル検知・伝達・応答の構造的基礎」共催,シンポジウム,第52回日本生物物理学会年会(2014年度))

2SBP-04 糖タンパク質品質管理システムにおける糖鎖修飾メカニズムの構造基盤(シグナル伝達機構における構造細胞生物学的新展開,新学術領域研究「細胞シグナリング複合体によるシグナル検知・伝達・応答の構造的基礎」共催,シンポジウム,第52回日本生物物理学会年会(2014年度))

Chemical Synthesis of Intentionally Misfolded Homogeneous Glycoprotein: A Unique Approach for the Study of Glycoprotein Quality Control

Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

Golgi localization of ERManI defines spatial separation of the mammalian glycoprotein quality control system

The Golgi complex has been implicated as a possible component of endoplasmic reticulum (ER) glycoprotein quality control, although the elucidation of its exact role is lacking. ERManI, a putative ER resident mannosidase, plays a rate-limiting role in generating a signal that targets misfolded N-linked glycoproteins for ER-associated degradation (ERAD). Herein we demonstrate that the endogenous human homologue predominantly resides in the Golgi complex, where it is subjected to O-glycosylation. To distinguish the intracellular site where the glycoprotein ERAD signal is generated, a COPI-binding motif was appended to the N terminus of the recombinant protein to facilitate its retrograde translocation back to the ER. Partial redistribution of the modified ERManI was observed along with an accelerated rate at which N-linked glycans of misfolded α1-antitrypsin variant NHK were trimmed. Despite these observations, the rate of NHK degradation was not accelerated, implicating the Golgi complex as the site for glycoprotein ERAD substrate tagging. Taken together, these data provide a potential mechanistic explanation for the spatial separation by which glycoprotein quality control components operate in mammalian cells.

Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man 9GlcNAc 2, providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.

Chemical approaches toward understanding glycan-mediated protein quality control

High-mannose-type oligosaccharides, which are cotranslationally introduced to nascent polypeptides during N-glycosylation, play critical roles in protein quality control. Involved in this process are a number of intracellular carbohydrate-recognizing proteins or carbohydrate-processing enzymes, including calnexin/calreticulin, malectin, glucosidase I (G-I) and II (G-II), UDP-glucose:glycoprotein glucosyltransferase (UGGT), cargo receptors (VIP36, ERGL, and ERGIC-53), ER 1,2-mannosidase I, ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) and ubiquitin ligase. Although all these proteins seem to recognize high-mannose glycans, their precise specificities are yet to be clarified. In order to conduct quantitative evaluation of the activity and specificity of these proteins, a comprehensive set of high-mannose-type glycans and their variously functionalized derivatives were synthesized and used to analyze enzymes involved in glycoprotein quality control system.