Publications by authors named "Siro Simizu"

81 Publications

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

Nat Commun 2021 02 24;12(1):1261. Epub 2021 Feb 24.

Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.
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http://dx.doi.org/10.1038/s41467-021-21396-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904790PMC
February 2021

Identification of madangamine A as a novel lysosomotropic agent to inhibit autophagy.

Bioorg Med Chem 2021 Mar 27;34:116041. Epub 2021 Jan 27.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Japan. Electronic address:

Madangamines are marine natural products isolated from Xestospongia ingens, and madangamine A-E with a different D-ring structure have been reported. We have reported that madangamine A has strong anti-proliferative activity against various human cancer cell lines. In this study, to clarify the anti-proliferative activity of madangamine A, we searched for molecular target of the madangamine A in human cells. Treatment with madangamine A increased the levels of LC3-II and p62, autophagy-related proteins, concomitant with growth inhibition. Moreover, madangamine A resulted in lysosome enlargement and increase in lysosomal pH, which are same phenomena observed in chloroquine-treated cells. These results suggest that madangamine A is a novel lysosome inhibitor, and the anti-proliferative activity of madangamine A is due to the inhibition of lysosome function.
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http://dx.doi.org/10.1016/j.bmc.2021.116041DOI Listing
March 2021

Regulation of N-glycosylation and secretion of Isthmin-1 by its C-mannosylation.

Biochim Biophys Acta Gen Subj 2021 03 4;1865(3):129840. Epub 2021 Jan 4.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address:

Background: C-mannosylation is a type of protein glycosylation. Human Isthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C-mannosylation sequences at Trp and Trp. In this study, we sought to examine the role of C-mannosylation in the secretion of ISM1.

Methods: We established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy.

Results: We found that ISM1 is C-mannosylated at Trp and Trp in the TSR domain. To determine the functions of the C-mannosylation of ISM1, we established a C-mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N-glycosylated only in these C-mannosylation-defective cells.

Conclusions: ISM1 is C-mannosylated in its TSR domain, and the status of the C-mannosylation of ISM1 affects its N-glycosylation.

General Significance: The C-mannosylation of ISM1 regulates its N-glycosylation status.
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http://dx.doi.org/10.1016/j.bbagen.2020.129840DOI Listing
March 2021

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4).

Biochim Biophys Acta Gen Subj 2021 03 29;1865(3):129833. Epub 2020 Dec 29.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Japan. Electronic address:

Background: C-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear.

Methods: We established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan.

Results: We identified that ADAMTS4 is C-mannosylated at Trp in the metalloprotease domain and at Trp, Trp, and Trp in the thrombospondin type 1 repeat (TSR). The replacement of Trp with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp, Trp, and Trp with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity.

Conclusions: Our results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity.

General Significance: Because C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.
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http://dx.doi.org/10.1016/j.bbagen.2020.129833DOI Listing
March 2021

Seven-Step Synthesis of All-Nitrogenated Sugar Derivatives Using Sequential Overman Rearrangements.

Angew Chem Int Ed Engl 2021 03 28;60(10):5193-5198. Epub 2021 Jan 28.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan.

All-nitrogenated sugars (ANSs), in which all hydroxy groups in a carbohydrate are replaced with amino groups, are anticipated to be privileged structures with useful biological activities. However, ANS synthesis has been challenging due to the difficulty in the installation of multi-amino groups. We report herein the development of a concise synthetic route to peracetylated ANSs in seven steps from commercially available monosaccharides. The key to success is the use of the sequential Overman rearrangement, which enables formal simultaneous substitution of four or five hydroxy groups in monosaccharides with amino groups. A variety of ANSs are available through the same reaction sequence starting from different initial monosaccharides by chirality transfer of secondary alcohols. Transformations of the resulting peracetylated ANSs such as glycosylation and deacetylation are also demonstrated. Biological studies reveal that ANS-modified cholesterol show cytotoxicity against human cancer cell lines, whereas each ANS and cholesterol have no cytotoxicity.
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http://dx.doi.org/10.1002/anie.202015141DOI Listing
March 2021

The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4.

Biochim Biophys Acta Gen Subj 2020 09 19;1864(9):129637. Epub 2020 May 19.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Japan. Electronic address:

Background: C-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation.

Methods: We established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope.

Results: We found that MFAP4 is C-mannosylated at Trp in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion.

Conclusions: Our results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion.

General Significance: These results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.
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http://dx.doi.org/10.1016/j.bbagen.2020.129637DOI Listing
September 2020

Cell-dependent regulation of vasculogenic mimicry by carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1).

Biochem Biophys Rep 2020 Mar 30;21:100734. Epub 2020 Jan 30.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Japan.

Vasculogenic mimicry (VM) promotes tumor migration, metastasis, and invasion in various types of cancer, but the relationship between VM and these phenotypes remains undefined. In this study, we examined carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) as a novel target of VM. We found that ectopic expression of CEACAM1 in HT1080 human fibrosarcoma cells suppressed the formation of a VM-like network. Further, cell migration and proliferation were abated by the introduction of CEACAM1 into HT1080 cells. Conversely, knockout (KO) of the CEACAM1 gene in SK-MEL-28 melanoma cells, which normally express high levels of CEACAM1, inhibited formation of a VM-like network, which was covered on reintroduction of CEACAM1. These results suggest that CEACAM1 differentially regulates formation of the VM-like network between cancer cell types and implicate CEACAM1 as a novel therapeutic target in malignant cancer.
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http://dx.doi.org/10.1016/j.bbrep.2020.100734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997815PMC
March 2020

Identification of vibsanin A analog as a novel HSP90 inhibitor.

Bioorg Med Chem 2020 01 9;28(2):115253. Epub 2019 Dec 9.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address:

Vibsanin A is the first natural product isolated from Viburnum awabuki and has several biological activities. We have reported that a vibsanin A analog, obtained from process of total synthesis of vibsanin A, has anti-proliferative activity against human cancer cell lines. In this study, we evaluated anti-proliferative effect of the vibsanin A analogs against various human cancer cell lines and examined molecular target of the analog in human cells. Among the vibsanin A analogs, vibsanin A analog C (VAC) showed anti-proliferative effect against various cancer cell lines, and the anti-proliferative activity was strongest among the vibsanin A analogs. Additionally, VAC fluctuated amounts of HSP90-related proteins in cells and inhibited HSP90-mediated protein refolding of luciferase in vitro. These results suggest that the anti-proliferative activity of VAC is due to HSP90 inhibition, and VAC has a potential as novel anti-cancer drug as HSP90 inhibitor.
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http://dx.doi.org/10.1016/j.bmc.2019.115253DOI Listing
January 2020

Identification and characterization of collagen-like glycosylation and hydroxylation of CCN1.

Glycobiology 2019 09;29(10):696-704

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.

CCN1 is a secreted protein and belongs to the CCN family of matricellular proteins. CCN1 binds to various cell surface receptors; thus, CCN1 has important functions in cell proliferation, migration and angiogenesis through a variety of signaling pathways. We have reported that CCN1 is O-fucosylated and that this O-fucosylation regulates the secretion of CCN1 into the extracellular region. In this study, we detected collagen-like glycosylation and hydroxylation at Lys203 of recombinant CCN1 by mass spectrometry. We then examined the role of collagen-like glycosylation in the functions of CCN1. As a result, we found that a deficiency in collagen-like glycosylation decreased the secretion of CCN1 using wild-type CCN1- and collagen-like glycosylation-defective mutant CCN1-overexpressing cell lines. Further, knockout of lysyl hydroxylase3, a multifunctional protein with hydroxylase and glucosyltransferase activities, impaired the secretion and glycosylation level of recombinant CCN1. Previous studies reported that collagen glycosylation of Lys residues mediated by lysyl hydroxylase3 is glucosyl-galactosyl-hydroxylation, presuming that this collagen-like glycosylation detected at Lys203 of recombinant CCN1 in this study might be glucosyl-galactosyl-hydroxylation. Taken together, our results demonstrate the novel function of the collagen-like glycosylation of CCN1 and suggest that lysyl hydroxylase3-mediated glycosylation is important for CCN1 secretion.
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http://dx.doi.org/10.1093/glycob/cwz052DOI Listing
September 2019

Identification of topoisomerases as molecular targets of cytosporolide C and its analog.

Bioorg Med Chem 2019 08 6;27(15):3334-3338. Epub 2019 Jun 6.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address:

Cytosporolide (Cytos) A-C, isolated from the fungus Cytospora sp., have anti-microbial activity, but their molecular targets in mammalian cells are unknown. We have previously reported the total synthesis of Cytos A by biomimetic hetero-Diels-Alder reaction. In this study, to examine the novel bioactivity of Cytos, we synthesized Cytos C and measured cell growth-inhibiting activities of 7 compounds, including Cytos A and C, in several human cancer cell lines. Among these compounds, Cytos C and tetradeoxycytosporolide A (TD-Cytos A), a model compound for the synthesis of Cytos A, had anti-proliferative effects on cancer cells, and TD-Cytos A exhibited stronger activity than Cytos C. In vitro topoisomerase-mediated DNA relaxing experiments showed that TD-Cytos A inhibited the activities of topoisomerase I and II, whereas Cytos C targeted only topoisomerase I. These data suggest that the anti-proliferative activities of Cytos correlate with the inhibition of topoisomerases and implicated TD-Cytos A as a novel anti-cancer drug that suppresses the activities of topoisomerase I and II.
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http://dx.doi.org/10.1016/j.bmc.2019.06.014DOI Listing
August 2019

C‑mannosylation of R‑spondin2 activates Wnt/β‑catenin signaling and migration activity in human tumor cells.

Int J Oncol 2019 Jun 1;54(6):2127-2138. Epub 2019 Apr 1.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa 223‑8522, Japan.

R‑spondin2 (Rspo2), one of the four members of the R‑spondin family of proteins, has agonistic activity in the Wnt/β‑catenin signaling pathway, and it is associated with normal development, as well as disease, such as cancer. The present study focused on the C‑mannosylation of Rspo2, which is a novel and unique type of glycosylation that occurs via a C‑C linkage between the tryptophan residue and an α‑mannose. Although Rspo2 has two putative C‑mannosylation sites at residues Trp150 and Trp153, it had not been reported to date whether these sites are C‑mannosylated. Firstly, results from mass spectrometry demonstrated that Rspo2 was C‑mannosylated at the Trp150 and Trp153 residues. Notably, while this C‑mannosylation of Rspo2 resulted in increased extracellular secretion in human fibrosarcoma HT1080 cells, in other human tumor cell lines it inhibited secretion. However, C‑mannosylation had consistent effects on the activation of Wnt/β‑catenin signaling in PANC1 and MDA‑MB‑231 cells, as well as HT1080 cells. Furthermore, overexpression of wild‑type Rspo2 significantly increased the migratory ability of A549 and HT1080 cells, whereas overexpression of a C‑mannosylation‑defective mutant enhanced migration to a lesser degree. These results suggested that C‑mannosylation of Rspo2 may promote cancer progression and that the inhibition of C‑mannosylation may serve as a potential novel therapeutic approach for cancer therapy.
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http://dx.doi.org/10.3892/ijo.2019.4767DOI Listing
June 2019

Integrin β1 is an essential factor in vasculogenic mimicry of human cancer cells.

Cancer Sci 2018 Aug 9;109(8):2490-2496. Epub 2018 Jul 9.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.

Vasculogenic mimicry (VM) formation by cancer cells is known to play a crucial role in tumor progression, but its detailed mechanism is unclear. In the present study, we focused on integrin β1 (ITGB1) and assessed the role of ITGB1 in VM formation. We used in vitro methods to seed cancer cells on Matrigel to evaluate the capability of VM formation. We carried out ITGB1 gene deletion using the CRISPR/Cas9 system, and these ITGB1-knockout cells did not show a VM-like network formation. Further, reintroduction of ITGB1 rescued VM-like network formation in ITGB1-knockout cells. In conclusion, ITGB1 is a critical factor in VM of human cancer cells, and inhibition of ITGB1 may be a novel therapeutic approach for malignant cancer.
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http://dx.doi.org/10.1111/cas.13693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113431PMC
August 2018

Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

Biochem Biophys Res Commun 2018 04 1;498(3):466-472. Epub 2018 Mar 1.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan. Electronic address:

Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling.
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http://dx.doi.org/10.1016/j.bbrc.2018.02.210DOI Listing
April 2018

Topological analysis of DPY19L3, a human C-mannosyltransferase.

FEBS J 2018 03 16;285(6):1162-1174. Epub 2018 Feb 16.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.

C-mannosylation is a rare type of protein glycosylation, the functions and mechanisms of which remain unclear. Recently, we identified DPY19L3 as a C-mannosyltransferase of R-spondin1 in human cells. DPY19L3 is predicted to be a multipass transmembrane protein that localizes to the endoplasmic reticulum (ER); however, its structure is undetermined. In this study, we propose a topological structure of DPY19L3 by in silico analysis and experimental methods such as redox-sensitive luciferase assay and introduction of N-glycosylation sites, suggesting that DPY19L3 comprises 11 transmembrane regions and two re-entrant loops with the N- and C-terminal ends facing the cytoplasm and ER lumen, respectively. Furthermore, DPY19L3 has four predicted N-glycosylation sites, and we have demonstrated that DPY19L3 is N-glycosylated at Asn and Asn but not Asn and Asn , supporting our topological model. By mass spectrometry, we measured the C-mannosyltransferase activity of N-glycosylation-defective mutants of DPY19L3 and isoform2, a splice variant, which lacks the C-terminal luminal region of DPY19L3. Isoform2 does not possess C-mannosyltransferase activity, indicating the importance of the C-terminal region; however, N-glycosylations of DPY19L3 do not have any roles for its enzymatic activity. These novel findings on DPY19L3 provide important insights into the mechanism of C-mannosylation.
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http://dx.doi.org/10.1111/febs.14398DOI Listing
March 2018

Therapeutic activity of plant-derived alkaloid conophylline on metabolic syndrome and neurodegenerative disease models.

Hum Cell 2018 Apr 16;31(2):95-101. Epub 2017 Dec 16.

Division of Gastroenterology and Hepatology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, 480-1195, Japan.

Increasing metabolic syndromes including type-2 diabetes mellitus, obesity, and steatohepatitis are serious problems in most countries in the world. Neurodegenerative diseases such as Alzheimer, Parkinson's, and Huntington's diseases are increasing in many countries. However, therapy for these diseases is not sufficient yet. Thus, effective chemotherapy for these diseases is being expected. Conophylline is an alkaloid isolated from the leaves of Ervatamia microphylla and related plants. It was found to induce beta-cell differentiation in the precursor pancreatic cells. Oral administration of this compound ameliorated type-2 diabetes mellitus model in mice and rats. Later, fibrosis of the pancreatic islets was found to be greatly reduced by conophylline in the pancreatic islets. It also inhibited chemically induced liver cirrhosis. Further study indicated that conophylline inhibited non-alcoholic steatohepatitis in the model mice. On the one hand, loss of autophagy often causes protein aggregation to give neural cell death. Conophylline was found to activate autophagy in cultured neural cells. Activation of autophagy ameliorated cellular models of Parkinson's and Huntington's diseases. Thus, conophylline is likely to be useful for the development of chemotherapy for metabolic and neurodegenerative diseases.
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http://dx.doi.org/10.1007/s13577-017-0196-4DOI Listing
April 2018

Synthesis and evaluation of biological activities of vibsanin A analogs.

Bioorg Med Chem Lett 2017 10 31;27(19):4536-4539. Epub 2017 Aug 31.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address:

Vibsanin A is an 11-membered vibsane diterpenoid and is reported to induce myeloid cell differentiation via activation of protein kinase C (PKC) without tumor-promoting activity. Therefore, vibsanin A is thought to be an attractive compound for acute myeloid leukemia (AML) therapy. In this study, we synthesized vibsanin A analogs and compared the activity of these compounds for PKC activation and myeloid cell differentiation. We found that the hydroxymethyl group in vibsanin A is an important substituent to induce differentiation of AML cells. Collectively, our results showed the biochemical features of vibsanin A and provided new insights into the development of new antileukemic drugs.
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http://dx.doi.org/10.1016/j.bmcl.2017.08.059DOI Listing
October 2017

Dpy-19 like 3-mediated -mannosylation and expression levels of RPE-spondin in human tumor cell lines.

Oncol Lett 2017 Aug 22;14(2):2537-2544. Epub 2017 Jun 22.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa 223-8522, Japan.

-mannosylation is a unique type of protein glycosylation with a mannose attached to the tryptophan residue via the linkage. Our previous study revealed that dpy-19 like 3 (DPY19L3) acts as a -mannosyltransferase in human cells. The present study hypothesized that RPE-spondin (RPESP) may be a substrate protein of DPY19L3-mediated -mannosylation. RPESP has unknown biological functions and has two putative -mannosylation sites at the W and W residues; however, to the best of our knowledge, -mannosylation of RPESP has not previously been investigated. The present study suggested that RPESP is -mannosylated at W and W in human cells, whereas gain-of-function experiments using S2 cells revealed that human DPY19L3 catalyzed the -mannosylation of RPESP at W but not W, which suggested substrate specificity. In addition, the present study detected mRNA expression levels of RPESP in various types of cancer cell lines and high expression levels of RPESP were revealed in certain colorectal cancer cell lines, suggesting that RPESP may have an association with the malignancy of colorectal cancers.
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http://dx.doi.org/10.3892/ol.2017.6465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530190PMC
August 2017

Regulation of secretion and enzymatic activity of lipoprotein lipase by C-mannosylation.

Biochem Biophys Res Commun 2017 04 19;486(2):558-563. Epub 2017 Mar 19.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address:

Lipoprotein lipase (LPL) is a crucial enzyme in lipid metabolism and transport, and its enzymatic deficiency causes metabolic disorders, such as hypertriglyceridemia. LPL has one predicted C-mannosylation site at Trp. In this study, we demonstrated that LPL is C-mannosylated at Trp by mass spectrometry. Furthermore, by using wild-type and a C-mannosylation-defective mutant of LPL-overexpressing cell lines, we revealed that both secretion efficiency and enzymatic activity of C-mannosylation-defective mutant LPL were lower than those of wild-type. These data suggest the importance of C-mannosylation for LPL functions.
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http://dx.doi.org/10.1016/j.bbrc.2017.03.085DOI Listing
April 2017

Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells.

J Cell Biol 2016 Oct 3;215(1):121-138. Epub 2016 Oct 3.

Laboratory of Molecular Traffic, Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan Department of Cell Biology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.
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http://dx.doi.org/10.1083/jcb.201604030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5057288PMC
October 2016

C-mannosylation of R-spondin3 regulates its secretion and activity of Wnt/β-catenin signaling in cells.

FEBS Lett 2016 08 13;590(16):2639-49. Epub 2016 Jul 13.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.

R-spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β-catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C-mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C-mannosylation sites at Trp(153) and Trp(156) ; however, it had been unclear whether these sites are C-mannosylated or not. We demonstrated that Rspo3 was C-mannosylated at both Trp(153) and Trp(156) by mass spectrometry. Using C-mannosylation-defective Rspo3 mutant-overexpressing cell lines, we found that C-mannosylation of Rspo3 promotes its secretion and activates Wnt/β-catenin signaling.
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http://dx.doi.org/10.1002/1873-3468.12274DOI Listing
August 2016

Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin.

Biochem Biophys Res Commun 2016 08 31;476(4):445-449. Epub 2016 May 31.

Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195, Japan. Electronic address:

15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity.
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http://dx.doi.org/10.1016/j.bbrc.2016.05.142DOI Listing
August 2016

-glycosylation of R-spondin1 at Asn137 negatively regulates its secretion and Wnt/β-catenin signaling-enhancing activity.

Oncol Lett 2016 May 7;11(5):3279-3286. Epub 2016 Apr 7.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.

N-glycosylation is a post-translational protein modification with a wide variety of functions. It has been predicted that R-spondin1 (RSPO1) is N-glycosylated, although this remains unknown. The present study identified that RSPO1 was N-glycosylated at Asn137, and that N-glycosylation of RSPO1 negatively influenced its secretion and enhancing effect on Wnt/β-catenin signaling. treatment with peptide-N-glycosidase F increased the electrophoretic mobility of RSPO1. Furthermore, treatment of wild-type (wt) RSPO1-overexpressing HT1080 cells with tunicamycin (TM), which inhibits N-glycosylation, resulted in a significant reduction in the molecular weight of RSPO1. However, TM treatment had no effect in the RSPO1 mutant whereby the Asn137 residue was replaced by Gln (N137Q). These results demonstrated for the first time that RSPO1 is N-glycosylated at Asn137. RSPO1 is a secreted protein that has Wnt/β-catenin signaling-enhancing activity and is expected to have therapeutic applications. The role of N-glycosylation in RSPO1 was evaluated by conducting comparative experiments with wt and N137Q RSPO1, which revealed that the N137Q mutant increased the secretion and Wnt/β-catenin signaling-enhancing effect of RSPO1, compared with wt RSPO1. These results suggest that N-glycosylation of RSPO1 has a negative influence on its secretion and Wnt/β-catenin signaling-enhancing effect.
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http://dx.doi.org/10.3892/ol.2016.4425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841080PMC
May 2016

Identification of seco-clavilactone B as a small-molecule actin polymerization inhibitor.

FEBS Lett 2016 04 6;590(8):1163-73. Epub 2016 Apr 6.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, Japan.

Phenotype-based chemical screening is an attractive strategy for the identification of bioactive compounds. We searched for a compound that induces cellular morphological change and identified a novel compound that we named seco-clavilactone B (Seco-CB). Treatment with Seco-CB decreased the ratio of filament actin (F-actin) to globular actin (G-actin). An in vitro actin polymerization assay revealed that Seco-CB inhibited actin polymerization directly. Further analysis demonstrated that the inhibitory effect of Seco-CB on actin polymerization was associated with Seco-CB binding to either Thr5 or Cys285 of actin. These data indicate that Seco-CB is a novel actin polymerization inhibitor.
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http://dx.doi.org/10.1002/1873-3468.12154DOI Listing
April 2016

Identification of a novel compound that inhibits osteoclastogenesis by suppressing nucleoside transporters.

FEBS Lett 2016 04 3;590(8):1152-62. Epub 2016 Apr 3.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.

We screened small-molecule compounds that inhibit osteoclast differentiation to find new anti-osteoporosis agents and found that a novel compound, SUKU-1, suppressed osteoclastogenesis. We also synthesized 38 derivatives of SUKU-1 and discovered that nine of them had inhibitory effects on osteoclastogenesis and that SUKU-33 was the most potent inhibitor. Next, we investigated the mechanisms by which SUKU-33 suppressed osteoclast differentiation. By measuring the uptake of [(3) H]-uridine in cells, we found that SUKU-33 suppressed both equilibrative nucleoside transporters and concentrative nucleoside transporters. These results suggest that SUKU-33 inhibits osteoclast differentiation by suppressing nucleoside transporters.
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http://dx.doi.org/10.1002/1873-3468.12146DOI Listing
April 2016

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells.

Mol Biol Cell 2016 Mar 13;27(5):744-56. Epub 2016 Jan 13.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein-protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W(153) and W(156). Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W(153) and W(156) by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W(156) but not W(153) based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W(156) and found that DPY19L3-mediated C-mannosylation of Rspo1 at W(156) is required for its secretion.
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http://dx.doi.org/10.1091/mbc.E15-06-0373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803301PMC
March 2016

C-Mannosylation of thrombopoietin receptor (c-Mpl) regulates thrombopoietin-dependent JAK-STAT signaling.

Biochem Biophys Res Commun 2015 Dec 4-11;468(1-2):262-8. Epub 2015 Oct 24.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address:

The thrombopoietin receptor, also known as c-Mpl, is a member of the cytokine superfamily, which regulates the differentiation of megakaryocytes and formation of platelets by binding to its ligand, thrombopoietin (TPO), through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. The loss-of-function mutations of c-Mpl cause severe thrombocytopenia due to impaired megakaryocytopoiesis, and gain-of-function mutations cause thrombocythemia. c-Mpl contains two Trp-Ser-Xaa-Trp-Ser (Xaa represents any amino acids) sequences, which are characteristic sequences of type I cytokine receptors, corresponding to C-mannosylation consensus sequences: Trp-Xaa-Xaa-Trp/Cys. C-mannosylation is a post-translational modification of tryptophan residue in which one mannose is attached to the first tryptophan residue in the consensus sequence via C-C linkage. Although c-Mpl contains some C-mannosylation sequences, whether c-Mpl is C-mannosylated or not has been uninvestigated. We identified that c-Mpl is C-mannosylated not only at Trp(269) and Trp(474), which are putative C-mannosylation site, but also at Trp(272), Trp(416), and Trp(477). Using C-mannosylation defective mutant of c-Mpl, the C-mannosylated tryptophan residues at four sites (Trp(269), Trp(272), Trp(474), and Trp(477)) are essential for c-Mpl-mediated JAK-STAT signaling. Our findings suggested that C-mannosylation of c-Mpl is a possible therapeutic target for platelet disorders.
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http://dx.doi.org/10.1016/j.bbrc.2015.10.116DOI Listing
March 2016

Identification of a molecular target of kurahyne, an apoptosis-inducing lipopeptide from marine cyanobacterial assemblages.

Bioorg Med Chem Lett 2015 Nov 21;25(22):5295-8. Epub 2015 Sep 21.

Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Yokohama, Kanagawa 223-8522, Japan. Electronic address:

In 2014, we isolated kurahyne, an acetylene-containing lipopeptide, from a marine cyanobacterial assemblage of Lyngbya sp. Kurahyne exhibited growth-inhibitory activity against human cancer cells, and induced apoptosis in HeLa cells. However, its mode of action is not yet clear. To elucidate its mode of action, we carried out several cell-based assays, and identified the intracellular target molecule of kurahyne as sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). In addition, we found that kurahyne inhibited the differentiation of macrophages into osteoclasts.
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http://dx.doi.org/10.1016/j.bmcl.2015.09.044DOI Listing
November 2015

O-Fucosylation of CCN1 is required for its secretion.

FEBS Lett 2015 Oct 28;589(21):3287-93. Epub 2015 Sep 28.

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan. Electronic address:

The matricellular protein CCN1, also known as Cyr61, is a secreted ligand and has numerous functions. Human CCN1 contains one predicted O-fucosylation site in the thrombospondin type-1 repeat (TSR1) domain at Thr(242). In this report, we demonstrated that CCN1 is O-fucosylated at Thr(242) using mass spectrometry. Deficiency of O-fucosylation resulted in the decrement of the cell surface localization and the secretion of CCN1. Furthermore, knockdown of protein O-fucosyltransferase 2, which modifies a specific Ser/Thr residue in the TSR1 domain, decreased secreted levels of CCN1. These results demonstrated that O-fucosylation of CCN1 at Thr(242) regulates its secretion.
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http://dx.doi.org/10.1016/j.febslet.2015.09.012DOI Listing
October 2015

The Mammalian Sterile 20-like 1 Kinase Controls Selective CCR7-Dependent Functions in Human Dendritic Cells.

J Immunol 2015 Aug 26;195(3):973-81. Epub 2015 Jun 26.

Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain; and

The chemokine receptor CCR7 directs mature dendritic cells (mDCs) to the lymph nodes where these cells control the initiation of the immune response. CCR7 regulates chemotaxis, endocytosis, survival, migratory speed, and cytoarchitecture in mDCs. The molecular mechanisms used by CCR7 to regulate these functions in mDCs are not completely understood. The mammalian sterile 20-like 1 kinase (Mst1) plays a proapoptotic role under stress conditions; however, recently, it has been shown that Mst1 can also control homeostatic cell functions under normal conditions. In this study, we show that stimulation of CCR7 in mDCs induces Gαi-dependent activation of Mst1, suggesting the involvement of this kinase in the control of CCR7-dependent functions. Analysis of the mDCs in which Mst1 expression levels were reduced with small interfering RNA shows that this kinase mediates CCR7-dependent effects on cytoarchitecture, endocytosis and migratory speed but not on chemotaxis or survival. In line with these results, biochemical analysis indicates that Mst1 does not control key signaling regulators of CCR7-dependent chemotaxis or survival. In contrast, Mst1 regulates downstream of CCR7 and, of note, independently of Gα13, the RhoA pathway. Reduction of Mst1 inhibits CCR7-dependent phosphorylation of downstream targets of RhoA, including cofilin, myosin L chain, and myosin L chain phosphatase. Consistent with the role of the latter molecules as modulators of the actin cytoskeleton, mDCs with reduced Mst1 also displayed a dramatic reduction in actin barbed-end formation that could not be recovered by stimulating CCR7. The results indicate that the kinase Mst1 controls selective CCR7-dependent functions in human mDCs.
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http://dx.doi.org/10.4049/jimmunol.1401966DOI Listing
August 2015

Inhibition of MMP-2-Mediated Mast Cell Invasion by NF-κB Inhibitor DHMEQ in Mast Cells.

Int Arch Allergy Immunol 2015 13;166(2):84-90. Epub 2015 Mar 13.

Innovation Center for Immunoregulation and Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Background: Stimulation with antigen and IgE is known to activate NF-κB in mast cells. In the present research, we studied the role of NF-κB on cellular migration in mast cell-like RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) using the NF-κB inhibitor (-)-DHMEQ.

Methods: A Matrigel invasion chamber was used to evaluate cell migration. A PCR array was used to screen the expression of 84 key genes involved in cell migration.

Results: (-)-DHMEQ inhibited antigen/IgE-induced NF-κB activation and expressions of its target genes such as IL-6 and TNF-α. (-)-DHMEQ was found to inhibit in vitro invasion toward the antigen without any toxicity. We then looked for NF-κB-dependent genes that would be important for mast cell invasion using the PCR array. (-)-DHMEQ was found to lower the expression of matrix metalloproteinase (MMP)-2. The MMP inhibitor GM6001 also inhibited cellular invasion toward the antigen. These effects of (-)-DHMEQ were obtained in both RBL-2H3 cells and BMMCs.

Conclusions: These findings indicate that (-)-DHMEQ suppressed mast cell migration via the inhibition of NF-κB-regulated MMP-2 expression.
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http://dx.doi.org/10.1159/000371419DOI Listing
July 2015