戸谷 希一郎

In molecular biology research, a vitamin E (VE) vehicle (VE dissolved in organic solvent) is often added to water media without a stabilizer. However, the detailed behavior of VE colloids in water media is unclear. In this study, we reveal that VE nanoemulsion readily forms in water-based media through the existing protocol. The colloid size was changed from 39 nm to the submicron scale by adjusting the initial concentration of the VE solution and adding a buffer. The radical scavenging effect of the dispersed nanosized VEs is comparable to that of the water-soluble antioxidant Trolox, providing excellent antioxidant performance in colloid form. The cytoprotection effect of the VE colloids under a lipid oxidation condition largely depends on the size of the nanodispersion. Smaller dispersed particles are more efficient radical scavengers than larger particles for a constant VE amount owing to sophisticated uptake behavior of cell. This unveiled fundamental knowledge pave the way for a preparative protocol of stabilizer-free VE vehicles, which are expected to become widely used in molecular biology research.

Glycoprotein folding plays a critical role in sorting glycoprotein secretion and degradation in the endoplasmic reticulum (ER). Furthermore, relationships between glycoprotein folding and several diseases, such as type 2 diabetes and various neurodegenerative disorders, are indicated. Patients’ cells with type 2 diabetes, and various neurodegenerative disorders induce ER stress, against which the cells utilize the unfolded protein response for protection. However, in some cases, chronic and/or massive ER stress causes critical damage to cells, leading to the onset of ER stress-related diseases, which are categorized into misfolding diseases. Accumulation of misfolded proteins may be a cause of ER stress, in this respect, perturbation of oligomannose-type glycan processing in the ER may occur. A great number of studies indicate the relationships between ER stress and misfolding diseases, while little evidence has been reported on the connection between oligomannose-type glycan processing and misfolding diseases. In this review, we summarize alteration of oligomannose-type glycan processing in several ER stress-related diseases, especially misfolding diseases and show the possibility of these alteration of oligomannose-type glycan processing as indicators of diseases.

The stereoselectivity of the 1,2-cis-alpha-glucosylation reaction has been reported to be enhanced by introducing a tert-butyldimethylsilyl (TBS) protecting group into the glucosyl donor. Herein, the origin of this silyl-assist effect was identified using chemical experiments and density functional theory (DFT) calculations. Glucosylation reactions using various glucosyl donors with different TBS group positions showed that alpha-selectivity decreased when the TBS group position was further from the anomeric position. These systematic experiments showed that the silyl-assist effect was mainly due to electronic factors. Comparing DFT calculations of oxocarbenium intermediates derived from typical glucosyl donors showed that the introduced TBS group contributed to enhanced electron density at the anomeric position. Chemical experiments and DFT calculations showed that introducing a TBS group near the anomeric position stabilized the cationic intermediate and promoted alpha-selectivity.

A tri-antennary Man(9)GlcNAc(2) glycan on the surface of endoplasmic reticulum (ER) glycoproteins functions as a glycoprotein secretion or degradation signal after regioselective cleavage of the terminal alpha-1,2-mannose residue of each branch. Four alpha-1,2-mannosidases-ER mannosidase I, ER degradation-enhancing alpha-mannosidase-like protein 1 (EDEM1), EDEM2, and EDEM3-are involved in the production of these signal glycans. Although selective production of signal glycans is important in determining the fate of glycoproteins, the branch-discrimination abilities of the alpha-1,2-mannosidases are not well understood. A structural feature of the Man(9)GlcNAc(2) glycan is that all terminal glycosidic linkages of the three branches are of the alpha-1,2 type, while the adjacent inner glycosidic linkages are different. In this study, we examined whether the alpha-1,2-mannosidases showed branch specificity by discriminating between different inner glycosides. Four trisaccharides with different glycosidic linkages [Man alpha 1-2Man alpha 1-2Man (natural A-branch), Man alpha 1-2Man alpha 1-3Man (natural B-branch), Man alpha 1-2Man alpha 1-6Man (natural C-branch), and Man alpha 1-2Man alpha 1-4Man (unnatural D-branch)] were synthesized and used to evaluate the hypothesis. When synthesizing these oligosaccharides, highly stereoselective glycosylation was achieved with a high yield in each case by adding a weak base or tuning the polarity of the mixed solvent. Enzymatic hydrolysis of the synthetic trisaccharides by a mouse liver ER fraction containing the target enzymes showed that the ER alpha-1,2-mannosidases had clear specificity for the trisaccharides in the order of A-branch > B-branch > C-branch approximate to D-branch. Various competitive experiments have revealed for the first time that alpha-1,2-mannosidase with inner glycoside specificity is present in the ER. Our findings suggest that exo-acting ER alpha-1,2-mannosidases can discriminate between endo-glycosidic linkages.

N-glycans are attached to newly synthesised polypeptides and are involved in the folding, secretion, and degradation of N-linked glycoproteins. In particular, the calnexin/calreticulin cycle, which is the central mechanism of the entry and release of N-linked glycoproteins depending on the folding sates, has been well studied. In addition to biological studies on the calnexin/calreticulin cycle, several studies have revealed complementary roles of in vitro chemistry-based research in the structure-based understanding of the cycle. In this mini-review, we summarise chemistry-based results and highlight their importance for further understanding of the cycle.

About half of all newly synthesized proteins have N-linked glycans. These glycans play pivotal roles in controlling the folding, sorting, and degradation of glycoproteins via several glycan-related proteins. The glycan-mediated protein quality control system is important for cellular homeostasis. In this review, we summarize recent advances in our understanding of the system and discuss structural insights from chemical and biological perspectives. In particular, we focus on the mechanisms by which these mediators respond to several folding states of glycoproteins.

We demonstrate the preferential orders of molecular chaperones glucose-regulated protein 94 (GRP94), binding immunoglobulin protein (BiP), and calreticulin (CRT) in an endoplasmic reticulum (ER) fraction from rat liver using columns conjugated with denatured myoglobin, RNase A, or β-lactoglobulin as client proteins in the presence or absence of ATP. The results showed that BiP, CRT, and GRP94 preferentially contributed myoglobin, RNase A, and β-lactoglobulin, respectively, in the presence of ATP. In the absence of ATP, GRP94 and CRT preferentially recognized misfolded myoglobin (α-helix-rich protein), whereas BiP preferentially recognized misfolded RNase A (α-helix/β-sheet mixed protein) and β-lactoglobulin (β-sheet-rich protein). The preferential order of ER chaperones may be dynamically regulated by ER conditions and the higher-order structure of client proteins.

Deglucosylation and reglucosylation of glycoproteins by glucosidase II and uridine diphosphate-glucose: glycoprotein glucosyltransferase 1 (UGGT1), respectively, are important steps in glycoprotein quality control. Misfolded glycoprotein accumulation is associated with endoplasmic reticulum stress and can lead to protein misfolding diseases such as metabolic syndrome. Here, we analyzed the expression and activities of glucosidase II and UGGT1 in rat models of obesity and obese type 2 diabetes, and phenotypes associated with moderate and severe metabolic syndrome, respectively. In obesity, the mRNA and protein levels of glucosidase II and UGGT1 are decreased and their activities are reduced. In obese type 2 diabetes, the mRNA and protein levels of these enzymes are increased, and glucosidase II activity is slightly recovered, although UGGT1 activity is reduced. Our findings suggest that metabolic syndrome affects deglucosylation/reglucosylation enzymes according to disease severity.

Cancer research is increasingly focused on discovering strategies to induce cancer cell apoptosis without affecting surrounding normal cells. One potential biocompatible method is mechanical vibration, which has been developed as part of the emerging field of mechanomedicine. Previous studies of mechanical vibration have employed high-frequency vibration, which damages healthy cells. In this study, we examined the effects of brief (1 h) low-frequency (20 Hz) mechanical vibration on glucose consumption and survival (apoptosis, necrosis, HMGB1 release) of the human epidermoid carcinoma cell line A431. We found that apoptosis, but not necrosis, was significantly increased at 48 h after mechanical vibration compared with cells maintained in static culture. In keeping with this, extracellular release of HMGB1, a necrosis marker, was lower in cultures of A431 cells subjected to mechanical vibration compared with control cells. Glucose consumption was increased in the first 24 h after mechanical vibration but returned to control levels before the onset of apoptosis. Although the precise intracellular mechanisms by which low-frequency mechanical vibration triggers apoptosis of A431 cells is unknown, these results suggest a possible role for metabolic pathways. Mechanical vibration may thus represent a novel application of mechanomedicine to cancer therapy.

Golgi endo-α-mannosidase (G-EM) catalyzes an alternative deglucosylation process for N-glycans and plays important roles in the post-endoplasmic reticulum (ER) quality control pathway. To understand the post-ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G-EM based on a fluorescence quenching assay. The probe was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and a 2,4-dinitrophenyl group as a quencher at the reducing end. This probe is hydrolyzed to disaccharide derivatives by G-EM, resulting in increased fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of disaccharide derivative present, allowing the G-EM activity to be evaluated easily and quantitatively.

Bioengineering research and applications are supported by cell culture technologies that produce a large number of homogeneous cells. However, trypsin used in the general culture procedure for cell detachment decreases cell activity and culture efficiency. Furthermore, manually conducted culture procedures, especially pipetting after trypsin treatment, can induce inhomogeneous mechanical stress in cells, which may influence cellular functions. Alternate detachment methods using specialized culture devices without trypsin and/or manual pipetting have been reported. However, conventional trypsinization is still widely used. Diluted trypsin increases culture efficiency. Therefore, we developed a cell-detaching method using diluted trypsin and ultrasonic vibration for cell detachment from ubiquitous culture vessels. To demonstrate our concept, we used a T25 flask. Vibration of the culture surface was excited by ultrasonic waves propagated from an ultrasonic transducer placed under the flask. Using the proposed method, cells were completely detached by diluted trypsin, whereas 8.6% of cells remained on the flask with manual pipetting. The viability and proliferation of cells detached by the proposed method were higher than those of cells detached by the conventional method, owing to the low concentration of trypsin. Furthermore, glucose consumption after detachment showed no abnormality, eliminating possible oncogenesis. Two membrane proteins were quantified immediately after detachment and at 24 h of culture, and there were no differences between the detachment methods. Thus, we conclude that our proposed method improves culture efficiency without any adverse effects and ensures homogeneous mechanical stress on cells.

Cellular aggregates that mimic cell-cell interactions in vitro are essential for biological research. This study introduces a method to form large scaffold-free 3-D aggregates in a clinically ubiquitous cell culture dish using kilohertz-order ultrasound standing wave trapping (USWT). We fabricated an aggregate formation system in which a 60-mm dish was set above a Langevin transducer via water. The transducer was excited at 110.8 kHz, and then C2C12 myoblasts were injected into the dish and trapped at the node position of the standing wave. The diameter and thickness of the formed aggregate were 8 and 2.7 mm, respectively, which are larger than those of aggregates formed previously by USWT. Moreover, we confirmed that >94% of cells constituting the aggregates survived 9 h, and the protein expression of cells was not altered significantly. This method can be applied to form aggregates with high functionality, which contributes to the development of biological research methodology.

<p>近年，細胞の医療応用に注目が集まっている．これに伴って効率的な細胞培養法が研究されており，とくに細胞剥離に関する研究が盛んである．一般的にはトリプシンに浸漬した細胞に対してピペッティング等の機械刺激を付与することで細胞を剥離する．トリプシンの毒性を鑑みると，低濃度のトリプシンを用いることが望ましいが，細胞の剥離率が低下してしまう．そこで，本研究では，超音波の併用よって低濃度のトリプシンによる細胞剥離を実現した．汎用フラスコに接着した接着細胞の下方から超音波を付与する装置（図）を開発した．さらに，バースト波を用いた超音波照射によって温度上昇を抑制しつつ，高強度の超音波を細胞に付与した．この結果，ピペッティングでは一部の細胞のみが剥離する低濃度トリプシンを用いた際にも，超音波の照射によって全細胞が剥離した．低濃度トリプシンで細胞剥離を実現したことで細胞の増殖性が向上したこと，超音波照射によるタンパク質発現の変化や代謝に異常がないことを確認した．</p>

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

Cell detachment is an essential process in adherent cell culture. However, trypsinization, which is the most popular detachment technique used in culture, damages cellular membranes. Reducing cellular membrane damage during detachment should improve the quality of cell culture. In this article, we propose an enzyme-free cell detachment method based on resonance vibration with temperature modulation. We developed a culture device that can excite a resonance vibration and control temperature. We then evaluated the cell detachment ratio and the growth response, observed the morphology, and analyzed the cellular protein of the collected cellsmouse myoblast cell line (C2C12). With the temperature of 10 degrees C and the maximum vibration amplitude of 2m, 77.9% of cells in number were successfully detached compared with traditional trypsinization. The 72-h proliferation ratio of the reseeded cells was similar to that with trypsinization, whereas the proliferation ratio of proposed method was 12.6% greater than that of trypsinization after freezing and thawing. Moreover, the cells can be collected relatively intact and both intracellular and cell surface proteins in the proposed method were less damaged than in trypsinization. These results show that this method has definite advantages over trypsinization, which indicates that it could be applied to subcultures of cells that are more susceptible to trypsin damage for mass culture of sustainable clinical use. Biotechnol. Bioeng. 2017;114: 2279-2288. (c) 2017 Wiley Periodicals, Inc.

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-alpha-mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-alpha-mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from alpha-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 Man alpha 1-2Man. The synthetic utility of the enzyme was demonstrated by generation of a high-mannose-type undecasaccharide (Glc(1)Man(9)GlcNAc(2)).

Within the endoplasmic reticulum, immature glycoproteins are sorted into secretion and degradation pathways through the sequential trimming of mannose residues from Man(9)GlcNAc(2) to Man(5)GlcNAc(2) by the combined actions of assorted -1,2-mannosidases. It has been speculated that specific glycoforms encode signals for secretion and degradation. However, it is unclear whether the specific signal glycoforms are produced by random mannosidase action or are produced regioselectively in a sequenced manner by specific -1,2-mannosidases. Here, we report the identification of a set of selective mannosidase inhibitors and development of conditions for their use that enable production of distinct pools of Man(8)GlcNAc(2) isomers from a structurally defined synthetic Man(9)GlcNAc(2) substrate in an endoplasmic reticulum fraction. Glycan processing analysis with these inhibitors provides the first biochemical evidence for selective production of the signal glycoforms contributing to traffic control in glycoprotein quality control.

Glycoprotein N-linked oligosaccharides in the endoplasmic reticulum function as tags to regulate glycoprotein folding, sorting, secretion and degradation. Since the N-glycan structure of a glycoprotein should reflect the folding state, N-glycan processing may be affected by the aglycone state. In this study, we examined the influence of aglycone structures on N-glycan processing using synthetic substrates. We prepared (Glc(1))Man(9)GlcNAc(2) linked to hydrophobic BODIPY-dye with a systematic series of different linker lengths. With these compounds, glucose transfer, glucose trimming and mannose trimming reactions of an endoplasmic reticulum fraction were examined. The results showed that substrates with shorter linkers between the N-glycan and hydrophobic patch had higher activities for both the glucose transfer and the mannose trimming reactions. In contrast, the glucose trimming reaction showed lower activity when substrates had shorter linkers. Thus, the reactivity for N-linked oligosaccharide processing of glycoproteins in the endoplasmic reticulum might be tunable by the aglycone structure, e.g., protein portion of glycoproteins. (C) 2016 Elsevier Ltd. All rights reserved.

Background: Peptide: N-glycanase is a deglycosylation enzyme releasing N-glycan from glycoproteins. Although glycan specificity analysis of this enzyme has been reported, recognition requirements for the peptide sequence have not been precisely elucidated. Objective: In this study, we carried out peptide specificity analysis of several peptide: N-glycanases. Methods: Using synthetic chitobiose-pentapeptide substrates having a systematic series of amino acid sequences composed of hydrophobic leucine and hydrophilic serine, we examined the peptide specificities of peptide: N-glycanases comprising yeast cytoplasmic PNGase, bacterial PNGase F, and plant PNGase A by ultra-performance liquid chromatography combined with electrospray ionization mass spectrometry. Results: We found that each of the PNGases had higher activity for the more hydrophobic (leucine-rich) chitobiose-pentapeptides, although the sensitivities of the PNGases for hydrophobicity varied. Cytoplasmic PNGase showed broad specificity. In contrast, PNGase A showed moderate specificity. PNGase F showed the highest specificity. Conclusion: PNGases from different origins had similar but significantly independent peptide specificities.

Background: Megalin is a 600-kDa single-spanning transmembrane glycoprotein and functions as an endocytic receptor, distributed not only in the kidney but also in other tissues. Structurally and functionally distinct ligands for megalin have been identified. Megalin has 30 potential N-glycosylation sites in its extracellular domain. We found that megalin interacts with its ligands in a glycoform-dependent manner. Methods: Distribution of megalin and glycans was histochemically analyzed in mouse kidneys. Kidney absorption of Cy5-labeled ligands was examined in vivo. Megalin-ligand interactions were analyzed using ligand blotting and ELISA. Results: Megalins expressed on renal proximal convoluted tubules (Pas) and proximal straight tubules (PSTs) have different N-glycans. PCT megalin stained with Lens culinaris agglutinin (LCA), which recognizes core-fucosyl N-glycans catalyzed by alpha 1,6-fucosyltransferase (Fut8). In contrast, PST megalin stained with wheat germ agglutinin (WGA), which recognizes hybrid-type N-glycans. Retinol-binding protein-Cy5 (RBP-Cy5) was endocytosed by megalin on PCTs but minimally endocytosed by PSTs. BSA-Cy5 was endocytosed nearly equally by both tubules. The purified LCA-positive glycoform megalin had higher binding activity for RBP and vitamin D-binding protein than did WGA-positive glycoform megalin. Both glycoforms had nearly the same BSA- and kanamycin-binding activities. RBP-binding analysis of megalin lacking core fucose, in Fut8(-/-) mouse kidneys, had significantly decreased binding activity. Conclusions: N-Glycosylation of megalin can modulate its ligand-binding activity. Core fucosylation, in particular, is a modification crucial for megalin-RBP interactions. General significance: Cell type-specific glycoforms of megalin exist in the proximal tubular cells and modulate ligand absorption capacity. (C) 2016 Elsevier B.V. All rights reserved.

BACKGROUND: Megalin is a 600-kDa single-spanning transmembrane glycoprotein and functions as an endocytic receptor, distributed not only in the kidney but also in other tissues. Structurally and functionally distinct ligands for megalin have been identified. Megalin has 30 potential N-glycosylation sites in its extracellular domain. We found that megalin interacts with its ligands in a glycoform-dependent manner. METHODS: Distribution of megalin and glycans was histochemically analyzed in mouse kidneys. Kidney absorption of Cy5-labeled ligands was examined in vivo. Megalin-ligand interactions were analyzed using ligand blotting and ELISA. RESULTS: Megalins expressed on renal proximal convoluted tubules (PCTs) and proximal straight tubules (PSTs) have different N-glycans. PCT megalin stained with Lens culinaris agglutinin (LCA), which recognizes core-fucosyl N-glycans catalyzed by α1,6-fucosyltransferase (Fut8). In contrast, PST megalin stained with wheat germ agglutinin (WGA), which recognizes hybrid-type N-glycans. Retinol-binding protein-Cy5 (RBP-Cy5) was endocytosed by megalin on PCTs but minimally endocytosed by PSTs. BSA-Cy5 was endocytosed nearly equally by both tubules. The purified LCA-positive glycoform megalin had higher binding activity for RBP and vitamin D-binding protein than did WGA-positive glycoform megalin. Both glycoforms had nearly the same BSA- and kanamycin-binding activities. RBP-binding analysis of megalin lacking core fucose, in Fut8-/- mouse kidneys, had significantly decreased binding activity. CONCLUSIONS: N-Glycosylation of megalin can modulate its ligand-binding activity. Core fucosylation, in particular, is a modification crucial for megalin-RBP interactions. GENERAL SIGNIFICANCE: Cell type-specific glycoforms of megalin exist in the proximal tubular cells and modulate ligand absorption capacity.

The metabolic syndrome including obesity and diabetes mellitus is known to be a major health problem worldwide. A recent study reported that obesity causes endoplasmic reticulum (ER) stress and subsequently leads to insulin resistance and type 2 diabetes. However, little is known about the alterations in the components of the calnexin/calreticulin (CNX/CRT) cycle, which promote glycoprotein folding in obese and diabetic conditions. To understand the operating status of the lectin-like chaperones related to the CNX/CRT cycle in the metabolic syndrome, we analyzed the chaperones for the activity, protein expression, and mRNA expression levels using Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rat models for obesity and diabetes, respectively. We demonstrated that misfolded proteins were gradually increased with progression of the syndrome, obesity to diabetes. The individual chaperone activities of CNX and CRT were both decreased in the ZF rat ER and, in contrast, were increased in the ZDF rat ER. The protein quantities and mRNA expressions of CNX and CRT were decreased in the ZF rats, but increased in the ZDF rats compared with those of the healthy model. Therefore, these results indicate that obesity down-regulates CNX and CRT expressions and their activities and diabetes up-regulates the expressions and activities of CNX and CRT. Our findings clearly suggest that metabolic syndrome affects the lectin-like chaperones in the CNX/CRT cycle at both the activity and expression levels. (C) 2016 Elsevier Inc. All rights reserved.

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-Bu-t) 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-Bu-t &gt; -Gly-NH2 &gt; -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. (C) 2015 Elsevier Inc. All rights reserved.

Calreticulin (CRT) has been described as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However whether CRT directly recognizes aglycone (protein portion) of glycoprotein remains controversial. Our previous study demonstrated that CRT competitively inhibited glucosidase II activity against a hydrophobic substrate not but that against a hydrophilic substrate, implying that CRT recognizes not only glycan structure but also aglycone hydrophobicity. Here to investigate the possibility we prepared ligands for CRT: G1M9-derivatives with different hydrophobicity on the aglycone and gradually denatured immunoglobulin Y (IgY), which harbors G1M9 glycan, and analyzed the interaction of a recombinant CRT with the ligands using thermal shift assay. These results demonstrate that CRT strongly binds to more hydrophobic G1M9-derivative and more denatured IgY, clarifying that CRT more strongly binds misfolded glycoprotein, and reversely releases folded glycoprotein by distinguishing the folding status of glycoprotein in the ER glycoprotein quality control system.

© The Royal Society of Chemistry 2015. A highly stereoselective 1,2-cis-α-glucosylation reaction was developed, in which the use of a strongly electron-donating tert-butyldimethylsilyl protecting group on the C-2 hydroxy group of a glycosyl donor enhanced the α-favoured transition state, and thus resulted in high yield and α-selectivity. Synthetic utility of this α-glucosylation was demonstrated by the generation of a triglucoside moiety in high-mannose-type oligosaccharides.

Investigating the relative efficiencies of molecular chaperones is important for understanding protein biosynthesis inside a cell. We developed an analytical method for estimating relative chaperone activity under physiological, multi-chaperone conditions using a protein-conjugated column. A chaperone mixture was subjected to chromatography on a column conjugated with denatured ovalbumin, and the elution positions of target chaperones were compared using western blotting to determine the relative affinity of each chaperone for the denatured protein. Because molecular chaperones should be eluted according to their strength of association with the denatured ovalbumin in the column, the elution position must accord with the chaperone activity and can be used as an indicator of relative chaperone activity. We found that the column procedure was effective in an assay of a mixture of calreticulin and BiP, the molecular chaperones in the endoplasmic reticulum; the assay showed that calreticulin associated with denatured ovalbumin more strongly than BiP. (C) 2014 Elsevier Inc. All rights reserved.

A systematic series of chitobiose-modified pentapeptides with sequence variations of hydrophobic leucine and hydrophilic serine were synthesized. The resulting glycopeptides were used as molecular probes to elucidate aglycon peptide specificity of the glycoprotein-folding sensor enzyme UGGT. Inhibitory experiments with a synthetic fluorescent glyco-substrate and the glycopeptides revealed that UGGT prefers a serine residue directly linked to C-terminal of the N-glycosylation site in its substrate recognition. (C) 2014 Elsevier Ltd. All rights reserved.

Compared with in vitro conditions, the intracellular environment is highly crowded with biomolecules; this has numerous effects on protein functions, including enzymatic activity. We examined the effects of macromolecular crowding on glycan processing of N-glycoprotein in the endoplasmic reticulum as a model sequential metabolic pathway. Experiments with synthetic substrates of physiological glycan structure clearly showed that the first half of the pathway (glucose trimming) was accelerated, whereas the second (mannose trimming) was decelerated under molecular crowding conditions. Furthermore, calreticulin, a lectin-like molecular chaperone, bound more strongly to a glycan-processing intermediate under these conditions. This study demonstrates the diverse effects of molecular crowding on sequential enzymatic processing, and the importance of the effects of macromolecular crowding on in vitro assays for understanding sequential metabolic pathways.

Glycoprotein oligosaccharides function as tags for protein quality control in the endoplasmic reticulum (ER). Since most of proteins are glycosylated and function only after they are properly folded, glycoprotein glycan profiles in the ER might be useful to analyze various cellular status including diseases. Here, we examined whether ER glycan-processing profiles in diabetic rats and osteoporotic mice as models might have different cellular status from those of normal controls. Direct analysis of glycoprotein-processing profiles in the ER is often hampered by glycoforms that are retro-translocated to the ER from other cellular compartments. Moreover, when we focus on the mixture of glycoproteins as the processing substrates, the glycan-processing efficiencies are influenced by the aglycon states including their polypeptide folding. To overcome this problem, we reconstructed glycan profiles using ER extracts as an enzymatic source and synthetic glycoprotein mimetic having homogeneous aglycon as a substrate, resulted in disease-specific glycan profiles. To understand such differences, we also analyzed the activity, and expression level, of each glycan-related enzyme. These glycan profiles are expected to be useful indexes for operational status of the ER glycoprotein quality control, and may also give information to classify some diseases. © The Author 2012. Published by Oxford University Press. All rights reserved.

The hyaluronan that is present in extracellular matrices forms chiral mesh-like structures in aqueous media. To understand the influence of specific environments on the stereoselectivity of chemical reactions, organocatalysis-mediated aldol reactions in the presence of hyaluronan were investigated in water. In these experiments, changes in the diastereomeric ratio were observed in an aldol reaction under hyaluronan conditions.

In this article, the authors summarize a variety of stereoselective carbon-carbon bond-forming reactions realized by the use of sugar-derived chiral templates, which have been one of the active concerns in the authors&#039; group in these fifteen years. Representative chiral templates prepared from D-glucose, D-galactose, or D-mannose are introduced first. Then, the utility of these templates for stereoselective carbon-carbon bond-forming reactions are outlined. These reactions include, 1)1,4-conjugate additions, 2)α-alkylations of esters, 3)Diels-Alder cycloadditions, 4)1,3-dipolar cycloadditions, and 5)α,α-dialkylation for the construction of an all-carbon asymmetric quaternary center. The use of a D-glucose derivative equipped with two bulky silyl ethers at C2 and C3 is emphasized throughout the article as the most effective chiral template.

Investigating unidentified weak-acting lectins is important for understanding glycan-related phenomena. We have developed an improved screening method for weak-acting lectins using glycan-conjugated magnetic beads (or glycobeads) involving a partial washing method and named it the mild enrichment procedure. Weak-acting lectins exist in equilibrium between bound lectin and free lectin produced by dissociation, whereas most tight-binding lectin exists in a bound state. The conventional washing step, in which the solution phase is replaced, may remove dissociated lectin from around the glycobeads; therefore, we attempted to leave a buffer space around the glycobeads to maintain the association-dissociation equilibrium of weak-acting lectins. Our results revealed that our mild enrichment procedure for screening for weak interactions, such as maltose-concanavalin A (K(a) similar to 10(4) M(-1)) and lactose-peanut agglutinin (K(a) similar to 10(3) M(-1)) interactions, was more effective than conventional batch methods. (C) 2010 Elsevier Inc. All rights reserved.

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(9)GlcNAc(2), providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase. (C) 2010 Elsevier Inc. All rights reserved.

Bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene; BCD) has been known as an inhibitor of glucosidase II (G-II), which plays pivotal roles in glycoprotein processing and folding in the ER. Previous works suggested that BCD specifically inhibits the cleavage of the innermost glucose (Glc) among two alpha 1-3 linked Glc residues (cleavage-2). This study addressed the mode of BCD&apos;s inhibition toward G-II by using fluorescently labeled substrates. Our analysis clarified that BCD inhibits both cleavage-1 and cleavage-2 activities of G-II. However, the inhibitory activity toward cleavage-2 was 6-fold higher than that toward cleavage-1. Inhibition against both of these activities was retained after dialysis, supporting that BCD exhibits inhibition through irreversible binding to G-II. (C) 2009 Published by Elsevier Ltd.

Nascent polypeptides emerging into the lumen of the endoplasmic reticulum (ER) undergo high-mannose-type glycan modifications to the asparagines in their Asn-Xaa-Ser/Thr motifs. The processing of these oligosaccharides has been shown to play important roles in protein quality control. This process is highly complex, involving a number of lectins, chaperones, and glycan-processing enzymes. For example, calnexin (CNX) and calreticulin (CRT) are molecular chaperones that recognize monoglucosylated high-mannose-type glycans. Glucosidase II has two independent Glc-trimming activities, which participate in glycoprotein delivery to CNX/CRT to modify their conformations and glycoprotein recovery from the molecular chaperones. The protein folding sensor enzyme UDP-glucose : glycoprotein glucosyltransferase (UGGT) specifically glucosylates high-mannose-type glycans attached to partially folded proteins to deliver the candidate glycoprotein to CNX/CRT. Although recent studies have clarified the properties of these proteins, most of them used oligosaccharides derived from natural sources, which show structural heterogeneity. In order to gain a more precise understanding of this process, we attempted to synthesize high-mannose-type glycans associated with the protein quality control system. Additionally, an investigation using artificial glycoproteins led us to discover the first nonpeptidic substrate of UGGT. These synthetic oligosaccharide probes have allowed us to conduct quantitative evaluations of the activity and specificity of UGGT, glucosidase II, and CRT.

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.

Glucosidase II (GII) is a glycan-processing enzyme that trims two alpha 1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first alpha 1,3-linked glucose from Glc(2)Man(9)GlcNAc(2) (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc(1)Man(9)GlcNAc(2) (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic alpha subunit (GII alpha) and a tightly associated beta subunit (GII beta) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GII beta (GII beta-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GII beta-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GII beta-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GII beta-MRH binds most strongly to the glycans with the alpha 1,2-linked mannobiose structure. GII with the mutant GII beta that lost the sugar-binding activity of GII beta-MRH hydrolyzes p-nitrophenyl-alpha-glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GII beta-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.