Cryostorage of unstable <i>N</i>-acetylglucosaminyltransferase-V by synthetic zwitterions

Hirata, Tetsuya; Takekiyo, Takahiro; Yoshimura, Yukihiro; Tokoro, Yuko; Ishizaki, Takeru; Kizuka, Yasuhiko; Kuroda, Kosuke

doi:10.1039/d2ra01575g

Cited by 7 publications

(4 citation statements)

References 28 publications

(35 reference statements)

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“…34,35 Many reports have been published on other proteins as well. 2,34,35,[42][43][44][45][46][47][48] This current review is focused on living organisms, in particular, cells, which are the smallest units of living organisms, and higher organisms. First, the toxicity of [C 8 mim] +based ionic liquids (mentioned in the introduction) is reviewed.…”

Section: Introductionmentioning

confidence: 99%

A simple overview of toxicity of ionic liquids and designs of biocompatible ionic liquids

Kuroda

2022

New J. Chem.

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Section: Introductionmentioning

confidence: 99%

A simple overview of toxicity of ionic liquids and designs of biocompatible ionic liquids

Kuroda

2022

New J. Chem.

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“…12,13 In a previous study, we showed that a dilute aqueous OE 2 imC 3 C solution up to 10 wt % can be used to freeze Nacetylglucosaminyltransferase-V (GnT-V), which is an enzyme branching asparagine-linked glycosylation, involved in the aggravation of cancer, and can be used as a cryopreservation medium for deep freezing. 19 The activity of GnT-V in 10 wt % OE 2 imC 3 C solution after cryopreservation was found to be higher than that of a sample stored in an aqueous glycerol solution (20 vol %), which is a commonly used CPA. Furthermore, the percentage of residual GnT-V protein after freezing was also slightly higher in the 10 wt % OE 2 imC 3 C solution than that in the 20 vol % glycerol solution, suggesting that the OE 2 imC 3 C solution prevents the degradation or aggregation of GnT-V during cryopreservation.…”

Section: ■ Introductionmentioning

confidence: 90%

“…In a previous study, we showed that a dilute aqueous OE 2 imC 3 C solution up to 10 wt % can be used to freeze N -acetylglucosaminyltransferase-V (GnT-V), which is an enzyme branching asparagine-linked glycosylation, involved in the aggravation of cancer, and can be used as a cryopreservation medium for deep freezing . The activity of GnT-V in 10 wt % OE 2 imC 3 C solution after cryopreservation was found to be higher than that of a sample stored in an aqueous glycerol solution (20 vol %), which is a commonly used CPA.…”

Section: Introductionmentioning

confidence: 99%

Protein Cryoprotectant Ability of the Aqueous Zwitterionic Solution

Takekiyo,

Yamada,

Uto

et al. 2024

J. Phys. Chem. B

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Protein cryopreservation is important for the long-term storage of unstable proteins. Recently, we found that N-acetylglucosaminyltransferase-V (GnT-V) can be cryopreserved in a deep freezer without temperature control using a dilute binary aqueous solution of 3-(1-(2-(2-methoxyethoxy)ethyl)imidazol-3-io)butane-1-carboxylate (OE 2 imC 3 C) [10 wt %, mole fraction of solute (x) = 7.75 × 10 −3 ], an artificial zwitterion. However, it is unclear which solvent properties are required in these media to preserve unstable proteins, such as GnT-V. In this study, we investigated the melting phenomena and solution structure of dilute binary aqueous OE 2 imC 3 C solutions [x = 0−2.96 × 10 −2 (0−30 wt %)] using differential scanning calorimetry (DSC) and Raman and Fourier transform infrared (FTIR) spectroscopies combined with molecular dynamics (MD) simulation to compare the cryoprotectant ability of OE 2 imC 3 C with two general cryoprotectants (CPAs), glycerol and dimethyl sulfoxide. DSC results indicated that aqueous OE 2 imC 3 C solutions can be melted at lower temperatures with less energy than the control CPA solution, with increasing x, primarily due to OE 2 imC 3 C having a higher content of unfrozen water molecules. Moreover, Raman and FTIR results showed that the high content of unfrozen water molecules in aqueous OE 2 imC 3 C solutions was due to the hydration around the ionic parts (the COO − group and imidazolium ring) and the OCH 2 CH 2 O segment. In addition, the MD simulation results showed that there were fewer structured water molecules around the OCH 2 CH 2 O segment than the hydration water molecules around the ionic parts. These solvent properties suggest that dilute aqueous OE 2 imC 3 C solutions are effective in preventing freezing, even in a deep freezer. Therefore, this medium has the potential to act as a novel cryoprotectant for proteins in biotechnology and biomedical fields.

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“…We have proposed zwitterions, including OE 2 imC 3 C (Figure ), as novel cryoprotectants. − Zwitterions are not permeable to cells and do not affect the cytoplasm, which results in low cytotoxicity, − and zwitterions strongly inhibit ice crystallization. , Therefore, a zwitterion aqueous solution can efficiently cryopreserve various cells . However, this did not work for some cell lines.…”

Section: Introductionmentioning

confidence: 99%

Cell Damage Mechanisms during Cryopreservation in a Zwitterion Solution and Its Alleviation by DMSO

Ishizaki,

Tanaka,

Ishibashi

et al. 2024

J. Phys. Chem. B

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Recently, zwitterions have been proposed as novel cryoprotectants. However, some cells are difficult to cryopreserve using aqueous zwitterion solutions alone. We investigated here the reason for cell damage in such cells, and it was the osmotic pressure after freeze concentration. Furthermore, the addition of dimethyl sulfoxide (DMSO) has been reported to improve the cryoprotective effect in such cells: the zwitterion/DMSO aqueous solution shows a higher cryoprotective effect than the commercial cryoprotectant. This study also clarified the mechanisms underlying the improvement in a cryoprotective effect. The addition of cell-permeable DMSO alleviated the osmotic pressure after the freeze concentration. This alleviation was also found to be a key factor for cryopreserving cell spheroids, while there has been no insight into this phenomenon.

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Cryostorage of unstable N-acetylglucosaminyltransferase-V by synthetic zwitterions

Abstract: We report biocompatible materials for cryostorage of unstable proteins such as cancer-related enzyme, N-acetylglucosaminyltransferase-V (GnT-V).

Cited by 7 publications

References 28 publications

A simple overview of toxicity of ionic liquids and designs of biocompatible ionic liquids

A simple overview of toxicity of ionic liquids and designs of biocompatible ionic liquids

Protein Cryoprotectant Ability of the Aqueous Zwitterionic Solution

Cell Damage Mechanisms during Cryopreservation in a Zwitterion Solution and Its Alleviation by DMSO

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