In vitro mannosidase activity of EDEM3 against asparagine-linked oligomannose-type glycans.

Oligomannose-type glycans on glycoproteins play an important role in the endoplasmic reticulum (ER)-protein quality control. Mannose trimming of the glycans triggers the ER-associated protein degradation pathway. In mammals, ER mannosyl-oligosaccharide 1,2-α-mannosidase 1 and three ER degradation -enhancing α-mannosidase-like proteins (EDEMs) are responsible for mannose trimming. However, the exact role of EDEMs as α-mannosidases in ERAD remains unclear. Here, we performed the biochemical characterization of EDEM3 using synthetic oligomannose-type glycan substrates. In vitro assays revealed that EDEM3 can convert an asparagine-linked M9 glycan to M8 and M7 glycans in contrast to glycine-linked M9 glycan, and the activity is enhanced in the presence of ERp46, a known partner protein of EDEM3. Our study provides novel insights into the enzymatic properties of EDEM3 and the use of artificial glycan substrates as tools to study ERAD mechanisms.

Dimerization of ER-resident molecular chaperones mediated by ERp29.

The lectin chaperones calnexin (CNX) and calreticulin (CRT) localized in the endoplasmic reticulum play important roles in glycoprotein quality control. Although the interaction between these lectin chaperones and ERp57 is well known, it has been recently reported that endoplasmic reticulum protein 29 (ERp29), a member of PDI family, interacts with CNX and CRT. The biochemical function of ERp29 is unclear because it exhibits no ERp57-like redox activity. In this study, we addressed the possibility that ER chaperones CNX and CRT are connected via ERp29, based on our observation that ERp29 exists as a dimer. As a result, we showed that CNX dimerizes through ERp29. These results endorse the hypothesis that ERp29 serves as a bridge that links two molecules of CNX. Also, we showed that similar complexes such as CNX-CRT were formed via ERp29.

Glycan dependent refolding activity of ER glucosyltransferase (UGGT).

BACKGROUND: In the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the "folding sensor" by virtue of its ability to discriminate folding states of client glycoproteins. However, besides its transferase activity, whether UGGT1 possesses any chaperone activity that facilitates protein folding is yet to be addressed. METHODS: We prepared oligomannose-type glycan modified RNase (M9GN2-RNase) by chemoenzymatic means using M9GN-oxazoline and glycan truncated RNase B and analyzed the effect of human UGGT1 (HUGT1) for refolding of the denatured M9GN2-RNase. Refolding was evaluated based on the RNase activity which was measured by the cleavage of the RNA substrate. RESULTS: HUGT1 slightly accelerated the folding of M9GN2-RNase and non-glycosylated RNase A as the same extent. However, HUGT1 remarkably accelerated the folding of M9GN2-RNase in the presence of UDP-Glc. In contrast, neither UDP nor UDP-Gal was effective in enhancing the folding. Additionally, an HUGT1 mutant which lacks the glucosyltransferase activity did not accelerate the protein folding of M9GN2-RNase. CONCLUSIONS: HUGT1has the ability to promote the refolding of denatured protein and the effect would be enhanced when HUGT1 tightly interacts with the client protein via glycan recognition. GENERAL SIGNIFICANCE: Our study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.

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-ß (IFN-ß), 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.

Substrate Recognition of Glycoprotein Folding Sensor UGGT Analyzed by Site-Specifically 15N-Labeled Glycopeptide and Small Glycopeptide Library Prepared by Parallel Native Chemical Ligation.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) distinguishes glycoproteins in non-native conformations from those in native conformations and glucosylates from only non-native glycoproteins. To analyze how UGGT recognizes non-native glycoproteins, we chemically synthesized site-specifically 15N-labeled interleukin 8 (IL-8) C-terminal (34-72) glycopeptides bearing a Man9GlcNAc2 (M9) oligosaccharide. Chemical shift perturbation mapping NMR experiments suggested that Phe65 of the glycopeptide specifically interacts with UGGT. To analyze this interaction, we constructed a glycopeptide library by varying Phe65 with 10 other natural amino acids, via parallel native chemical ligation between a glycopeptide-α-thioester and a peptide library consisting of 11 peptides. UGGT assay against the glycopeptide library revealed that, although less hydrophobic glycopeptides could be used as substrates for UGGT, hydrophobic glycopeptides are preferred.

PDI family protein ERp29 recognizes P-domain of molecular chaperone calnexin.

Endoplasmic reticulum (ER) resident lectin chaperone calnexin (CNX) and calreticulin (CRT) assist folding of nascent glycoproteins. Their association with ERp57, a member of PDI family proteins (PDIs) which promote disulfide bond formation of unfolded proteins, has been well documented. Recent studies have provided evidence that other PDIs may also interact with CNX and CRT. Accordingly, it seems possible that the ER provides a repertoire of CNX/CRT-PDI complexes, in order to facilitate refolding of various glycoproteins. In this study, we examined the ability of PDIs to interact with CNX. Among them ERp29 was shown to interact with CNX, similarly to ERp57. Judging from the dissociation constant, its ability to interact with CNX was similar to that of ERp57. Results of further analyses by using a CNX mutant imply that ERp29 and ERp57 recognize the same domain of CNX, whereas the mode of interaction with CNX might be somewhat different between them.

Endo-α-Mannosidase-Catalyzed Transglycosylation.

In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo-α-mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-α-mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α-glycosyl-fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1- 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high-mannose-type undecasaccharide (Glc1 Man9 GlcNAc2 ).

Non-enzymatic reaction of glycosyl oxazoline with peptides.

Recently, a number of chemoenzymatic strategies have been explored for achieving preparation of homogeneous glycopeptides and glycoproteins, especially by using endoglycanases and glycosyl oxazolines. However, concomitant occurrence of non-enzymatic reactions has been reported, but no further characterization of the byproducts was conducted. In this work, we made an attempt to identify the side product by using model substrates. Analysis of the product allowed us to propose that the oxazoline ring was attacked by the amino group of lysine, leading to the formation of disubstituted acetamidine.

Direct assay for endo-α-mannosidase substrate preference on correctly folded and misfolded model glycoproteins.

We previously reported a unique assay system for UDP-glucose glycoprotein glucosyltransferase (UGGT) toward glycoprotein folding intermediates during the folding process. The assay involved the in vitro folding of both high-mannose type oligosaccharyl crambin, which yielded only the correctly folded glycoprotein form (M9-glycosyl-native-crambin), and its mutant, which yielded misfolded glycoproteins (M9-glycosyl-misfolded-crambin), in the presence of UGGT. The process successfully yielded both mono-glucosylated M9-glycosyl-native-crambin (G1M9-glycosyl-native-crambin) and M9-glycosyl-misfolded-crambin (G1M9-glycosyl-misfolded-crambin). Here, we report the use of our in vitro folding system to evaluate the substrate preference of Golgi endo-α-mannosidase against G1M9-native and -misfolded glycoprotein forms. In our assay Golgi endo-α-mannosidase removed Glc-α-1-3-Man unit from G1M9-native and -misfolded-crambins clearly proving that Golgi endo-α-mannosidase does not have specific preference for correctly folded or misfolded protein structure.

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.

Synthesis of misfolded glycoprotein dimers through native chemical ligation of a dimeric peptide thioester.

Glycoprotein quality control processes are very important for an efficient production of glycoproteins and for avoiding the accumulation of unwanted toxic species in cells. These complex processes consist of multiple enzymes and chaperones such as UGGT, calnexin/calreticulin, and glucosidase II. We designed and synthesized monomeric and dimeric misfolded glycoprotein probes. Synthetic homogeneous monomeric glycoproteins proved to be useful substrates for kinetic analyses of the folding sensor enzyme UGGT. For a concise synthesis of a bismaleimide-linked dimer, we examined double native chemical ligation (dNCL) of a dimeric peptide-α-thioester. The dNCL to two equivalents of glycopeptides gave a homodimer. The dNCL to a 1 : 1 mixture of a glycopeptide and a non-glycosylated peptide gave all the three possible ligation products consisting of two homodimers and a heterodimer. Both the homodimer bearing two Man9GlcNAc2 (M9) oligosaccharides and the heterodimer bearing one M9 oligosaccharide were found to be good substrates of UGGT.

Structural Analysis of Free N-Glycans in α-Glucosidase Mutants of Saccharomyces cerevisiae: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the N-Glycans.

Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc3Man9GlcNAc2) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.

Synthesis of Glc1Man9-Glycoprotein Probes by a Misfolding/Enzymatic Glucosylation/Misfolding Sequence.

Glycoproteins in non-native conformations are often toxic to cells and may cause diseases, thus the quality control (QC) system eliminates these unwanted species. Lectin chaperone calreticulin and glucosidase II, both of which recognize the Glc1 Man9 oligosaccharide on glycoproteins, are important components of the glycoprotein QC system. Reported herein is the preparation of Glc1 Man9 -glycoproteins in both native and non-native conformations by using the following sequence: misfolding of chemically synthesized Man9 -glycoprotein, enzymatic glucosylation, and another misfolding step. By using synthetic glycoprotein probes, calreticulin was found to bind preferentially to a hydrophobic non-native glycoprotein whereas glucosidase II activity was not affected by glycoprotein conformation. The results demonstrate the ability of chemical synthesis to deliver homogeneous glycoproteins in several non-native conformations for probing the glycoprotein QC system.

Endoplasmic Reticulum (ER)-Targeted, Galectin-Mediated Retrograde Transport by Using a HaloTag Carrier Protein.

Investigations into metabolic processes within the cell have often relied on genetic methods such as forced expression and knockout or knockdown techniques. An alternative approach would be introducing a molecule into the desired location inside the cell. To translocate compounds from outside cells into the endoplasmic reticulum (ER), we constructed a delivery carrier protein. This comprised N-terminal galectin-1 for cell-surface binding (G1), a protease cleavable sequence (ps), a HaloTag domain for attaching exogenous compounds (Halo), and a C-terminal KDEL sequence for ER retention. Fluorescently labeled G1-ps-Halo-KDEL passed through the Golgi apparatus and reached the ER. By using Man9 GlcNAc2 -BODIPY as a cargo compound, the carrier protein was also delivered into the ER with concomitant processing of mannose to Man5,6, by the ER-resident α1,2-mannosidase. G1-ps-Halo-KDEL might serve as a new type of delivery carrier protein to direct compounds into the ER.

Approaches toward High-Mannose-Type Glycan Libraries.

Asparagine-linked (N-linked) sugar chains are widely found in the rough endoplasmic reticulum (ER), which has attracted renewed attention because of its participation in the glycoprotein quality control process. In the ER, newly formed glycoproteins are properly folded to higher-order structures by the action of a variety of lectin chaperones and processing enzymes and are transported into the Golgi, while terminally misfolded glycoproteins are carried into the cytosol for degradation. A group of proteins related to this system are known to recognize subtle differences in the high-mannose-type oligosaccharide structures of glycoproteins; however, their molecular foundations are still unclear. In order to gain a more precise understanding, our group has established a strategy for the systematic synthesis of high-mannose-type glycans. More recently, we have developed "top-down" chemoenzymatic approaches that allow expeditious access to theoretically all types of high-mannose glycans. This strategy comprehensively delivered 37 high-mannose-type glycans, including G1M9-M3 glycans, and opened up the possibility of the elucidation of structure-function relationships with a series of high-mannose-type glycans.

Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics.

In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality-control systems composed by various proteins. Among them, UDP-glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure. This study used pseudo-misfolded glycoproteins, comprising a modified dihydrofolate reductase with artificial pyrene-cysteine moiety on the protein surface (pDHFR) and Man9 GlcNAc2 -methotrexate (M9-MTX). All five M9-MTX/pDHFR complexes, with a pyrene group at different positions, were found to be good substrates of UGGT, irrespective of the site of pyrene modification. These results suggest UGGT's mode of substrate recognition is fuzzy, thus allowing various glycoproteins to be accommodated in the folding cycle.

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.

Cooperative role of calnexin and TigA in Aspergillus oryzae glycoprotein folding.

Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G1M9GN2-proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae, which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G1M9GN2-ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G1M9GN2-RNase was chemoenzymatically synthesized from G1M9GN-oxazoline and GN-RNase. Denatured G1M9GN2-RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae, TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX.

Preparation of asparagine-linked monoglucosylated high-mannose-type oligosaccharide from egg yolk.

Monoglucosylated high-mannose-type glycan (Glc1Man9GlcNAc2: G1M9) is well-known as a key glycoform in the glycoprotein folding process, which is specifically recognized by lectin chaperones calnexin (CNX) and calreticulin (CRT) in the endoplasmic reticulum (ER). In this work, we developed an efficient method for the preparation of G1M9-Asn. The G1M9-Asn was obtained from the IgY-rich fraction derived from hen egg yolk by the digestion with pronase. The α-amino group of asparagine in G1M9-Asn was protected with the 9-fluorenylmethyloxycarbonyl (Fmoc) group and the labeled glycans were subsequently purified using high performance liquid chromatography (HPLC). This method will provide useful substrates for analysis of the glycoprotein folding cycle in the ER.

The relationship between glycan structures and expression levels of an endoplasmic reticulum-resident glycoprotein, UDP-glucose: Glycoprotein glucosyltransferase 1.

In this article, we report a relationship between glycan structures and expression levels of a recombinant ER-resident glycoprotein, uridine 5'-diphosphate-glucose: glycoprotein glucosyltransferase (UGGT1). The function of glycan structures attached to a glycoprotein is actively studied; however, the glycan structures of recombinant, and not endogenous, glycoproteins have not been examined. In this study, we indicate a relationship between the glycan structure and the level of protein expression. Expression levels were controlled utilizing a series of vectors (pFN21K, pFN22K, pFN23K, and pFN24K HaloTag CMV Flexi Vectors). Qualitative and semi-quantitative confirmation of glycan structures was achieved with tandem mass spectrometry. The results of this study indicate that glycan structures are similar to endogenous glycans at low expression levels.