Identification of a novel glycan processing enzyme with exo-acting β-allosidase activity in the Golgi apparatus using a new platform for the synthesis of fluorescent substrates

Hakamata, Wataru; Miura, Kazuki; Hirano, Tetsufumi; Nishio, Takeshi

doi:10.1016/j.bmc.2014.11.023

Cited by 9 publications

(11 citation statements)

References 20 publications

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“…CaPR-1 was localized mainly in the cytosol and the nucleus, thougho ther fluorescences ignals, possibly in the Golgi apparatus, were also observed ( Supplementary Figure S2 b). [17] The oscillation of the intracellular Ca 2 + concentration in individual cells was clearly visualized (Figure 4f). We then added ionomycin, aC a 2 + ionophore, and the resultingC a 2 + influx into the cells was also detected (Figure 4f and Supplementary Movie S1).…”

Section: Resultsmentioning

confidence: 95%

Rational Design of a Near‐infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Takahashi

Hanaoka

Okubo

et al. 2020

Chemistry An Asian Journal

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Fluorescence imaging in the near‐infrared (NIR) region (650–900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)‐mediated fluorescence quenching of silicon‐ and phosphorus‐substituted rhodamines (SiRs and PRs) in order to guide the development of improved far‐red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+, CaPR‐1, and its membrane‐permeable acetoxymethyl derivative, CaPR‐1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far‐red to NIR fluorescence probe based on the SiR scaffold, CaSiR‐1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR‐1 showed longer‐wavelength absorption and emission (up to 712 nm) than CaSiR‐1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

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Section: Resultsmentioning

confidence: 95%

Rational Design of a Near‐infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Takahashi

Hanaoka

Okubo

et al. 2020

Chemistry An Asian Journal

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“…9) Previously, we developed fluorogenic substrates for a β-galactosidase based on the quinone methide cleavage (QMC)-substrate design platform. [10][11][12][13][14] and discovered unreported β-galactosidase activity in the Golgi apparatus in three human cell lines. 12) This newly discovered β-galactosidase activity is entirely different from that of GLB1 isoform 1 and galactocerebrosidase; however, we could not identify the protein responsible for this activity.…”

Section: Introductionmentioning

confidence: 99%

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi β-Galactosidase

Miura

Onodera

Takagi

et al. 2020

CHEMICAL & PHARMACEUTICAL BULLETIN

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The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit β-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its β-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated β-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel β-galactosidase activity in the Golgi apparatus of human cells; however, the protein responsible for this activity could not be identified. Inhibitor-derived chemical probes can serve as powerful tools to identify the responsible protein. In this study, we first constructed a cell-based high-throughput screening (HTS) system for Golgi β-galactosidase inhibitors, and then screened inhibitors from two compound libraries using the HTS system, in vitro assay, and cytotoxicity assay. An isoflavone derivative was identified among the final Golgi β-galactosidase inhibitor compound hits. Molecular docking simulations were performed to redesign the isoflavone derivative into a more potent inhibitor, and six designed derivatives were then synthesized. One of the derivatives, ARM07, exhibited potent inhibitory activity against β-galactosidase, with an IC 50 value of 14.8 µM and competitive inhibition with K i value of 13.3 µM. Furthermore, the in vitro and cellular inhibitory activities of ARM07 exceeded those of deoxygalactonojirimycin. ARM07 may contribute to the development of affinity-based chemical probes to identify the protein responsible for the newly discovered Golgi β-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.

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“…16 Furthermore, we previously reported that acetyl modification of substrate hydroxyl groups effectively improved membrane permeability without influencing the detection of glycosidase activity in living cells. 14,15 To assess the affinity of the designed substrates for the tFuc active site, we performed molecular docking simulations using the crystallographic structure of Thermotoga maritima α-Lfucosidase (PDB ID: 2ZXD 17 ) and three types of ligands: QMC platform-based substrates 4 through 6, α-1,6-fucoselinked GlcNAc as a natural substrate, and 2MeTG α-Lfucopyranoside (a model compound with 2MeTG instead of a commercial substrate). Due to the lack of a tFuc crystal structure, we used the T. maritima enzyme from the same glycoside hydrolase family as tFuc.…”

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confidence: 99%

“…Compound 10 was synthesized by Schmidt glycosylation 18 of compound 9, and the tertbutyldimethylsily group of 10 was subsequently deprotected to obtain compound 11. Compound 11 and a fluorophore (resorufin, TFMU, or 2MeTG) were reacted under Mitsunobu reaction conditions 14,15 to obtain acetylated fluorogenic substrates 1 through 3, after which substrates 4 through 6 were synthesized by deacetylation of substrates 1 through 3, respectively. Synthesis details, including 1 H NMR, 13 C NMR, MS, and elemental analysis (C, H, N, and F), are provided in the Supporting Information.…”

mentioning

confidence: 99%

“…For use as tFuc-specific fluorogenic substrates, it is common to conjugate these fluorophores at the C1 position of α- l -fucopyranoside similar to the commercial substrates. However, we previously raised a concern about the poor binding affinity of such designed substrates for the active site of tFuc, which is an exo -acting glycan hydrolase. , The active sites of hydrolases are bound by their respective substrates at the subsite −1, which recognizes a sugar residue at the nonreducing end of the substrate, and the subsite +1, which recognizes the penultimate sugar residue at the nonreducing end. Therefore, fluorophores representing these substrates might be incompatible with the subsite +1 due to steric hindrance, thereby requiring a different approach to design fluorogenic substrates for tFuc.…”

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confidence: 99%

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Development of Fluorogenic Substrates of α-l-Fucosidase Useful for Inhibitor Screening and Gene-expression Profiling

Miura

Tsukagoshi

Hirano

et al. 2019

ACS Med. Chem. Lett.

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Inhibitors of human α-l-fucosidases, tissue α-l-fucosidase (tFuc), and plasma α-l-fucosidase reportedly play roles in multiple diseases, suggesting their therapeutic potential for gastric disease associated with Helicobacter pylori and fucosidosis. Terminal fucose linkages on glycoproteins and glycolipids are a natural substrate for both enzymes; however, there are currently no fluorogenic substrates allowing their cellular evaluation. Here, we described the development of novel three-color fluorogenic substrates for lysosome-localized tFuc that exhibited excellent specificity and sensitivity in three human cell lines. Additionally, we developed a cell-based high-throughput inhibitor screening system in a 96-well format and a cell-based inhibitory activity evaluation system in a 6-well format for tFuc inhibitors using this substrate, which allowed accurate quantification of the inhibition rate. Moreover, analysis of significant changes in gene expression resulting from 30% inhibition of tFuc in HeLa cells revealed potential roles in gastric disease.

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Identification of a novel glycan processing enzyme with exo-acting β-allosidase activity in the Golgi apparatus using a new platform for the synthesis of fluorescent substrates

Cited by 9 publications

References 20 publications

Rational Design of a Near‐infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Rational Design of a Near‐infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi β-Galactosidase

Development of Fluorogenic Substrates of α-l-Fucosidase Useful for Inhibitor Screening and Gene-expression Profiling

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Identification of a novel glycan processing enzyme with exo-acting β-allosidase activity in the Golgi apparatus using a new platform for the synthesis of fluorescent substrates

Cited by 9 publications

References 20 publications

Rational Design of a Near‐infrared Fluorescence Probe for Ca2+ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Rational Design of a Near‐infrared Fluorescence Probe for Ca2+ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi β-Galactosidase

Development of Fluorogenic Substrates of α-l-Fucosidase Useful for Inhibitor Screening and Gene-expression Profiling

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Rational Design of a Near‐infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer

Rational Design of a Near‐infrared Fluorescence Probe for Ca²⁺ Based on Phosphorus‐substituted Rhodamines Utilizing Photoinduced Electron Transfer