Kagoshima University · Faculty of Fisheries · 教授

Research keywordsビテロゲニン・トランスポーター・ゼブラフィッシュ・GFP・アオコ・ケミカルバイオロジー・アポトーシス・細胞死・発がん・活性酸素

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• Ishii M., Tsurusaki A., Komatsu M., Shiozaki K. . Neu1-deficient zebrafish cells exhibit reduced Edwardsiella piscicida infection due to altered lysosomal exocytosis and membrane dynamics2025 · 記述言語： 英語 出版者・発行元： Fish and Shellfish Immunology Edwardsiella piscicida is a Gram-negative intracellular pathogen causing Edwardsiellosis, leading to economic losses in aquaculture. While phagocytosis is its primary infection route, alternative entry pathways remain largely unexplored. Neu1 sialidase, a lysosomal enzyme in glycoconjugate degradation, was investigated for its role in E. piscicida infection using primary cultured cells derived from Neu1-KO zebrafish fin (Neu1-KO cells). Compared to wild-type (WT) cells, Neu1-KO cells exhibited lower infection rates, which were associated with enhanced lysosomal exocytosis. Infection was restored by the intracellular calcium chelator BAPTA-AM, highlighting the role of exocytosis. Leupeptin, a cysteine/serine protease inhibitor, increased E. piscicida infection in Neu1-KO cells. Neu1-KO cells exhibited lower rab10 expression and reduced membrane ruffling, which was restored by BAPTA-AM and leupeptin. Given the role of epidermal growth factor receptor (EGFR) signaling, we assessed its phosphorylation, which was reduced in Neu1-KO cells but restored by treatment with BAPTA-AM and leupeptin. This suggests that inhibiting lysosome exocytosis or extracellular protease activity may enhance EGFR phosphorylation. These findings indicate that the decreased E. piscicida infection in Neu1-KO cells resulted from enhanced lysosomal exocytosis, leading to increased extracellular protease secretion, subsequent EGFR inactivation by extracellular protease, and reduced EGFR-regulated ruffling. This study provides novel insights into the regulatory mechanisms of bacterial infection and lysosomal exocytosis, informing potential therapeutic strategies against intracellular pathogens. DOI： 10.1016/j.fsi.2025.110273 Scopus PubMed
• Takumi S., Tomioka M., Yunoki Y., Eto R., Komatsu Y., Shiozaki K., Komatsu M. . Microcystin-LR-induced epithelial-mesenchymal transition-like cells acquire resistance to multi-toxins .2023 · 担当区分： 最終著者, 責任著者 記述言語： 英語 掲載種別： 研究論文（学術雑誌） 出版者・発行元： Toxicon The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation. DOI： 10.1016/j.toxicon.2023.107592 Scopus PubMed
• Hyodo T., Honda A., Yamate S., Kubo Y., Komatsu M., Shiozaki K. . Elucidation of the mechanism of nuclear localization of Mexican tetra Neu4 via bipartite nuclear localization signal and less2023 · 記述言語： 英語 出版者・発行元： Biochimie Nuclear sialoglycans are minor components in the nucleus, and their biological significance was not well understood. Recently, Nile tilapia Neu4 sialidase (OnNeu4) was identified and reported as the first nuclear sialidase in vertebrates. Although OnNeu4 possesses the nuclear localization signal (NLS) required for nuclear localization, other fish Neu4 sialidases, such as zebrafish and Japanese medaka, also possess NLS, but their subcellular localizations are not nucleus. To understand the nuclear localization mechanism of fish Neu4, we focused on Mexican tetra Neu4 (AmNeu4), which, unlike Neu4 in other fishes, has a bipartite NLS. AmNeu4 exhibited a wide range of optimal pH and substrate specificity, and its gene expression was specifically detected in the liver, spleen, and gut in adult fish. AmNeu4, like OnNeu4, exhibited nuclear localization, which was attenuated by importin inhibitor, and deletion of the bipartite NLS completely reduced the nuclear localization. In addition, the conjugation of the bipartite NLS of AmNeu4 made GFP show nuclear localization. To understand the mechanism of nuclear localization of AmNeu4 and OnNeu4, we compared fish Neu4 amino acid sequences and focused on the less conserved region of Neu4 sialidase (LCR). LCR-deletion mutants of AmNeu4 and OnNeu4 showed significantly reduced the nuclear localization. The LCR region in AmNeu4 and OnNeu4 possessed consecutive Ser/Thr. The Neu4 mutants in which consecutive Ser/Thr in LCR were changed to Ala or deleted significantly suppressed the nuclear localization. These results suggest that the nuclear localization of Neu4 in Nile tilapia and Mexican tetra may be regulated by NLS and LCR. DOI： 10.1016/j.biochi.2023.04.012 Scopus PubMed
• Sahashi D., Kubo Y., Ishii M., Ikeda A., Yamasaki C., Komatsu M., Shiozaki K. . Neu1 deficiency increases the susceptibility of zebrafish to Edwardsiella piscicida infection via lysosomal dysf2022 · 記述言語： 英語 出版者・発行元： Gene Neu1 is a lysosomal glycosidase that catalyzes the removal of sialic acids from glycoconjugates. Although Neu1 sialidase is highly conserved among vertebrates, the role of fish Neu1 is not fully understood because of its unique aquatic living situation. Compared to land animals, fish have a higher chance of bacterial infection, and to understand the role of fish Neu1, the susceptibility of Neu1 knockout zebrafish (Neu1-KO) was evaluated using Edwardsiella piscicida, a fish pathogen. Neu1-KO larvae showed high susceptibility to E. piscicida, despite the activation of macrophages, and presented increased lysosomal signals induced by the accumulation of Sia α2-3 linked oligosaccharides. The accumulation coincided with the signal of the macrophage marker, suggesting that the dysfunction of lysosomes in macrophages would result in a high susceptibility of Neu1-KO to E. piscicida. Chloroquine, an inhibitor of lysosomal degradation, induced high mortality of wild type zebrafish with E. piscicida infection accompanied by increased lysosomal accumulation, similar to Neu1-KO zebrafish. This study revealed that Neu1 sialidase plays a crucial role in the lysosomal degradation of macrophages with a bacterial infection. DOI： 10.1016/j.gene.2022.146667 Scopus PubMed

• アオコ由来肝臓毒Microcystin-LRの形質転換細胞誘導機序の解明と毒性抑制食材の探索
• 産業化学物質の感作性予知評価およびリスク評価への応用に関する研究
• アオコ由来マイクロシスチンLRの新奇な上皮間葉転換誘導能の生理的意味の解明
• ウナギビテロゲニン分解産物中のプロテアーゼ断片の精製及びその酵素化学的性質

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