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.

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.

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.

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.

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.

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.